The Involvement of Second Messenger Signalling in ... - FreiDok plus

Aus dem Institut für Umweltmedizin und Krankenhaushygiene der Albert-Ludwigs-Universität Freiburg i.Br.

The Involvement of Second Messenger Signalling in impaired Integrin Function of HIV-1 infected Macrophages

INAUGURAL-DISSERTATION Zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i.Br.

Vorgelegt 2005 Von Daniel Doischer Geboren in Bergneustadt

Dekan:

Prof. Dr. med. J. Zentner

1. Gutachter:

PD Dr. med. U. Frank

2. Gutachter:

PD Dr. med. A. Clad

Jahr der Promotion 2005

für Wenzel und Walther

Table of contents

i

Table of contents Table of contents

i

Abstract

v

Zusammenfassung

vi

Note of thanks

vii

Curriculum vitae

ix

Annexes

x

◊

Material

◊

Index of Pictures

xvi

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Index of Figures

xvii

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Index of Tables

xviii

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Scientific Abbreviations

xix

◊

References

xxii

xi

Table of contents

1

ii

Introduction 1.1

Pandemic

2

Literature review 2.1

The innate Immunosystem 2.1.1 2.1.2

2.2

The Complement: Steady-state components of non-specific immunity

Macrophages – members of the innate immune system 2.2.1 2.2.2 2.2.3

2.3

Ontogeny of monocytes The macrophage - an antigen presenting cell Effects of impaired phagocyte function

The importance of macrophages for HIV evolution 2.3.1 2.3.2

2.4

A sanctuary and “Trojan horse” Receptor expression by MDM

Integrin biology 2.4.1

2.5

Integrin configuration, activation and inside-out signalling

Phagocytosis 2.5.1 2.5.2

2.6

Basic elements in phagocytosis Cytoskeletal rearrangement and small GTPases

General concerns addressing second messengers 2.6.1

2.7

Cyclic AMP

HIV – Properties of a lentivirus 2.7.1 2.7.2 2.7.3 2.7.4

3

Etiology & taxonomy HIV-1: Morphology and Physiology Virus life cycle Viral proteins and enzymes

Material and Methods 3.1

Preparation of primary cell isolates 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5

Peripheral blood mononuclear cells (PBMCs) Monocyte isolation by adherence Isolate purity and viability Primary cell culture Micro reverse transcriptase assay

1 1

4 4 4 4

5 5 8 9

10 10 12

13 13

16 16 18

19 20

21 21 25 29 32

34 34 34 34 35 35 36

Table of contents

3.2 3.2.1 3.2.2 3.2.3 3.2.4

iii

Determination of Complement-mediated phagocytosis

36

Standardising contents of hemoglobin in sheep erythrocytes Preparation of human Complement Complement opsonization Phagocytosis assay

36 37 37 38

3.3

cAMP measurements

39

3.4

Pharmacological intervention

39

3.4.1 3.4.2

Augmentation of cAMPi Decrease of cAMP

39 40

3.5

Prostaglandin measurements

40

3.6

C’-mediated phagocytosis in the absence of retroviral replication

40

3.7

Statistics and Analysis

41

3.8

Immuno-fluorescence microscopy

42

3.8.1 3.8.2 3.8.3

4

Assessment of ratio HIV1-infected to non-infected macrophages Intracellular antibody-labeling of MDM on 96-well plates Fluorescence microscopy and digital imaging

Results 4.1

Isolation of human monocytes 4.1.1

4.2

Cell Purity

Phagocytosis assays 4.2.1

4.3

An in-vitro model for C’-mediated phagocytosis

Refinement of Phagocytosis Assay 4.3.1 4.3.2 4.3.3 4.3.4

4.4

Phagocytic capacity correlates with time of adherence Optimization of experimental variables Determination of autocrine factors contributing to the magnitude of Complement-mediated phagocytosis Solvent DMSO negatively affects phagocytosis by MDM

Long-term tissue culture 4.4.1 4.4.2

Purity control on plates by fluorescence microscopy Evaluation of HIV-infection in tissue culture

42 42 43

44 44 44

46 46

49 49 50 51 52

53 53 55

4.5

HIV-1 infection of MDM elevates intracellular cAMP levels

58

4.6

Pharmacological intervention

59

4.6.1 4.6.2 4.6.3

Rapid elevation of intracellular cAMP effectively inhibits Complementhowever not Fc-mediated phagocytosis Restoration of Complement-mediated phagocytosis by inhibition of cyclic AMP Specific and irreversible inhibition of adenyl cyclase with MDL-12 entirely reverses HIV-induced impairment of phagocytosis

60 61 64

Table of contents 4.7

iv

Delineating Mechanisms of Defective CR Function 4.7.1 4.7.2

5

Impaired phagocytosis by HIV-1-infected MDM does not result from altered prostaglandin secretion Suppression of C’-mediated phagocytosis is independent of retroviral replication

Discussion 5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7

Long-term adherent MDM as a model for tissue-resident macrophages Adoption and refinement of a colourimetric assay for C’-mediated phagocytosis of long-term adherent, HIV-1 infected MDM Expression of CR in the course of HIV-infection Second messenger & cytokines cAMP alterations induced by HIV impose on C’-mediated phagocytosis Bridging link: Rho and PKA pathways Disentanglement: HIV, cAMP and phagocytosis

66 66 67

68 69 71 72 72 74 75 78

Introduction

1

Pandemic

1 Introduction 1.1 Pandemic Twenty-four years ago, on June 5, 1981, the Morbidity and Mortality Weekly Report, (1981) the Bulletin of the Center for Disease Control (CDC), published a leading article about dengue fever infections in vacationers returning from the Caribbean to the USA. As it was not the major focus of the issue, a report by the University of California, Los Angeles, describing five cases of Pneumocystis carinii pneumonia in young, formerly healthy homosexuals, was generally overlooked. Unfortunately, it was impossible to foresee the ultimate extent of this crucial finding. That same year, several groups (Durack 1981, Gottlieb et al. 1981, Masur et al. 1981, Siegal et al. 1981) published their investigations into the sudden increase of a previously unknown and peculiar pattern of opportunistic infections. Simultaneously epidemiological studies provided evidence for clustering of this novel syndrome within the homosexual and intravenous drug-using communities. Thus, as early as then, the general consensus was to assume a common underlying cause, rendering particular groups susceptible to these diseases. Soon, transfusion recipients, infants, female contacts of infected men, prisoners, Africans and Haitians were also included in the group at increased risk (Sepkowitz 2001). Little was known about retroviruses, the most recent phenomenon in the field of virology. Only a decade earlier, their discovery had lead to the revelation of reverse transcriptase and the classification of human T cell leukemia/lymphoma virus types I and II (HTLV I, 1979 and II, 1981) (Poiesz et al. 1981, Kalyanaraman et al. 1982). Together with cytomegalovirus, Esptein-Barr virus and hepatitis B, concommitantly isolated from patients (Gottlieb et al. 1981), a novel viral particle was only one of many plausible hypotheses. In fact, a considerable number of attempts were made to unravel the causative agent for this syndrome. Creatively, causality was attributed to chemicals such as amyl nitrite, a prescription drug used recreationally by homosexual men for leisure purposes to increase sexual pleasure, or isobutyl nitrite, a room deodorizer. Other ‘sophisticated’ ideas included a chronic graft-versus-host reaction, suggested to be provoked by repeated exposure to ‘foreign’ sperm, or a general overloading of the immune system merging into physiological battle fatigue and wearing out biological defense (Sepkowitz 2001).

Introduction

2

Pandemic

The confusion caused by this new phenomenon, depicted in other non-scientific suggestions, supporting the notion of a punishment for homosexual men and injection-drug users. It was a simple intellectual leap to assume that a human analogue to the feline leukemia virus (described in the 1970s for its role in acquired immunodeficiency in cats) accounted for the syndrome (Hardy et al. 1973). In 1983, following of a complicated and rivalrous two-years race between ambitious research teams, the conundrum was solved by the detection, isolation and propagation of a novel virus (Barre-Sinoussi et al. 1983, Gallo et al. 1984, Levy et al. 1984). Its structure and properties were confirmed using new virological techniques and proved to markedly resemble a typical type-C RNA tumor virus. The requirement of magnesium for reverse transcriptase activity and an internal antigen (p25), similar to HTLV p24, implied that the new virus belonged to the family of HTLV I and II. However, the virus was distinctly new and was first classified as HTLV III, with regard to its morphological, biological and immunological characteristics. It is not far-fetched to assume that various interests such as the safety of health care workers, commercial interests of blood banks and overall public health highly influenced the development of HIV research, and certain groups might have impinged on revelations made in this field. The delay in accepting a virus as the agent truly provoking the syndrome may appear puzzling and is still matter of conjecture. Until 1998, publications claimed an antiviral drug (commonly administered to decrease viral load) was to account for the disease (Duesberg & Rasnick 1998). Until today, the symptoms previously described have not changed and in their combination are considered to be pathognomonical for the progress of a slowly augmenting exhaustion of the immune system. This development inevitably climaxes in its ultimate stage as The Acquired Immunodeficiency Syndrome, commonly known by its acronym “AIDS”. Introduced in 1986, the primary working definition of AIDS, as defined by the Center for Disease Control, has required only a single revision (1986). As early as this, a number of opportunistic microbes (Masur et al. 1981, Siegal et al. 1981) that successfully overrun the immune system of infected individuals were identified. Their names, as well as the resulting complications have gained essential importance. Most commonly mentioned in this context are names such as Pneumocystiis carinii, Candida albicans, Mycobacterium avium complex, Toxoplasma gondii, Herpes simplex virus (HSV1 and 2), Cytomegalovirus, Kaposi’s sarcoma, and angioimmunoblastic lymphadenopathy.

Introduction

3

Pandemic

Aside from discussion of a vast number of social implications, commercial interests, psychological interferences, mysticism and stigma originating from the discovery and investigation of HIV, epidemiological facts provided by the WHO (2000) attribute to the horrific number of 21.8 million deaths to the virus. Although most recent developments of modern medicine on the field of antiviral therapy have proved their efficacy in inhibiting the progression towards AIDS after infection with HIV, these achievements are only available in areas of the world where therapy is not limited by socio-economic restrictions. It is of ultimate significance to ensure that the majority of the 42 million people currently infected with HIV (WHO, 2003), but devoid of access to appropriate medication, will also benefit from new types of treatment in order to prevent worsening of the pandemic. Modern research is “in the line of fire” and it is our obligation, as well as in our interest, to fight the progressing outbreak.

Literature review

4

Innate Immunosystem

2 Literature review 2.1 The innate Immunosystem 2.1.1 The Complement: 2.1.2 Steady-state components of non-specific immunity Complement is the general term for a group of serum proteins that, in a delicately balanced manner, activate each other through cleavage, forming a cascade of perfectly orchestrated proteolytic events. First identified as a heat-labile principle in serum that ‘Complemented’ anti-bodies in the killing of bacteria, they amount to more than 3g per liter of plasma and constitute approximately 15 percent of the globulin fraction., The entire group of Complement proteins can be subdivided into three major systems, according to their function denominated (a) the classical pathway, (b) the mannose-binding lectin pathway, and (c) the alternative pathway (reviewed in (Walport 2001). Due to close cooperation at several stages, these mechanisms integrate 3 well-defined strategies: firstly, neutralization and lysis of pyogenic bacteria by opsonization, chemotaxis and activation of leukocytes; secondly, bridging of innate and adaptive immunity by means of augmentation of anti-body response and enhancement of immunologic memory; and lastly, clearance of apoptotic cell debris and of immune complexes from tissues. In general, all pathways merge on the level of Complement protein C3. Apart from its central role in particle opsonzation, C3 is the trigger and gatekeeper for the oligmerization of the lipophilic membrane-attack complex (MAC), resulting in penetration and disruption of the target cell’s membrane. Defective Complement pathways, both hereditary and acquired, may conflict with the delicate equilibrium in these systems. In this regard, hereditary Complement deficiencies can be subdivided into 3 classification: (i) deterioriation of opsonic activities causing pyogenic bacterial infections accompanied by distinctive rashes, (ii) impairment of the lytic activity leading to neisserial infections, and (iii) deficiency of mannose-binding pathway (Lachmann 1987, Lehner et al. 1992, Wurzner et al. 1992).

Literature review

5

Innate Immunosystem

DAF, (CD55/ decay-accelerating factor), is protein found on host cell membranes and is actively implemented in the regulation of autorecognition and host cell detoriation. With regard to the increase of mycobacterial spread in HIV-infected individuals, investigators provided evidence demonstrating that the virus is able to successfully evade Complementmediated virolysis. HIV incorporates DAF into its own cell membrane, thereby ensuring its survival by abusing the intrinsic protective property of this originally host-derived protein (Marschang et al. 1995, Saifuddin et al. 1995).

2.2 Macrophages – members of the innate immune system Although an extensive review of the immune response goes beyond the scope of this chapter, a brief overview seems indispensible in order for one to appreciate the pivotal role of macrophages in the background of the immune defense, and to dissect a compromisingly complex and intricately regulated sequence of events involving a multitude of cell types.

Immunogens of any kind (including viruses), having defeated and penetrated either external or internal defense barriers, are thought to be initially detected by a specialized class of professional cells called the antigen-presenting cell (APC). These APCs intercept and phagocytose minute amounts of the intruding particle, process it internally and display it to antigen-specific T-helper lymphocytes. These become activated and, in turn, promote the involvement of B-lymphocytes and cytotoxic T-cells. Communication at each stage of this process is mutually provided by secretory cytokines.

2.2.1 Ontogeny of monocytes Primordial classification of leukocytes into two basic subtypes was proposed by Metchnikoff, who coined the definition of macrophages and microphages to differentiate a multitude of cells preferentially characterized by their ability to internalize particles (Metchnikoff 1905). Although the clearance function of phagocytic cells was emphasized, leading to the description of the ‘reticulo-endothelial system’, in recent years the Metchnikovian division into mononuclear phagocytes and neutrophil polymorphs has been recovered (Gordon Reeves 1996). The cell-turnover of of the hematopoietic system in a man weighing 70kg is estimated to be close to 1×1012 cells per day, including 200×109 erythrocytes and 70×109 neutrophilic leukocytes.

Literature review

6

Innate Immunosystem

This marked process of cell renewal is only provided by a small population of bone marrow cells termed hematopoietec stem cells (HSC), which are generally defined by their unique, long-term ability to reconstitute all the various mature blood cell types (Uchida et al. 1993).

After several steps of differentiation leading to an intermediate promonocytic state, the cell emerges as a relatively large (12-20µm) monocyte prepared to leave the bone marrow after approximately 24h and circulate in the blood Monocytes are not very abundant in the blood, accounting for only 1-6% of all nucleated cells. They migrate into the tissue, having circulated in the vessel system for up to 70 hours. Nearly all tissues, organs, and serosal cavities harbour a population of resident phagocytes, most of which contain only a diffuse scattering of individual phagocytic cells (Picture 1). Regardless of their locations or appearance, all tissue-associated phagocytes are referred to as mononuclear phagocytes. In some organs, however, phagocytes, such as osteoclasts in the bone, brain tissue glial cells, and Kupffer cells in the liver are especially abundant and have distinct morphologic features (Stites 1997). Once having left the vessel into their designated tissue, macrophages live for 2-4 months. These phagocytes, however, do not comprise a static pool of cells being captured in the respective compartment but rather undergo a closed cycle of constant monocyte influx, macrophage migration via draining lymph nodes into the blood vessel, and cell death in the tissue.

Literature review

Picture 1

7

Cell differentiation of the macrophage lineage

Innate Immunosystem

Literature review

8

Macrophages

2.2.2 The macrophage - an antigen presenting cell APCs can be subdivided into two principle types the professional and non-professional APCs. Professional APCs are hematopoietic in origin and comprise macrophages, monocytes, dendritic cells and B lymphocytes which, by definition, are both capable of expressing MHC class II and of phagocytosis. Non-professional APC are less voracious than macrophages, tending to instead rely on receptor-mediated endocytosis or on pinocytosis. Exogenous immunogens become enclosed within phagosomes, and, after merging with a lysosomal vesicle, are processed via denaturation and partial proteolytic digestion. A limited number of the resulting peptides associate with non-covalently bound with MHC class II proteins for presentation on the cell surface. In the next stage, MHC II charged with antigenic peptides is recognized by THcells via a complex of CD3 T-cell receptor and its CD4 coreceptor. Throughout the entire sequence of immune activation, cytokine production is of ultimate importance for the balance between enhanced or decreased levels of response. In the case of a TH cell-mediated response, the lymphocytes can develop a partiality in cytokine expression that preferentially expands and activates either Tc or B cells. Although not applicable under all circumstances, a paradigm has been established which distinguishes between a Th1/cell-mediated immune response and a Th2/antibody-mediated response (Mosmann et al. 1986). As a consequence of the ratio of pro-Th1 cytokines (IL-12, IFN-γ) versus pro-Th2 cytokines (Il-4, IL-10), unprimed Th0 CD4+ T cells in the draining lymph node differentiate into either Th1 or Th2 cells. Once this polarization has occurred, it is self-reinforcing, as IFN-γ suppresses Th2 development and, conversely, IL-4 and IL-10 prevent Th1 development. This paradigm is clearly relevant in chronic infections, where Th1 and Th2 represent extremes of a continual dynamic by nature, rather than a categorical response to infection. With regard to HIV infection, there is circumstantial evidence that the progression of disease to correlates with a shift from Th1 towards Th2 cytokine profiles, (Clerici & Shearer 1994) although this view is still matter of conjecture (Graziosi et al. 1994). Interpretation of the data is difficult however, as there is (a) a constant destruction of CD4+cells by the virus and (b) considerations suggesting CD8+ Tc lymphocytes are probably involvement in the prevention of disease progress.

Literature review

9

Macrophages

2.2.3 Effects of impaired phagocyte function Macrophages are known to be multiply involved in the response against a marked variety of microbes, either directly via phagocytosis or indirectly by contributing to the microenvironmental pattern of cytokines. To delineate elements that, in their combination, are thought to confer the detrimental effects of HIV on phagocytes, it is required to examine the establishment of opportunistic infections arising from specific primary phagocytic defects. These defects may help to shed light on a scenario in which the immune system is impaired on specific levels by ablation of the phagocytic mechanism. Depending on their character, several defects can be categorized into groups comprising congenital neutropenia, defects of adhesion, defects of signalling, formation and function of neutrophil granules, and intracellular killing (reviewed in (Lekstrom-Himes & Gallin 2000)). In 1980, a report described a 5-year old boy with an augmented number of neutrophils, in association with recurrent infections and the absence of neutrophils in inflammatory foci (Crowley et al. 1980). The lack of a 110-kD surface glycoprotein in his neutrophils seemed to be profoundly related to an impairement in this cell type to adhere properly. Subsequently, it became clear that the process of aggregation and attachment of neutrophils to endothelial surfaces was mediated by a group of molecules, so-called integrins and selectins and that these molecules are essential for a normal inflammatory response (Larson & Springer 1990). Leukocyte adhesion deficiency type 1, an autosomal recessive disorder resulting from lack of β2 integrin adhesion molecules on neutrophils, may provide another excellent model for an individual devoid of a phagocytic immune response (Etzioni et al. 1999). Clinical features comprise severe periodontitits, recurrent infections of the oral and genital mucosa, intestinal and respiratory tracts, and skin. Pathogens shown to take advantage of this include the gramnegative enteric bacteria Staphylococcus aureus, as well as Candida and Aspergillus species (Brown 1997). Approximately 30 years ago, several cases of fatal infections following vaccination against Mycobacterium tuberculosis with an attenuated strain of BCG (Bacille Calmette-Guérin) unraveled the pivotal role of the interferon-γ-interleukin-12-axis against this intracellular parasite (Rosenberg et al. 1974). INF-γ, regularly secreted by T-cells and natural killer cells activates neutrophils and macrophages, which, in turn, would destroy the pathogen internally by an amplified production of hydrogen peroxide (Newport et al. 1996).

Literature review

10

Macrophages

In summary, apart from their involvement in Th1 and Th2-related immune response, (Chensue et al. 1995) activated macrophages have been shown to largely govern acute phase reactions, protective immunity against bacteria, fungi, parasites such as protozoa and helminthes, as well as limiting viral replication. Furthermore, it is noteworthy that antibodydependent cell toxicity of macrophages fundamentally contributes to detoriation and expelling of opsonized virus-infected cells.

2.3 The importance of macrophages for HIV evolution On the basis of current knowledge in disease progression, research in macrophage biology has gained crucial importance eliciting an ambiguous role in HIV cell tropism, presumed impairment of phagocytic response and cytokine signalling. Given the fact that HIV strongly impedes Fc-mediated ingestion, it was tempting to (i) assess whether or not this observation of phagocytic deficiency can be transposed to Complement-mediated response and (ii) if it is feasible to unravel and antagonize the pathological mechanisms.

2.3.1 A sanctuary and “Trojan horse” For more than a decade, the T-lymphocyte was in the focus of AIDS research. As a consequence, researchers have underestimated the vital role of macrophages for a long time, thereby delaying investigations in a number of pivotal questions regarding HIV. The erroneous position of macrophages is being re-evaluated (reviewed in (Khati et al. 2001)) following the revelation that primary HIV isolates essentially require a seven-transmembranedomain receptor (CCR5) for cell access while working in unison with CD4. Along with the classification of these receptors on macrophages and T-cells, HIV strains were described as M-tropic (via CCR5) or T-tropic (via CXCR4) strains (Berger et al. 1998) according to their preferential target of infection. Unlike T-tropic strains that emerge along with disease progression presiding over the fatal phase of AIDS, M-tropic isolates were found to be present predominantely early following infection (Schuitemaker et al. 1992, Zhu et al. 1993). The separate analysis of coreceptor evolution in sequential isolates from infected individuals (Connor et al. 1997, Scarlatti et al. 1997) markedly extended current knowledge and allowed the delineation of the scenario depicted hereafter (review in (Peden & Farber 2000). The life cycle of HIV naturally begins with host exposure to high titer body fluids such as blood or semen, typically via sexual transmission at mucosal tissues, the genital or colonic locations being the most common and likely, however not the only gateway into the host.

Literature review

11

Macrophages

Langerhans cells, expressing CCR5 in the submucosa, are assumed to be the initial target and major carrier of HIV to the nearest lymphoid station. Although Langerhans and dendritic cells can capture virus through CD4- and coreceptor-independent mechanisms, only R5 envelopes of HIV have the unique ability to mediate an activation signal to CD4+ T-cells. Thus, the preferential transmission of R5 strains ensures rapid recruitment of potential target cells and spreading in the lymph organ (Graziosi et al. 1998).

Despite mathematical-based calculations on the decay of various HIV-infected compartments (Perelson et al. 1997), the postulated eradication of the virus by HAART within 2 to 3 years has yet to be seen in practice (Crowe & Sonza 2000). The macrophage, a long-life phagocyte (Thomas et al. 1976), not suffering from injurious effects to cell structure (Crowe et al. 1987) when latently infected, provides a gateway into the brain, a sanctuary of decreased susceptibility to antiviral drugs. Together with dendritic cells, these phagocytes may fuse with T cells due to their approximity during antigen presentation (Crowe et al. 1992). Thus, many researchers have come to regard macrophages as “Trojan horses”, passing HIV by fusion to previously uninfected cells (Balter 1996). Albeit a consideably negligible source of plasma viral load (Perno et al. 1997), the macrophage is highly likely to drive disease development. It provides a continuous pool of viral particles which progresses, constantly under selective pressure, into the more virulent CXCR4 strain. This stage inadvertently results in the depletion of CD4+ T-cells (Brodie 2000) and the death of the host organism.

Literature review

12

Macrophages

2.3.2 Receptor expression by MDM Generally, the evaluation of receptor expression on cells requires further insight into the particular phase of maturation, differentiation, and state of activation. Thus, the number as well as the type of receptors on macrophages changes during the course of evolution from a hematopoietic stem cell into an active and tissue-resident phagocyte. At least 24 surface proteins have been elicited to be expressed by this cell species, some of which are indispensible in orchestrating pathogen recognition, binding, and internalization. The process of phagocytosis (greek, phagein = to eat), generally referred to as the clathrinindependent ingestion of particles greater than 0.5 micron in diameter, is induced via two principle clusters of surface proteins. The

former

group

is

constituted

by

vitronectin,

mannose,

scavenger

(CD36),

phosphatidylserin, β-glucan receptors, β1-integrins, and TLR2, a toll-like receptor, which resembles IL2 receptor and is stimulated by LPS (Aderem & Underhill 1999). The latter, in contrast, capable of opsonin-mediated recognition comprises various types of surface proteins, commonly known by their denomination as Fc- and Complement receptors. Four different Complement receptors have been discovered in mammals (CR1 to CR4), three of which CR1(CD35), CR3(CD11b/CD18), CR4(CD11c/CD18) and C1q receptor are expressed on human macrophages.

Literature review

13

Integrin biology

2.4 Integrin biology It is not intended here to depict all facets of the pathways involved in Complement receptor signalling. Instead, but to chiefly introduce the major players and roughly delineate the events that supposedly rule integrin activity preceding the issue of interest – Complement-mediated phagocytosis. In regard to the level of complexity, the following emphasizes the enormous number of crosslinks, theoretically available for retroviral interference at several stages of integrin function.

2.4.1 Integrin configuration, activation and inside-out signalling CR3 and CR4 exhibit common β2-subunit (Picture 2), whereas they diverge in the α subunit counterpart (αM and αX respectively). Integrins of the β2 family are involved in virtually all aspects of leukocyte function, including the immune response, adhesion to and transmigration through the endothelium, phagocytosis of microbes, and leukocyte activation (Arnaout 1990). As damage is likely to arise from a disbalance in integrin function, much effort was made towards the elucidation of mechanisms imperative in receptor efficacy and net effect. Unlike Fc Receptors, which are constitutively active, Complement receptors require supplementary stimulation to execute phagocytosis (Bianco et al. 1975, Griffin et al. 1984). They undergo a series of alterations during activation while simultaneously communicating with the cytoskeleton and the extracellular matrix. Phorbol ester derivatives, TNFα, GM-CSF, fibronectin (Wright et al. 1983), and differentiation of macrophages by adherence on plastic (Newman et al. 1980) may stand for the diversity of candidate stimuli capable of rendering the phagocyte standing by for particle uptake. It is striking, however, that Fc and CR are closely linked, since engagement of CR3 has been demonstrated to exert an accessory effect on FcR-mediated phagocytosis (Jones et al. 1998).

Literature review

Picture 2

14

Integrin biology

Molecular structure of the β2- Intgrin

Two common features of regulation, namely receptor affinity and avidity, were intensively scrutinized for their physiological relevance in integrin biology. In summary, receptor affinity is thought to be regulated by arresting the I domain (α-chain) in a steady state by means of the I-like domain of the β subunit. If triggered by changes to the receptor’s quaternary structure by external stimuli, the MIDAS1 is uncovered (first described for αM as a locus of coordination for divalent cations such as Mg2+ and Mn2+ (Michishita et al. 1993)) and exposes its iC3b-binding site (Harris et al. 2000).

Metal-ion dependent adhesion site1

Literature review

15

Integrin biology

Unlike other receptors, CR3 is rather promiscuous in favor of versatile interactions with an array of ligands such as neutrophil inhibitory factor, ICAM-1, factor X, Candida albicans and iC3b.

As a second principal mechanism, modulation of receptor avidity by segregation of integrins into clusters of adhesive patches was concluded from experiments on various cell and receptor models. It describes the cell’s unique property of directly responding to the environment via a mechanism called inside-out signalling. The process of receptor clustering, thought to basically adhere to paradigms outlined for the extensively studied integrin αLβ2 (LFA-1), is highly likely to be also applicable for CR3, an integrin of the same family (Lee et al. 1995, Loftus & Liddington 1997, Li et al. 1998). Nevertheless, it is not yet clear as to what extent these findings can be generalized among various families of integrins, as β1, β2 and β7 tend to slightly diverge at the level of intracellular regulation. Interestingly, there is substantial evidence implicating RhoA, a protein of the family of small GTPases, to be deeply involved in cytoskeletal rearrangement, thereby contributing to integrin function (Laudanna et al. 1996, 1997). Moreover, the ability of phorbol esters to activate CRs (Wright & Meyer 1986) appears to be associated with RhoA and, further downstream, with protein kinase C (PKC) to regulate inside-out signalling of αLβ2 in B-lymphblastoid cells. Jones et al. found that fusion of at least two previously independent signalling cascades is required for CR3 activation (Jones et al. 1998). Whereas CR3 activation by Fcγ-R ligation requires PI3-K and the actin cytoskeleton, both of these factors are irrelevant for G-protein linked receptors (recognizing ligands such as C5a, fMLP, Il-8) in triggering CR3. Although yet not confirmed, PAK1, a serine-threonine kinase, is likely to be in the final common pathway. Likewise, alterations in the phosphorylation state of the β2 tail differ depending on the trigger, therefore promoting the idea of two pathways. More precisely, both fMLP and PMA are readily able to confer integrin activation, the latter albeit only inducing β2 phosphorylation.

Literature review

16

Integrin biology

Other studies have shed light on two additional features whose importance for CR function has yet to be entirely identified. Firstly, the group of Sengelov investigated subcellular localization of integrin molecules. Intracellular clusters of CR1 and CR3, supposedly staying alert in the cytoplasm as a reserve pool, have been detected, at least in neutrophils. In response to an appropriate stimulus, however, they can be rapidly mobilized and ascend to the cell surface (Sengelov 1995). However, the emerging picture is that despite the number of studies into integrin biology and a better understanding of intracellular signalling cascades, the issue remaines largely obscure and increasingly intricate.

2.5 Phagocytosis Prior to the emergence of higher life forms, active internalization and destruction of foreign particles was of critical importance for driving evolution by providing primitive organisms with both defense and nutrition. Phagocytosis represents a highly optimized function in the multicellular organism, albeit still preserved among few monocellular organisms such as Entamobia histolytica. Generally, the process of phagocytosis is reserved to a confined spectrum of cells with the intrinsic ability to ingest particles greater than 5 micron. Particularly with mammals, this function is mainly carried out by a vast amount of macrophages which, by either circulating or residing in the tissues, work in concert with various types of less professional phagocytes.

2.5.1 Basic elements in phagocytosis To better comprehend the interplay of various elements which, in the case of Complementmediated phagocytosis constitute the biomolecular machinery and drive the process, it is feasible to further divide the process into three phases: (i) a pre-phagocytic stage comprising particle opsonization and integrin activation, (ii) particle uptake, mainly conducted by actincytoskeletal rearrangement and cell membrane movement, (iii) a post-phagocytic stage involving phagosome denuding from actin fibers followed by microtubulin-mediated propulsion in the cytoplasm and intravesicular maturation. Particle opsonization induces the first stage with Complement compounds, rendering the pathogen surface, usually less hydrophobic, susceptible to external recognition. In detail, C3b and C3bi deposition, resulting from both the classical and the alternative pathways, is able to confer both pathogen disruption via MAK or particle opsonization to facilitate phagocytosis, as described earlier.

Literature review

17

Phagocytosis

The precise and gradual follow-up to integrin activation is still one of the most troublesome and controversial issues. However, as for the major hallmarks, (defined by outside-in signalling and inside-out response,) the mechanisms adhere to a fundamental scheme, as depicted in the previous chapter. Most of the knowledge acquired in particle uptake is derived from extensive studies on Fcreceptors. It is therefore not recommended to transpose this data uncritically into a model for Complement-mediated phagocytosis, especially as CRs posess an elaborate aptitude for crosstalk and synergy. Comparing FcRs and CRs appears, however, to be advantageous at certain stages, whenever, rather than similarities, disparities among both pathways unravel apparent attributes of Complement receptors. Two models, namely a “trigger” and a “zipper” model, have been delineated to describe phagocytosis, current popular opinion generally favoring the latter (Griffin & Silverstein 1974, Griffin et al. 1975, Griffin et al. 1976). The “trigger” mechanism is based on the idea of a universal, all-or-none phagocytic response ensuing particle binding. According to the “zipper model”, in contrast, ingestion occurs by means of sequential engagement of a phagocyte’s membrane against the particle surface, whereas protrusions of pseudopods always remain confined to the perimeter arising from receptor-ligand interactions. In detail, sequential ligation of opsonins deposited on the pathogen to receptors in the phagocytic membrane precedes polymerization of actin microfilaments in the cytoplasm beneath the site of particle attachment. In the following step, the leading edge of the phagocyte advances, circumferentially protruding and enclosing the opsonized particle. The outcome is a phagosome internally embedded in the cytoplasm, prepared to further degrade its contents. In an elegant manner using electromicroscopy CR-mediated phagocytosis has been highlighted to be a relatively passive process which largely adheres to a variation of the classical zipper model (reviewed in (Aderem & Underhill 1999), unlike Fc-mediated phagocytosis, which is driven by active membrane protrusion, particles ligated by CRs appear to sink into the phagocyte, related to the formation of only small pseudopodia if any. Moreover, the phagosome membrane is less tightly opposed to the Complement-opsonized particle within. Instead, point-like areas, enriched with cytoskeletal proteins such as F-actin, vinculin, α-actinin, paxillin, appear to bridge ingested target and cytoskeletal scaffold beneath the phagosomal membrane. Together with these foci, numerous vesicles accumulate beneath the forming phagosome, supposedly indicating the magnitude of membrane trafficking (Allen & Aderem 1996a, b).

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18

Second messengers

2.5.2 Cytoskeletal rearrangement and small GTPases The small GTPases are thought to play a key role in the integration of signals among motor proteins, scaffold and second messengers to ensure a temporally and locally coherent orchestration of membrane traffic. A triplet of these GTPases, designated Cdc42, Rac1, and RhoA (in the order of their physiological function) has garnered special interest for its sequential involvement in Fc-Receptor pathways (Chimini & Chavrier 2000). Using embryo fibroblast migration assays, Cdc42 has been found to organize filopodin, thereby providing polarity, Golgi re-orientation and, in a microtubule-dependent manner, the directing of Rac against the leading edge during migration. Due to its ability of actin polymerization, Rac exerts a protrusive force on the leading edge and promotes lamellipodia formation. Stress fibers resulting from actin bundling, traverse the cell while binding to focal adhesions and cause integrin clustering (reviewed in (Schoenwaelder & Burridge 1999, Ellis & Mellor 2000, Hall & Nobes 2000). RhoA, commonly located in the cytoplasm, is translocated to the plasma membrane upon activation, where it is thought to (a) confer bundling of these stress fibers and (b) to drive actinomyosin-based contractility. It would be illusive to presume a functional redundancy among the group of Rho proteins. In fact, using models of monocytes and macrophages, data has been acquired in favor of these elements, along with PI3-kinases, to uniquely collaborate and to play distinct roles in cell motion (Ridley 2001). Yet, the function of Rac and Cdc42 seems antagonistic to those of RhoA. Whereas Cdc42 and Rac promote cell extension, RhoA supports contractility. This in itself can contribute to extension if strong adhesion is given. However, such strong adhesion to the substrate is not possible or in the interests of the cell. RhoA-induced contractility retracts the leading edge and opposes the extension induced by Rac or Cdc42 (Schoenwaelder & Burridge 1999). As a consequence, several investigators raised the question of whether each particular subtype of Rho proteins is differentially involved in Fc- and Complement receptor function (Caron & Hall 1998, May et al. 2000). Inhibitor studies demonstrated Fc-mediated phagocytosis to necessitate both Rac1 and Cdc42, as opposed to CRs, which showed no impairment. On the other hand, integrins proved highly dependent on the presence of functional RhoA leading to stress fiber contractility and Arp2/3 accumulation. The same appears to hold true for tyrosine phosphorylation and for Ca2+.

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19

Second messengers

Both these elemental players that are ubiquitously found and involved in a plethora of biomolecular pathways were not found to be critical for Complement receptors, however required for the FcR function. As for Ca2+, however, early studies suggested that an increase in concentration of this divalent cation is more likely to occur the result of rather than the cause of phagocytosis, representing a negative regulator or terminator of ongoing particle engulfment (Herlin & Borregaard 1983, Lew et al. 1985). The actual number of enzymes and second messengers, as well as the extent of their involvement in the biochemical machinery described above has yet to be entirely understood. Thus, the excerpt depicted represents only a mechanistical view and remains a superficial fragment of the true occurences behind the scenes.

2.6 General concerns addressing second messengers The identification and exploration of a multitude of second messenger molecules has revealed an exceptional level of complexity. In general, the information conveyed must be transferred intracellularly, evaluated, integrated, and either canalized focally or further allocated to several targets (reviewed in (Di Marzo et al. 1991). Dissection of these pathways is the focus of interest in molecular biology, and, although new techniques with higher specifity have evolved in this field, the data acquired is difficult to interpret and not rarely subject to heated debates. Some suggestions should be made to address these complications. Firstly, data should always be seen in the context of the system used, as models diverge among species, between cell lines, and the state of cell activation and differentiation. Secondly, the simplistic view of these signals to be solely distributed in the cytoplasm according to stochastic rules in a cell has to be reconsidered. In fact, investigations in second-messenger signalling have often unveiled ambiguous and contradictory properties, depending on the pathway they were implemented in. Compartmentalization might be the explanation for these prodigious habits, as it provides a model for locally confined messenger concentrations to account for a precisely regulated impact on the cell behaviour (Pryzwansky et al. 1998). The race for the detection of new second messengers and their targets is not yet over, and only some classes of chemical reagents might be mentioned hereafter: phophoinositides (IP, PIP2, IP3), nucleotidephosphates (GTP, GDP, ATP, cAMP, ADP), divalent cations (Ca2+), dissolved gases (NO).

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Second messengers

2.6.1 Cyclic AMP The importance in cell signalling of cAMP, an adenosine derivative, has been implicated since its discovery in the 60’s. Adenylate cyclase (AC) facilitates the transformation of ATP into cAMP and is ubiquitously present at basal levels of 65-950fmol per 106 cells with an average of ca. 308 fmol per 106 cells. Together with PDE (class IV), a rolipram-sensitive phosphodiesterase, AC negotiates alterations of cAMP concentrations temporally and locally required for a sudden response and a quick adaption to the microenvironment. Due to an intrinsic ambivalence, potential involvement of cAMP has garnered special interest in a plethora of biochemical pathways. Thus, extensive studies lead to the discovery and development of a great variety of agents, including VIP, adenosine, forskolin (AC activator), IBMX (PDE-inhibitor), PGE2 and PGE1, Cholera toxin, pentoxifylline, isoproterenol (βadrenergic agonist), and milrinone. Endogenous or synthetic by origin, these molecules trigger cAMP oscillations either up-stream (on the level of receptor transduction and AC activity) or further down-stream through PDE4, thereby enhancing or inhibiting cAMP degradation. As a net determinant, cAMP elevation in phagocytes (conveyed by means of protein kinase A) has been proposed to generally impede on immunoresponse, possibly in order to limit the damage arising from phagocyte dysfunction or an inappropriate reaction. This effect has already being accounted for in contemporary drug administration as represented by the implementation of two anti-inflammatory drugs, namely methotrexate and sulfalazine, both of which are known to increase cAMP. Although the precise pathways involved beyond the stage of PKA activation are largely obscure, several lines of evidence independently support the notion of cAMP as an immunoregulator. Adenosine, for instance, decreases the ability of neutrophils to adhere, aggregate, and originate a respiratory burst. It is therefore reasonable to assume that adenosine inhibits phagocytosis even more effectively in vivo, where the involvement of β2 integrins is probably more important, due to the fact that extravascularly located neutrophils phagocytosing invading pathogens are invariably in a state of adherence (Zalavary & Bengtsson 1998). As for Fc-receptor-mediated phagocytosis in a U937 pro-monocytic cell line, the inhibitory effect of cAMP has been established (Thomas et al. 1997). Likewise, impairment has been demonstrated in macrophage phagocytosis of apopotic cells (Rossi et al. 1998).

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Second messengers

Highly intriguing was data obtained by Nokta et al. who investigated the generation of cAMP and cGMP in several cell models using various strains of HIV-1. In fact, they were able to verify a significant increase of cAMP within 8 days, which declined to sub-control levels on day 14 post-infection (Nokta & Pollard 1991). However, whether or not these results are physiologically or clinically relevant with regard to phagocytosis, and the extent to which they can be generalized and transposed to other cell models (such as monocyte-derived macrophages) remains to be determined.

2.7 HIV – Properties of a lentivirus This review may be recognized as only a brief survey, meant to present key parameters of the HIV structure and life cycle and does by no means claim to be complete. The mechanisms depicted herein are based on current scientific knowledge of this issue. However, as some of these findings are still under discussion, certain assumptions may require confirmation.

2.7.1 Etiology & taxonomy The family of retroviridae is extensive, and currently comprises a total of seven genera, three of which, the spumaviruses, BLV-HTLV viruses, and lentiviruses, also include human tropic strains. The latter is represented by a great number of simian strains (SIV) as well as HIV-1 and 2, both recognized to be causative agents of AIDS and currently thought of as archetypical models of a retroviral infection (Murray 1999). All primate strains of this kind are genetically related, however distinctly different, and classified according to morphologic, antigenic, and enzymatic features. Since their discovery, however, these primate species have gained significant importance among all other strains and currently seem to constitute an entity of their own within the genus of lentivirus. In the late 1970s, the chapter of retrovirology was opened with the identification of HTLV I and II, referred to hereafter as Oncoviruses. Initial studies in HIV benefited greatly from knowledge acquired this far back. In summary, the picture represented by the genera of Spuma, Lenti, and HTL viruses along with the large group of non human-tropic species (mammalian and avian retroviruses of different types) seems to be complete and comprehensive, although the taxonomy in this field is still evolving (Picture 3).

Literature review

Picture 3

Taxonomy of the family retroviridae

22

HIV-Properties of a lentivirus

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23

HIV-Properties of a lentivirus

The more virulent strain of HIV-1, isolated in 1983 (Barre-Sinoussi et al. 1983, BarreSinoussi 1996) is predominant in Asia, Europe, America, and some parts of Africa, whereas HIV-2, identified in 1986, is found almost exclusively in West Africa. Following evolution of molecular biological techniques, such as gene nucleotide sequencing, homology between both types was revealed to be approximately 50%. This is remarkable, considering phylogenetic relations and the etiology among different species of lentiviruses. Notably, HIV-1 and HIV-2 are less closely related to each other than to their simian analogues SIVCPZ/SIVAGM, and SIVSMM/SIVSYK, respectively (Picture 4). As early as two years ago, Gao et al. contributed another piece to the understanding of lentiviral etiology (Gao et al. 1999). With Cercocebus atys, a mangabey, the origin of HIV-2 had already been elucidated. Following investigations in HIV-1 using mitochondrial DNA analysis, Gao detected a second independent case of cross-species transmission suggesting another primate, namely the chimpanzee Pan troglodytes troglodytes, to account for the original source. An event of ancestral genetic recombination is likely to have occurred, upon which SIVCPZ initiated a further endemic distribution of HIV-1 (subgroups M, N, and O). Apart from characteristics generally found in retroviruses which also define this family, the genus of lentiviruses possesses specific properties. Completely exogenous, non-oncogenic, and able to infect non-dividing cells, they cause chronic infections after a rather long period of incubation. As reflected in their denomination (lenti, lat. = slow) (Mandell 2000) these infections are not cleared by the immune system, and eventually lead to the accumulation of damage over the course of many years.

Literature review

Picture 4

Retroviral phylogeny

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HIV-Properties of a lentivirus

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25

HIV – Properties of a lentivirus

2.7.2 HIV-1: Morphology and Physiology 2.7.2.1

Virion Structure

The mature HIV-1 virion is spheric, and enveloped by a host cell-derived, cholesterol-rich, lipid bilayer including various host surface proteins (Arthur et al. 1992) such as HLA I, HLA II DR, actin, ubiquitin, microglobulin and, most importantly, two glycoproteins (SU, TM) which together comprise ENV (= envelope) protein, originating from the viral genome itself. While protruding from the lipid layer, the spiked-shaped trimer of Env (gp120) is anchored into the membrane, thereby tightly interacting with a transmembrane adapting protein TM (gp41) as demonstrated in Picture 5. A matrix shell, containing approximately 2000 copies of the myristoylated MA (p17) protein, lines the lipid bilayer (Mandell 2000). Each viral particle has a dense, conical-shaped core (CA = capsid), suggested to be attached to the lipid envelope. Consisting of an outer and inner leaflet comprising the viral proteins p6 and p24 respectively, the capsid surrounds a viral genome of 9 to 10kb, additionally stabilized by p7, another HIV product, in form of a dimeric ribonucleoprotein complex. Three viral enzymes, protease PR, integrase IN and reverse transcriptase RT are encapsidated by CA, thereby contributing to the concept of a highly organized and complex biological structure. In the mature virion, these enzymes are proportedly accompanied by only three of six accessory viral proteins, namely Nef, Vif and Vpr, whereas Rev, Tat and Vpu seem to be excluded (reviewed in (Turner & Summers 1999)). Lastly, cyclophylin A, a host cell-derived peptidyl-prolyle isomerase in complex with p17, is incorporated into the mature viral particle as well, facilitating virion disassembly and promoting replication (Agresta & Carter 1997).

Literature review

Picture 5

HIV-1 virion structure

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HIV – Properties of a lentivirus

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27

HIV – Properties of a lentivirus

The HIV-1 RNA contains two identical positive sense single-strands, 9.7kb in length and encoding nine open reading frames. Three of these frames, encoding Gag, Pol and Env polyproteins, are readily proteolyzed into individual proteins common to all retroviruses (Table 1). To produce many proteins from a single primary RNA transcript, retroviruses have evolved several elaborate techniques, namely (a) generation and proteolytic processing of precursor polyproteins, (b) alternative splicing of the primary transcript, (c) bicistronic mRNAs producing two proteins, and (d) suppression of translation termination or ribosomal frameshifting.

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RNA

core structural proteins

gag

env

enzymes

regulatory proteins

accessory proteins

LTR long terminal repeat

Table 1

Pol

HIV – Properties of a lentivirus

presumed function

designations MA CA NC p1 p3 p6 SU TM PR IN RT

matrix capsid nucleocapsid

p17 p24 p7

contains NLS2

surface transmembrane protease integrase reverse transciptase (heterodimer)

gp120 gp41 p10 p32 p66/51

binds CD4 binds CCR5/CXCR4

regulates RNA transport and splicing, binds RRE upregulates viral transcription via TAT disease induction CD4 downregulation infectivity and maturation arrests cell proliferation

Rev

regulator of viral protein synthesis

Tat

trans-activator of viral transcription

Nef

negative factor

Vif

virion infectivity factor

Vpr

viral protein regulatory

Vpu

viral protein U

virion release

U3

unique 3’ region

Promotor

U5

unique 5’ region

R

terminal redundancy

Tar

Tat responsiveness

RRE

Rev response element

Designation of viral structures

2

NLS (nuclear localization signal)

cooperates with TAT

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Virus life cycle

2.7.3 Virus life cycle 2.7.3.2

Replication cycle

The replication cycle of HIV (reviewed in (Turner & Summers 1999)) can be subdivided into two stages. The early phase is induced by particle binding, followed by membrane fusion, virus disassembly, uncoating, reverse transcription and translocation of the DNA into the nucleus, whereas the late phase is governed by the establishment and regulation of virus replication leading to virion budding and maturation, resulting in completion of the life cycle (Picture 6). A typical feature of HIV is the temporary regulation and expression of various RNA species. Differential splicing of synthesized mRNA has been implicated to be driven by Rev, a regulatory viral protein. In consequence to the accumulation of Rev protein during the initial phase, it is postulated to switch between the synthesis of highly spliced mRNAs (for Tat, Nef, and Rev itself) shortly after infection and the synthesis of unspliced, genomic (encoding Gag and Gag-Pol precursor) and singly spliced mRNA (encoding Env, Vpu, Vif, and Vpr) in advanced stages of the life cycle.

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Picture 6

HIV-1 replication cycle

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Virus life cycle

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2.7.3.3

31

Virus life cycle

Virus entry

Several independent lines of evidence reveal CD4as the major surface receptor molecule for HIV-1. This molecule, a coreceptor for MHC class II, is expressed on the surface of a range of cells including CD4 positive B- and T-lymphocytes, monocytes, macrophages, dendritic and Langerhans cells. Although thought to be imperative for infection with HIV-1, other receptors such as sphingolipid galactosyl ceramide on glial and neuroblastoma cell lines and Fc- receptors on macrophages binding to anti-body-coated virus, may substitute for CD4. In general, CD4 binds in a recessed pocket of gp120, initiating conformational changes in this receptor. The remodeling results in exposure of specific N-terminal fusion peptides in the associated subunit gp41, subsequently further promoting the process of fusion. Similarly to the mechanism described above involving gp41, the V3 loop of SU (gp120) is uncovered due to ligation by CD4 and may contribute to post-binding steps. Noteworthy, as alterations in V3 negatively impose on gp120 affinity to CD4 providing a novel concept of cell tropism of HIV.

2.7.3.4

Tropism

Earlier experiments have demonstrated that, as with other lentiviruses, macrophages could also be infected by HIV. Interestingly, preferences among distinctive strains of HIV in their capacity to enter T-cell lines or monocytes indicated that the interaction of Env with CD4 is not sufficient to allow entry. A group of chemokines, however, isolated from CD8+ T-cells and inhibiting infection with macrophage-tropic but not with T-cell line-adapted strains, have lend credence to the potential and unexpected involvement of additional receptor species in viral entry. Further studies have led to the identification of chemokine receptors, namely CXCR4, to account for T-cell tropism and formation of cell-syncytia, whereas CCR5 was detected shortly thereafter in a rapid series of investigations as the principal ligand for macrophage-tropic and non-syncytia forming strains of HIV. Previously, CXCR4 and CCR5 were only described for their physiological role in recognition of the chemokines SDF-1, and RANTES, MIP-1α and MIP-1β, respectively. For some viral isolates, however, CCR3 and CCR-2b are considered to serve as accessory proteins for viral entry in monocytes.

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Virus life cycle

At present, two potential scenarios have been proposed for CD4- and chemokine receptorinduced fusion of viral and cellular membranes: a) a spring-loaded mechanism, similar to that suggested for hemagglutinin (influenza virus), in which conformational changes in the TM ectodomain lead to displacement of its N-terminal fusogenic peptide toward the cellular membrane; b) a shedding mechanism, where binding of HIV to CD4 and chemokine receptor yields loss of SU proteins, enabling reorientation of the TM and subsequently permitting insertion of the fusogenic peptide into the cellular membrane.

2.7.4 Viral proteins and enzymes Interaction between the virus and its host varies greatly depending on the state of cell activity. A simplistic approach to understanding the mechanisms impinges on a great number of extracellular and intracellular components that contribute to the complexity of this system.

2.7.4.5

Integrase

Integrase, a 31kD multimer, is derived from the Pol fragment of Gag-Pol precursor, and is thought to drive formation of the preintegration complex. Due to its 3’ processing and DNA strand-transfer activity, it carries out random cleavage of the cell genome before conferring integration of the proviral DNA plus-strand.

2.7.4.6

Protease

HIV-1 protease (PR), shaped as a symmetrical homodimer, is released from Gag-pol precursor by an autocatalytic mechanism during maturation in collaboration with the adjacent p6 and p2 sites. Protease has been a principal target for drug design and with the description of its tertiary structure rational design became possible, leading investigations further towards the development of specific inhibitors In this respect, several classes of pharmaceuticals have been synthesized and are already available for administration of highly active antiretroviral therapy (HAART). After the release of Saquinavir, a protease-inhibitor of the first generation, new drugs including Ritonavir and Indinavir have followed, while other promising medicaments are currently undergoing clinical trial.

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2.7.4.7

33

Virus life cycle

Reverse transcriptase

RT possesses three basic enzymatic activities: RNA-dependent DNA polymerase, DNAdependent DNA polymerase and ribonuclease H, all of which are essentially implemented in reverse transcription.

Since their discovery, all three viral enzymes have been considered potential objects for antiviral drug design. Reverse transcriptase however, was the first protein striven for, and has been the unit most successfully targeted by therapeutical intervention ever since. This was principally achieved in two classes of enzyme-inhibiting drugs, namely NRTIs and NNRTIs. According to their denomination, NRTIs (nucleoside RT inhibitors) are nucleoside analogues comprising Didanosine (ddI), lamivudine (3TC), and Zidovudine (AZT). The latter, as the first drug of its kind, introduced a new era of therapy. The underlying mechanism is based on termination of the growing DNA chain during RNA reverse transcription and requires the host cell phosphorylation pathway in order to converte the inactive nucleoside into the active triphosphate. NNRTIs such as Nevirapine, Efavirenz, or Delavirdine, as opposed to nucleoside analogues, are specific for HIV-1 and intervene in a unique, however not yet entirely understood manner. As a standard anti-retroviral regimen, three drugs are used in combination – two NRTIs together with either a protease inhibitor or a NNRTI. There are many other possible combinations, none of which prove to be clearly superior. Other drugs, such as extracts from Chinese medicinal herbs Prunella vulgaris and Rhizoma cibotta, have been identified and studied for their disruptive effect on HIV-1 gp41 six-helix bundle formation, preventing virus fusion with its target cell (Liu, 2002). With Enfurvitide (T-20) a new class of drug recently appeared on the market, likewise extracellularly intercepting the virus prior to fusion. In unison with the conventional anti-viral regimen, the application of T-20 in therapy is promising and dampens reverse side effects on the level of cross-resistance and toxicity (O'Brien, 2003).

Material and Methods

34

Cell culture and Virus detection

3 Material and Methods 3.1 Preparation of primary cell isolates 3.1.1 Peripheral blood mononuclear cells (PBMCs) Under PC2 conditions, human PBMC’s were extracted from whole blood by Ficoll-Paque density gradient centrifugation as described elsewhere. Freshly obtained venous blood donations (supplied by the Red Cross Blood Bank, Melbourne) were diluted 1:1 with PBS(-) (M04) and 25mL were carefully layered onto a 15mL Ficoll-Paque (M01) cushion using conical tubes (50mL). Erythrocytes were pelleted by centrifugation (D01) at 914×g at room temperature for 20min and the leukocyte buffy layer collected from the Ficoll/plasma interface. For further purification, cells were washed in PBS(-) and pelleted at 10°C (585×g for 10min, 500×g for 7min, and 200×g for 10min), thereby extracting cytotoxic Ficoll and other contaminating cells such as erythrocytes and thrombocytes. Harvests were either spun at 500×g for 5min and left over night in 20mL of RF10 (M02) at 4 °C or immediately used for monocyte isolation. As determined by the Blood Bank on the following day, each sample was confirmed to be seronegative for HIV, HBV, HTLV and TPAK.

3.1.2 Monocyte isolation by adherence Briefly, the property of monocytes to strongly adhere to plastic dishes after only a short time of incubation was used to separate these cells from other less adherent peripheral blood mononuclear cells, such as lymphocytes and granulocytes. Extraction of monocytes was obtained by diluting cell harvest in adherence medium (M06) at a concentration of 2x107cells/mL (ca. 45mL). Approximately 3x108cells were transferred on 15cm Petri dishes (S04) and incubated for 1h (humidified air, 37°C, 5% CO2). By vigorously washing each plate 6-8 times in pre-warmed PBS(+) (M05), contaminating non-adherent granulocytes, platelets and lymphocytes were removed (as confirmed by quick microscopical observations (D11)). Following a last rinse with ice-cold PBS(-), adherent cells were left on ice for 15 to 30min, covered by approximately 20mL PBS(-).

Material and Methods

35

Cell culture and Virus detection

Using transfer pipettes (S03) to detach monocytes from the plastic surface, cells were recovered in PBS(-) supplemented with 5% FCS (M03) and pelleted at 400×g for 7min. Resuspended in 10mL of macrophage medium (M06), total monocyte number was assessed and viability determined by Tripan blue exclusion.

3.1.3 Isolate purity and viability As determined by Tripan Blue exclusion, viability of freshly isolated cells was confirmed to be greater than 95% immediately after isolation. Isolate purity was tested by flowcytometry on the day of isolation or on the day of plating on well plates. In contrast to the elutriation protocol, monocytes isolated by adherence were additionally analyzed for expression of CD14-PE (R01)(monocytes), and CD3-FITC (lymphocytes) (including no-antibody control). Singlicates of 2×105cells were aliquoted in FACS tubes (S06) and pelleted in 4mL of PBS(-) at 330×g for 5min, 10°C. Supernatant was aspirated, leaving 200µL in the tube. Anti-CD14PE Mab, anti-CD3 FITC Mab or no antibody were then added (0.7µg) to respective tubes followed by a quick vortex. An incubation of 30min on ice was followed by washes in 4mL of cold PBS (-), as described above. Supernatant was removed and cells fixed by adding 200µl of 1% ultrapure formaldehyde (R02). The average monocyte purity (depending on donor and method of isolation) was 60 to 85%.

3.1.4 Primary cell culture Monocytes were cultured in Teflon jars (S05), nourished by macrophage medium (at an initial concentration of 106cells/mL) and cultured at 37°C, 5%CO2 until use. Monocytes were allowed to differentiate in Teflon jars for 4 days, then centrifuged at 500×g for 7 min and resuspended in 5mL of its previous medium for counting. Approximately 3×105cells per quadruplicate were seeded on 96-well plates in 200µl of culture medium without any additional replenishment. On the day on infection, (i.e. after 1 day of adhesion or 5 days post monocyte isolation), nonadherent cells were washed off with 100µl of PBS(+) and 40µl of HIVBa-L was added, ascertaining a multiplicity of infection (MOI) of 0.1 to 1.0 virion particles per macrophage. As negative control we introduced a mock infection with 40µl of untreated macrophage medium (M06).

Material and Methods

36

Cell culture and Virus detection

Cells were incubated for 2 – 4h (100% humidified air, 37°C, 5%CO2) and 200µl of fresh macrophage medium replenished. The plates were left in the incubator, sealed and without medium changes, to provide viral replication and achieve both macrophage differentiation and strong adhesion on the surface. A colourimetric phagocytosis assay was conducted 7 days after infection.

3.1.5 Micro reverse transcriptase assay Levels of HIV replication in culture are thought to correlate with viral protein reverse transcriptase activity in virus culture supernatant. Commonly used in laboratories, the RT assay determines this activity by measuring the integration of isotope 33P -labeled TTP into the DNA product during reverse transcription. A sample of 10µl of each of the supernatants (gathered either during TCID50 determination or from experimental cultures) was transferred in triplicates into 96-round bottom well plates (S09). The virus was inactivated and the enzyme was released by adding another 10µl of 0.3% Nonident P-40 per well for 30min at 4°C. In the following, transcription was allowed to occur for 2h at 37°C in the presence of 40µl RT assay mixture (M10), 5µg/ml of template-primer p.An.dT12-18, and 3µCi

33

P-dTTP (R07).

8µl of the product were spotted on chromatography paper (S11), and air-dried for 30min. Surplus radioactive nucleotide was removed by 6-8 washes in 2×SSC buffer (M11). The paper was rinsed twice in 95% ethanol, dried in a microwave oven for 1 min and covered with melted scintillant (R08). Protected between two transparent plate sealers, scintillation of the sample was quantified [CPM] using a “LKB micro betacounter” (D09).

3.2 Determination of Complement-mediated phagocytosis On day 7 post infection with HIV-1, the phagocytic capacity of macrophages was measured using a lab-adapted colourimetric assay as described by (Gebran et al. 1992). The concentration of internalized SRBC correlates with the amount of hemoglobin in the supernatant following complete lysis of macrophage and erythrocyte. The oxygen carrier exhibits pseudoenzyme activity and reacts with DAF (R11), resulting in a blue product which absorbs light at 620nm, as assessed by spectrophotometry.

3.2.1 Standardising contents of hemoglobin in sheep erythrocytes A standard curve for each batch of erythrocytes was prepared, allowing for intrapolation of the amount of cells in suspension via the concentration of hemoglobin.

Material and Methods

37

Determination of Phagocytosis

A sample of 2×108 cells was washed in cold PBS(+) at 1430×g for 7 min, 4°C and hypotonically lysed in dH2O. The absorbance of the sample in 2fold dilutions was measured with a spectrophotometer at 541nm. A spreadsheet program (D10) was used to process data acquired in the following steps of particle opsonization.

3.2.2 Preparation of human Complement Active Complement components for particle opsonization were obtained from serum of human AB negative blood donations. Providing sterile conditions, 50mL of blood was aspirated by venipuncture using a butterfly catheter and 60ml syringes. Immediately thereafter, blood was transferred in a 50mL tube (S01) and placed in a waterbath at 37°C for 1 hour, promoting clot formation. Clotted erythrocytes and fibrin fibers were spun down at 3,000rpm, 4° for 10min and supernatant (containing Complement) gently aspirated. We aliquotted half of the obtained serum in Eppendorf tubes (S10) for inactivation on a heatblock at 56°C. Precipates resulting from protein coagulation were centrifuged at 20,000×g, 4°C). Supernatant was recovered in aliquots of 500µL and stored at -70°C until use for mock opsonization. The second half of the Complement remained untreated, and 50µl aliquots were snap-frozen in liquid nitrogen ensuring high activity of Complement proteins. Samples of heat-inactivated serum were reused, undergoing several cycles of thawing and freezing, whereas normal serum was only thawed once shortly prior to opsonization and discarded thereafter.

3.2.3 Complement opsonization Complement opsonization was performed by preparation of 2x108 sheep red blood cells immediately prior to phagocytosis assay. Storage medium was removed through three washes in cold PBS(+) at 1430×g for 7 min (4°C). Cells were then incubated with either 5% untreated or heat-inactivated human serum (Mills J, group AB neg.) for 1 hour at room temperature. Lysis usually occurred during incubation, providing a rough estimate for the amount of Complement components in the serum and confirming the strength of opsonization expressed by cell rupture through spontaneous assembly of the membrane attack complex.

Material and Methods

38

Determination of Phagocytosis

Therefore, after terminating opsonization with three quick washes in cold PBS(+) at 660×g, pellets were resuspended in 1100µL of PBS(+) and 100µl taken for determination of absorbance in a total of 1500µL of dH2O at 541nm. Concentration of opsonized and control cells was adjusted by dilution in an according volume of Iscove’s medium derived from Excel® program calculations.

3.2.4 Phagocytosis assay In order to minimize variations of medium volumes due to evaporation in the incubator during culture, the medium was changed two hours prior to the experiment and adjusted to 80 or 85µL per well. Samples of culture medium were recovered for assessment of infection by RT assay determination, debris spun down in Eppendorf tubes, (20,000×g for 5min) and stored frozen. We used a working solution of 5µl phorbol-12-myristate-13-acetate (R09) dissolved in a vehicle solution of 10% DMSO(R10)/Iscove’s medium to pretreat the macrophages, with a final concentration of 200nM PMA and 0.5% DMSO for 10mins. Phagocytosis was initiated by adding 10µL of SRBC’s, (1x106cells) at the ratio of 33 SRBCs per macrophage, briefly sedimented at 47×g for 5 min at 4°C. Ingestion of opsonized particles was allowed to proceed for 1 hour at 37°C, 5%CO2. Instant termination of particle engulfment was achieved multipipetting (D07) 100µl cold PBS(+) into the well. Non-internalized erythrocytes were first thoroughly removed by hypotonical lysis in 0.2% NaCl(aq) for 3min, whereas internalized SRBC’s were protected in the phagoyte from temporary variations of extracellular conditions. Three washes with 100µl of pre-warmed Macrophage Medium (37°C) were performed to withdraw the hemoglobin released by the burst erythrocytes. Macrophages (including ingested SRBCs) were exposed for 10-20min to 100µl of 8M Urea buffered with 0.2MTris-HCl (pH 7.4), concomitantly inactivating virions in the wells. In a modified version of a previously published procedure, we took advantage of the pseudoenzymatic property of haemoglobin to generate different tints of a blue product according to its concentration in the well by adding a volume of 100µL buffer containing 100mg DAF, and 100µL H2O2 dissolved in 10mL 10% glacial acetic acid. Reaction was allowed to occur for 5min, and absorbance evaluated at 620nm with an ELISA plate reader (D08).

Material and Methods

39

Determination of Phagocytosis

Absolute numbers of internalized SRBCs were estimated by interpolation from a 4-parameter standard curve derived from a series of 2fold dilutions of the erythrocyte working solution ranging from 1×106 to approximately 8×103 cells per well

3.3 cAMP measurements A total of 2x106 MDM per condition were lysed for determination of cAMP content. To extract cAMP and inactivate HIV-1, MDM were incubated with 50mM Tris/HCl containing 4mM EDTA (pH 7.5) for 20mins at 100°C. After incubation, cells were centrifuged at 20,000×g for 10mins, and supernatant collected. Measurements of intracellular cAMP were performed

using

a

commercially

available

assay

according

to

manufacturer’s

recommendations (Cyclic AMP 3H assay system, Amersham, International, Buckinghamshire, UK).

3.4 Pharmacological intervention Our primary investigations were aimed at manipulating biochemical pathways involved in Complement-mediated phagocytosis in HIV-infected or non-infected macrophages. Various commercially available drugs such as Forskolin (R14), MDL-12 (R12) or 2’5’ ddAD (R13) (known for their specific inhibition or activation of second messengers) were introduced in the assay in order to verify the role and importance of these proteins in the invitro system described above. In each case, the order of use and time of incubation for all implemented drugs were optimized and the reaction initiated by adding 5µl of the respective reagent 30min prior to the treatment with PMA (R09), representing the final step of the phagocytosis assay.

3.4.1 Augmentation of cAMPi 3.4.1.1

Forskolin

Forskolin was purchased from Calbiochem (R14) and stored at -20°C in 200µl aliquots of 10µM dissolved in 100% DMSO (R10). On the day of the assay, working solutions were prepared with Iscove’s medium (M12) in log10 dilutions and kept on ice until use. Next, 5µL of Forskolin and DMSO were added obtaining concentrations of the drug and the vehicle control ranging from 0.1 to 100µM and 1% respectively.

Material and Methods

40

Pharmacological effects

3.4.2 Decrease of cAMP 3.4.2.2

2’5’dideoxyadenosine

The cAMP analogue 2’5’ddAD (R13), provided by Calbiochem was diluted in 100% DMSO for stock at a concentration of 1mg/mL and stored in fractions of 20µL per Eppendorf tube at -20°C until use. Working solutions were prepared and treated as described earlier. The concentrations used for macrophage manipulations ranged from 0.1 to 10µg/mL and included a 1%DMSO vehicle control. Final levels of 1% DMSO in the medium on the plate rendering a vehicle control indispensable for the experiments subsequently prevented more extensive investigation at higher drug concentrations.

3.4.2.3

MDL-12, 330A, hydrochloride

MDL-12 (R12) was dissolved in water (50µL volumes, 5mM) and stored at –20°C. Further dilutions were prepared briefly in Iscove’s medium (M12) prior to the set up of the experiment. Dose-response experiments were done at drug levels of 0, 0.2, 1.0, and 5µM.

3.5 Prostaglandin measurements In selected experiments, the supernatant of HIV-1-infected and uninfected MDM was collected on day 7 post-infection, and stored at -70°C until analysis. PGE2 was measured by radioimmunoassay (Amersham) using manufacturer’s instructions.

3.6 C’-mediated phagocytosis in the absence of retroviral replication In selected experiments, MDM on day 7 post-isolation were incubated with the antiretroviral drug lamivudine (100µM; GlaxoSmithKline, Hertfordshire, UK), and cultured in the presence of these compounds until the phagocytosis assay was performed. All compounds were used at non-toxic concentrations, as assessed by Trypan blue exclusion (viability of MDM >95%).

Results

41

Pharmacological effects

3.7 Statistics and Analysis Raw data of absorbance were converted into values of internalized SRBC per 100 macrophages using standard Labsystem ELISA plate reader software. Unless mentioned otherwise, results are expressed as the means ± sem of at least 5 independent experiments set up in quadruplicates for each condition. To verify a statistically significant difference between experimental and control groups, we applied a paired two-tailed Student’s t-test.

Material and Methods

42

Immuno-fluorescence microscopy

3.8 Immuno-fluorescence microscopy 3.8.1 Assessment of ratio HIV1-infected to non-infected macrophages The advantages of combining both the technique fluorescence microscopy and digital imaging made it feasible to obtain a precise picture of the HIV status of each single macrophage in a culture which is only roughly confirmed to be HIV-positive on the basis of RT assay data. CD68 labelling was conducted to verify the purity of the cell culture, whereas staining for the viral p24-antigen provided a distinction between infected macrophages actively replicating virus and those and non-infected by-standers. Unless described otherwise, a multichannel pipette was used for all washes (100µl each) in this protocol in order to minimize delay of time and intra-experimental variations.

3.8.2 Intracellular antibody-labeling of MDM on 96-well plates On day 7 post-infection on 96-well plates (S09) with HIV1Ba-L, monocyte-derived macrophages were prepared for analysis under the fluorescence microscope by fixation, permeabilization of the cell membrane and antibody labelling. In short, specimens of supernatant were recovered from wells for RT assay determinations and the monolayer of adherent MDM’s washed twice with prewarmed PBS(+). Fresh fixative consisting of 3.5% paraformaldehyde in dH2O (pH 7.2-7.4) was first dispensed into the wells for 15min, followed by reaspiration and a gentle rinse with PBS(+). Immediately thereafter, cell membranes were permeabilized for intracellular labeling with chilling-cold acetone/methanol (-20°C) for 10min and unspecific binding of primary antibody was blocked using 2%FCS/PBS(+) for 15min. Serial dilutions of various primary and secondary antibodies in 2%FCS in PBS(+) were examined to minimize background fluorescence and permit microscopical analysis of both the presence of virion particles and macrophages purity in the culture. An incubation on ice over night using volumes of 40µL of the primary markers, antip24Mab(R06) (1:10) and anti-CD68Mab (R05) (1:50) were found to yield optimal identification of the viral protein and the phagocyte respectively. Secondary labelling was carried out at room temperature for 1 h using Alexa 568 goat antimouse antibody (R04) (1:50). HIV-negative controls for p24-labelling and no-primary antibody controls were implemented in the template to quantify unspecific antigen recognition.

Material and Methods

43

Immuno-fluorescence microscopy

Following each incubation, plates were placed on a vortex mixer equipped with an adaptor table for 96-well for 5min and intermittently washed three times with PBS(+) to gently detach and remove unbound antibodies. Hereafter, wells were replenished with 100µl of PBS(+), sealed with parafilm, and stored at 4°C for fluorescence microscopy.

3.8.3 Fluorescence microscopy and digital imaging At least 5 randomly chosen frames per well were examined and photos acquired with a high resolution digital camera (D05) connected to a phase contrast/fluorescence microscope (D04). The data was processed using the digital photo software V++® (D06) to superimpose and match pictures of equal frames taken either as phase contrast or fluorescent image. A total of 400 cells per well were counted and investigated for CD68 surface antigen and status of HIV infection.

Results

44

Isolation of human monocytes

4 Results 4.1 Isolation of human monocytes To maximize the relevance of the findings made in this project, cell experiments were carried out using primary cultures of human macrophages, rather than immortalized cell lines. Preparations were obtained by culturing monocytes, isolated from individual donors. This meant, however, that experimental results depended on the method used for initial monocyte isolation and were subject to the inherent variation due to differences among donors.

4.1.1 Cell Purity Given that optimal conditions are maintained throughout isolation, monocyte purity by adhesion often reaches peak levels of appoximately 90% in the hands of our laboratory (Table 2/Figure 1). Surface staining of monocyte preparations with CD3, CD19 and CD56 showed that the remaining cells largely comprised NK cells, T-lymphocytes, and B-lymphocytes. As opposed to total cell numbers, isolate purity increased during culture. Under the laboratory conditions, the contaminating cells appear to be significantly less resistant to sudden and harsh alterations of the environment they had undergone with isolation and culture (data not shown). Together with the predominant macrophages, these cells are known to secrete a cocktail of messenger molecules. Nevertheless, it is reasonable to assume that the concentration of growth factors and lymphocyte-specific cytokines contributed is insufficient to prevent apoptosis of the latter. stained for

monocyte fraction [%]

CD14 - PE = monocytes

82.0

no antibody

96.4

mean ± SEM

89.2 ± 7.2

CD3 - FITC = lymphocytes

4.6 [lymphocytes]

Table 2

Representative results

of monoycte purity determination by flowcytometry on day of isolation.

Results

Figure 1

45

Isolation of human monocytes

Monocyte purity

Analyzed by flowcytometry on day of isolation. To estimate the amount of monocytes and contaminating lymphocytes, anti- CD14 mAB-PE and anti-CD3mAB-FITC conjugates were used respectively. Without additional staining, however, cells may be distinguished roughly by size and granularity through forward scatter and sideward scatter height. Raw data was computed with WinMDI® Version 2.8

Results

46

Phagocytosis assay

4.2 Phagocytosis assays 4.2.1 An in-vitro model for C’-mediated phagocytosis Our aim of measuring particle internalization in HIV-infected macrophages was limited by safety requirements imposed by the PC3 containment necessary for HIV cultures. Generally, these demands led to the adoption of an accurate, expediend, and convenient colourimetric assay for the specific determination of both Fc-receptor and Complementmediated phagocytosis (Chan et al. 2001) using opsonized SRBC’s as targets. The specificity of this assay was verified in our in vitro model, based on properties published of Complement and its receptor. Firstly, deposition of C3b and iC3b on the surface of sheep erythrocytes, required for particle recognition by Complement receptors, occurs during incubation with untreated human serum, whereas treatment with heat-inactivated human serum, devoid of any active Complement components, results in un-opsonized SRBC, unable to be phagocytosed via CR’s. Secondly, receptors of the integrin family are not constitutively active and do not phagocytose C’-opsonized particles unless specifically activated. Under physiological conditions CR’s are activated by chemokines and cytokines produced in response to infection. Short-term activation of protein kinase C by phorbol esters (such as PMA), however, by-passes physiological activation signals and results in specific up-regulation of Complement-mediated internalization. Preliminary experiments were undertaken to optimize phagocytosis and interexperimental variability. These experiments defined the following parameters: (a) prestimulation with 200nm PMA for 10min at 37°C, (b) Complement opsonization with 5% human serum at room temperature for 1h, (c) all subsequent experiments were carried out with one preparation of Complement in serum, which, stored in small aliquots at -80°C, have been reported to remain stable for at least 6 months under these conditions (Newman et al. 1985), (d) each batch of SRBC was used for no longer than 1 month and standardized for the concentration of hemoglobin per erythrocyte (Figure 3); (e) all experiments included a DMSO vehicle control for PMA to quantify any effects of this solvent on phagocytosis; (f) the extent of phagocytosis in response to incubation with non-opsinized SRBC was considered to be mediated by mechanisms other then CR’s and hence subtracted from the experimental values, (g) erythrocyte concentration after opsonization was reassessed, as deposition of Complement components led to a small but variable lysis of SRBC due to the membrane attack complex (MAC) activity present in untreated serum.

Results

47

Phagocytosis assays

The degree of hemolysis, however, was found to predict whether the opsonized SRBC were phagocytosed by macrophages efficiently, as both lysis and opsonization are assumed to depend on the concentration of active Complement components in the serum. In practice, this meant that the time and serum concentration for opsonization had to be optimized to allow sufficient C3b deposition without excessive lysis (Figure 2/Table 3). Table 4 illustrates results of an opsonization procedure and the Excel®-based data sheet used to determine lysis and to re-assess the concentration of SRBCs as described above. The presence of human anti-sheep xenoantibodies, potentially able to opsonize SRBC with IgG, has been reported (Strokan et al. 1998), but appears to be negligible as confirmed by the low values obtained with the HI Serum negative control. These findings, in unison with the PMA negative controls (see above), demonstrate that phagocytosis does not occur via FcR’s and further underlines the specificity of this assay.

Figure 2

Loss [%] of initial SRBC during opsonization

35

Distinct difference between

preparations of SRBC using normal or heat inactivated human serum.

30 25

Concentrations

20

supernatant

of

free

directly

hemoglobin

after

in

opsonization

resulted from MAC-caused cell lysis and was

15

quantified by spectro-photometry. Cell lysis

10

is depicted as loss [%] of the initial number of SRBC. Data represents means ± SEM of

5

19 independent experiments.

0 1 Serum Normal

Heat inactivated

loss [%] of initial SRBC

heat-inactivated serum

normal serum

1.9 ± 0.3

28.5 ± 2.5

Table 3

Cell lysis during SRBC

opsonization with normal versus heatinactivated human serum.

Pooled data obtained in 19 independent experiments illustrates the difference in the ability of heat-treated and untreated human serum causing SRBC hemolysis following one hour of incubation.

Results

48

Phagocytosis assay

Figure 3 3,5

Hb concentrations per SRBC. y = 1E-08 x

3

OD [541nm]

Standard curve for assessment of

Each batch of SRBC was examined for its

2,5

concentration of hemoglobin per SRBC in

2

order to determine the relationship between

1,5

absorbance at 541nm (due to Hb) and the SRBC

1

number.

trendline

0,5

A

equation

standard was

curve and

obtained

and

implemented in an excel®-programme. Loss

0 0,E+00

1,E+08

2,E+08

3,E+08

4,E+08

SRBC

of cells during opsonization was interpolated from the pre-determined standard curve correlating with the content of dissolved Hb in the supernatant.

Table 4

Data sheet for calculation of loss of SRBC during opsonization and washing steps.

linear regression for SRBC

OD[541nm] = [SRBC] a= b= x= y=

x = (y-a)/b y = x b+a

0 1 x 10-8 # of SRBC in SN OD[541nm] measured

Re-suspension volume without lysis

2000

Initial SRBC taken

2.00 x 108

Temperature during opsonization [°C]

21.9

net OD:

# SRBC in 1.5mL [cuvette]

# of SRBC in 1mL [sample]

% loss during washes and opsonization

normal serum:

0.121

1.34 x 107

1.34 x 108

32.78

HI-human serum:

0.153

1.70 x 107

1.70 x 108

15.00

normal serum

HI serum

Volume (µl) of supernatant aspirated

100

100

Volume of PBS(+) added to SN for OD reading

1400

1400

Insert here measured OD[541] of SNin 1.5 ml)

0.121

0.153

Re-suspension volume in a total of (ml)

1344

1700

Results

49

Refinement of Phagocytosis Assay

4.3 Refinement of Phagocytosis Assay After leaving the blood circulation, the monocyte differentiates after 3 days to become a mature macrophage of and is, by definition, a resident tissue phagocyte in a steady state of adherence (Zalavary & Bengtsson 1998). The argument that the majority of, if not all, properties of phagocytes are directly or indirectly linked to adherence, might find its correlate in the close association of adhesion to cell differentiation and activation. In order to investigate phagocytosis of adherent cells and to better evaluate the in vivo situation, we reviewed our previous cell culture system and allowed macrophages to adhere for 7 days (i.e. from day 4 to day 12 post isolation) on 96-well plates instead of culturing them in suspension in hydrophobic Teflon jars. We therefore initially compared phagocytosis by macrophages adhered to plastic tissue culture plates for varying lengths of time.

4.3.1 Phagocytic capacity correlates with time of adherence Figure 4 shows the relative efficacy of phagocytosis by day 7 MDM adhered to plastic for 1 to 5 days. Macrophages from the same monocyte preparation were kept in suspension in Teflon jars. Equal numbers were then sequentially plated out on days 4, 5, and 6 postisolation, thus allowing them to adhere for the designated times prior to the phagocytosis assay (performed on day 7). We observed considerable enhancement of Complementmediated phagocytosis correlated with time of adherence.

relative PI

Particle internalization exhibited a 4.7 fold (± 1.6) increase after 72h.

7

Figure 4

6

mediated phagocytosis in infected MDM.

5

Data represents means and SEM of 3

4

independent

Adhesion improves C’-

monocyte

preparations

normalized and shown as relative values

3

of phagocytic index.

2

(PI [2h] considered 1)

1 0

20

40 Time [h]

60

80

Results

50

Refinement of Phagocytosis Assay

4.3.2 Optimization of experimental variables Initial experiments were conducted using macrophages seeded at a density of 50,000 per well of a 96 flat-bottom well plate. At this level, cultures were not confluent and no overcrowding was observed microscopically (Picture 7). It is, however, possible that cell density affects phagocytosis by adherent macrophages, thus determining the effect of seeding density on the phagocytic index. Cells were plated at 30,000 or 50,000 cells per well and assayed for Complement-mediated phagocytosis keeping the target : MDM ratio constant at 20:1. The results (Figure 5) show a 35% enhancement of phagocytosis at lower cell density. All of the following experiments were therefore conducted at this concentration.

Picture 7

Phase contrast microscopy of adherent MDMs on day 5 post-isolation

plated at a concentration of 30,000 cells/per well confirmed the phagocytes to be evenly spread and equally distributed. Neither confluence nor overcrowding was observed, leaving ample space for further cell growth and maturation.

relative PI [%] of Control

Figure 5

Effect of MDM monolayer cell density on

1,6

C’-mediated phagocytosis

1,4

As cells were studied in preliminary experiments at a concentration of 50,000 per well, data is normalized

1,2

for this condition and set as control. The figure is

1

representative of 4 independent cell preparations.

0,8

Values are shown as mean ± sem.

0,6

50,000 cells 30,000 cells

0,4

[%] PI of Control

0,2 0 No.cells of cells per well cells 50,000 30,000

1.00 ± 0.05 1.35 ± 0.18

Results

51

Refinement of Phagocytosis Assay

4.3.3 Determination of autocrine factors contributing to the magnitude of Complement-mediated phagocytosis As plating density was found to have an effect on phagocytosis, it was taken into consideration that accumulation of secreted products into the culture by the MDM (such as cytokines) may influence the assay. Cells were plated on day 5 and medium changed after days 3, 4, and 6 with particle internalization measured on day 7. As seen in Figure 6, replenishment of the medium did not affect the phagocytic index for either PMA-stimulated or unstimulated cells. Thus, no evidence was found for either stimulatory or inhibitory cytokines to be present in the culture medium at levels high enough to interfere with our assay.

Figure 6

No effect of medium replenishing on C’-mediated ingestion following long-term adherence

Interestingly and unexpectedly, under these experimental conditions C’-mediated phagocytosis exhibited rather stable results in both the total number of internalized particles and non-specific background activation. Results were obtained in two sets of experiments applying quadruplicates for each condition. Cells were seeded on day 5 post-isolation and medium replenished on day 3, 4, or 6. HIS controls were subtracted, and data was normalized for day 3 phagocytosis (set as 100%). Any slight alteration in cell performance is statistically negligible and within the standard deviations as illustrated by the error bars.

120%

relative PI

100% 80% DAY3 DAY4 DAY6

60% 40% 20% 0% PMA stimulated phagocytosis

unspecific background stimulation

Results

52

Refinement of Phagocytosis Assay

4.3.4 Solvent DMSO negatively affects phagocytosis by MDM DMSO was applied as a solvent for all water-insoluble agents used in this study. Although a vehicle control was included in all of the experiments, the effect of DMSO on phagocytosis was examined (Figure 7). Inhibition occurred in a concentration-dependent manner, however impairment of phagocytosis was not significant with the levels of DMSO used in this study (0.5 – 1.0%).

1,25

Relative PI

1,00 0,75 0,50 0,25 0,00 0

2

4

6

8

10

DMSO [%]

Figure 7

Effect of various DMSO concentrations (ranging from 0 to 10%) on Complement-mediated

phagocytosis of SRBC. MDM seeded for 24 hours on 96-well plates were incubated for 15min with the respective concentrations of DMSO supplemented with 200nm PMA. Data points and error bars represent the mean and SEM of three independent cell isolates.

Results

53

Long-term tissue culture

4.4 Long-term tissue culture 4.4.1 Purity control on plates by fluorescence microscopy Monocyte purity was examined by flow cytometry on the day of isolation (Figure 1/Table 2). It wasalso important, however, to determine macrophage purity after long-term adherence in order to facilitate interpretation of the experiments in relation to the effect of HIV-1 infection of MDM on Complement-mediated phagocytosis. On day 7, purity of adherent macrophage cultures was measured by staining cells for expression of intracellular CD68. Preliminary experiments determined the concentration of αCD68 hybridoma supernatant to achieve saturating levels of Ab (data not shown). Bound Ab was detected by counterstaining with Alexa 568-conjugated goat anti-mouse Ab. The use of this fluorescent probe had the advantage of being resistant to photo-bleaching caused by UVlight than traditional fluorophores and has a higher fluorescent yield. Bright field phase contrast and UV-light photos were obtained with a digital camera, superimposed with digital photo processing software, and examined for CD68 expression. Picture 8 demonstrates sections of the same microscopic frame (A-C) in a single experiment. The well containing the negative control was examined at a higher magnification (50×) and showed minor background noise. As can be seen, all cells stained positive for intracellular CD68, leading to the conclusion that the cell culture consisted of a 100% pure macrophage monolayer.

Results

54

Long-term tissue culture

Exposure A. MDM adhered for 12 days Phase contrast - bright light Magnification 20x

Exposure B. The same

section stained for

macrophage marker CD68 Texas red channel – fluorescence microscopy

Exposure C. Layer A+B offset

Exposure D. Negative control A different well of the same culture stained

as

above

primary antibody. Magnification 40x

Picture 8

Verification of macrophage purity in long-term adherent cell monolayer.

but

without

Results

55

Long-term tissue culture

4.4.2 Evaluation of HIV-infection in tissue culture HIV-infection of macrophages in cell cultures varies and is most commonly assessed by detecting the activity of the viral enzyme reverse transcriptase. The RT assay, however, solely provides a semi-quantitative answer about into the true underlying infection of the culture. Thus, in addition to testing culture supernatant for HIV RT-activity, a representative experiment fluorescence microscopy was used to determine the proportion of MDM, expressing intracellular p24 viral antigen. This provided a direct measure of the actual number of macrophages infected with HIV. After 7 days of infection, a strong fluorescent signal was detected from p24 viral core antigen showing a typical pattern of intracellularly labeled hot spots within the infected cells (Picture 9). Counterstaining, including a minus-primary antibody control, was performed using Alexa 568-labelled goat anti-mouse anti-body and a series of photos (A-C showing an example of a single section) was taken randomly to assess status of infection. Based on staining for p24, the finding that only a fraction of cells (46.7% (± 9.8) or 199 out of 436) were infected productively, is in perfect agreement with results from other laboratories using a MDM culture system (Glienke et al. 1994). An RT assay value of 807 CPM obtained for this experiment (Figure 8) estimates the mean values of all infections of MDM conducted under equal conditions (836.7 ± 85.6) and is within the range expected for efficient viral reproduction.

Results

56

Long-term tissue culture

Exposure A. HIV-1 infected MDM, day 7 on plates day 12 post isolation phase contrast - bright light Magnification 40x

Exposure B. The same section depicting infection marker p24 viral core antigen Texas red channel – fluorescence microscopy

Exposure C. Layer A+B offset

Exposure D. HIV negative control

Picture 9

Assessment of HIV-1 infection in long-term-adherent MDMs

(Magnification (40x).

Results

57

Long-term tissue culture

100%

Percent infected cells

90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

p24 positive cells

Figure 8

Determination of ratio HIV-1Ba-L-infected to non-infected MDM after 7 days of infection and

long-term adherence on 96well plates. MDMs were counted and examined for “hot spots” corresponding with HIV p24 antigen. In a represenative experiment, data represents means and STDEV of 436 cells counted in 7 randomly chosen frames.

Results

58

Determination of cAMP

4.5 HIV-1 infection of MDM elevates intracellular cAMP levels To determine whether cAMP may mediate the inhibition of C’-phagocytosis by HIV-1 infection, we measured intracellular levels of cAMP in HIV-1 infected and uninfected adherent MDM. Intracellular cAMP, measured in cell lysates 7 days post-infection, had an increase of 167% in HIV-1-infected MDM compared to uninfected MDM (n=5, p=0.005). This increase was observed in all experiments, but did not correlate with the level of infection as indicated by RT activity (Table 5).

Table 5

HIV-infection of MDM elevates intracellular cAMP

[cAMP]i pmol

RT

Exp

uninfected

HIV+

% increase [cpm/µl]

1

6,7

9,3

39

615

2

6,8

14,8

118

454

3

7,61

12,04

58

1777

4

3

11,78

293

430

5

1,75

7,55

331

3100

Mean

167,80

SEM

60,59

p-value

0,005

activity

MDM on day 5 post-isolation were infected with HIV-1, and cultured for the following 7 days. Intracellular cAMP was extracted and measured from 2×106 MDM on day 7 post-infection and measured by the use of a radioimmunoassay kit according to the manufacturer’s instructions.

Results

59

Pharmacological intervention

4.6 Pharmacological intervention Having established and optimized the phagocytosis assay and demonstrated HIV-1 infection of MDM, we then combined both techniques to investigate (a) whether HIV-1 downregulates Complement-mediated phagocytosis and (b) whether this inhibition occurred via elevation of cAMPi. Data recently published by our laboratory concerning properties of HIV-1 infected, short-term (24 hours) adherent macrophages, focused on the effect of 8-Bromo-cAMP on Complement and Fc-receptors. These experiments showed a specific impairment of Complement-mediated phagocytosis by cAMP analogues, thus corresponding to the literature. In this regard, however, this data does not indicate how HIV-1 might take its effect on MDM.

Results

60

Pharmacological intervention

4.6.1 Rapid elevation of intracellular cAMP effectively inhibits Complement- however not Fc-mediated phagocytosis In order to extend the observation that cAMP analogues inhibit Complement-mediated phagocytosis, we determined the effect of Forskolin, (a derivative isolated from the plant diterpene and known for its strong activation of adenylate cyclase), to expediently and specifically raise intracellular cAMP. Complement-receptors, as opposed to Fc-receptors, are strongly inhibited by cAMP, decreasing C’-mediated phagocytosis by ca. 80% at [100µM] forskolin. (n = 2 or 3, respectively, p < 0.0016) (Figure 9/Table 6). Thus, both the use of a cAMP analogue and an agent which elevates endogenous cAMP demonstrate the specific sensitivity of CRs to this second messenger. 1,2

relative PI

1,0 0,8 0,6 0,4 0,2 0,0 0

20

40

60

80

100 120 Complement Ig

Forskolin [µM] Figure 9

Effect of forskolin on C’-mediated and Fc-mediated phagocytosis

Table 6

Inhibition of Complement receptors by forskolin Unlike Fc-receptors, cAMP

Forskolin [µM]

C’-med. PI

Fc PI

0

1.00 ± 0.00

1.00 ± 0.13

mediated phagocytosis with

0.1

0.93 ± 0.06

0.85 ± 0.03

[100µM] forskolin. (n = 2 or

1

0.77 ± 0.03

0.90 ± 0.01

3, respectively, p < 0.0016)

10

0.55 ± 0.09

0.72 ± 0.02

100

0.20 ± 0.08

0.68 ± 0.07

stronlgy

inhibits

C’-

a decrease of ca.80% at

Results

61

Pharmacological intervention

4.6.2 Restoration of Complement-mediated phagocytosis by inhibition of cyclic AMP Various lines of evidence suggest that HIV, known for its destructive impact on the immune system, exerts this effect, at least in part, by interfering with specific pathways which execute particle internalization. Two independent observations - the elevation of cyclic nucleotriphosphates in HIV-infected cell lines and the cAMP-induced, specific down-regulation of Complement-mediated phagocytosis in macrophages - prompted us to examine the possibility of HIV inhibiting Complement-mediated phagocytosis on cAMP-induced pathways. The in vitro model and colourimetric assay established in our laboratory enabled us to both investigate macrophage development by microscopy under the conditions of HIV infection and, subsequently, to precisely quantify phagocytosis in combination with pharmaceutical manipulation. Two sets of experiments were conducted to address this notion.

4.6.2.1

Competitive inhibition of adenyl cyclase by 2’5’ddAD reestablishes C’-mediated phagocytosis in HIV 1-infected macrophages

MDM were cultured in Teflon pots for 5 days prior to infection and mock-infection on 96well plates with HIV-1Ba-L as described in “Material and Methods”. After 7 days of culture, both experiment and controls were incubated for 30 min with designated concentrations of 2’5’ddAD, a non-metabolizable competitive inhibitor of adenyl cyclase, and particle internalization assayed as described. Infection was confirmed by assessing RT-activity of supernatants obtained on the day of colourimetric assay (Table 7). The data from 8 independent experiments using cell preparations from various donors were pooled (Table 8/Figure 10), demonstrating that ddAD enhances Complement-mediated phagocytosis by MDM. Markedly, the effect of ddAD on HIV-infected cells is more pronounced than HIV negative controls with identical treatment. These findings are consistent with the model that HIV infection inhibits Complementmediated particle engulfment by elevating cAMPi. As the data plotted in Figure 11 shows, the treatment of HIV-infected MDM with ddAD restored phagocyte efficacy to levels comparable with those in uninfected MDM (Table 9).

Results

62

Table 7

Pharmacological intervention

Inhibition of C’-mediated phagocytosis in relation to HIV-1 infection.

mean ± sem RT [CPM]

836 ± 85.6

Inhibition relative to uninfected control

0.66 ± 0.06

1,8 1,7

relative PI

1,6 1,5 1,4 1,3 1,2 1,1 1,0 0

2

4

6 ddAD [µg/mL]

Figure 10

8

10

12

HIVHIV+

Effect of 2’5’ddAD on C’-mediated phagocytosis in HIV-infected MDM.

Longterm-adherent HIV-infected MDM and controls were incubated for 1h at designated concentrations of 2’5’ddAD, a non-metabolizable cAMP analogue. Results shown are the means (± sem) of 8 assays and are expressed as phagocytic indices relative to basal values of untreated controls at [0µg/mL] ddAD.

Table 8

Effect of 2’5’ddAD on C’-mediated phagocytosisrelative in HIV-infected MDM. PI

ddAD[µg/mL]

HIV -

HIV +

0.00

1.00

1.00

0.10

1.06 ± 0.02

1.11 ± 0.04

1.0

1.20 ± 0.05

1.27 ± 0.13

10.0

1.30 ± 0.06

1.57 ± 0.20

Results

63

Pharmacological intervention

1,1

relative PI (% uninfected control)

1,0 0,9 0,8 0,7 0,6 0,5 0

2

4

6

8

10

ddAD [µg/mL] Figure 11

12

HIV+ HIV- control

Restoration of phagocytosis in HIV-infected MDM.

Values expressed relative to untreated HIV negative controls (considered 1 and depicted as solid line). Results confirm (p< 0.0164) that the cAMP inhibitor is able to confer elevation of phagocytosis. At concentrations of 10µg/mL ddAD, particle uptake by infected macrophages reaches 97% (±7) of uninfected controls.

ddAD [µg/mL]

relative PI [HIV+]

0.00

0.68 ± 0.08

0.10

0.70 ± 0.09

1.0

0.80 ± 0.07

10.0

0.97 ± 0.07

Table 9

Restoration of C’-mediated phagocytosis in

HIV 1-infected MDM by competitive inhibitor 2’5’dideoxy adenosine

Two considerations made it appear reasonable to verify the data acquired in the first set. First, the inhibitor, 2’5’ddAD was dissolved in DMSO and, together with PMA, also contributed to the concentration of this cytotoxic solvent in the system. However, the final concentration still remained within the assessed limits (1.5%). Second, little is known about the kinetics of ddAD with regard to membrane penetration and its property as a reversible, competitive inhibitor. Further side effects on viral replication or lifecycle relating to its structural similarity to Didanosine (ddI), a drug commonly used in combination with other anti-retrovirals, seem unlikely. The time frame of ddAD incubation would be inadequate for these mechanisms to play a role in our experiments.

Results

64

Pharmacological intervention

4.6.3 Specific and irreversible inhibition of adenyl cyclase with MDL12 entirely reverses HIV-induced impairment of phagocytosis An irreversible, membrane-permeable and water-soluble AC inhibitor, MDL-12, structurally unrelated to ddAD was implemented in a second set of experiments to emphasize the correlation between inhibition of adenylate elevation of phagocytosis in HIV 1-infected macrophages. On day 7 post-infection, cells were pre-incubated for 30 min with MDL-12 at concentration levels ranging from 0 to 5µM and then allowed to internalize Complementopsonized SRBC for 1h. Figure 12 and Table 10 demonstrate the strong effect (p<0.035) at [1µM] of the inhibitor with relative PI (1.02±0.07) intersecting threshold levels defined by uninfected control cells.

HIV+ HIV- control

1,2

relative phagocytosis (% of uninfected control)

1,1

1,0

0,9

0,8

0,7

0,6 0

1

2

3

4

5

MDL [µM] Figure 12

Effect of irreversible and selective adenyl cyclase inihibitor MDL-12 on HIV 1-infected, long-

term-adherent MDM. On day 7 post-infection, cell preparations were treated with increasing concentration levels of MDL-12 for 1h prior to colourimetric assay determinations. Data is representative of three different cell isolates and is shown as the means (± sem) of PI relative to untreated, HIV negative controls (straight threshold line, considered 1).

Results Table 10

65

Pharmacological Intervention

Effect of irreversible and specific adenylate cyclase inhibitor MDL-12 on C’-mediated

phagocytosis in HIV 1-infected MDM.

MDL-12 [µM]

relative PI HIV+ [% of control]

0

0.68 ± 0.03

0.1

0.76 ± 0.03

1

1.02 ± 0.07

5

0.90 ± 0.06

Results

66

Delineation of mechanisms

4.7 Delineating Mechanisms of Defective CR Function 4.7.1 Impaired phagocytosis by HIV-1-infected MDM does not result from altered prostaglandin secretion HIV-1 infection has been shown to increase prostaglandin (PG) secretion in vitro (Mastino et al. 1993, Hayes et al. 2002) and prostaglandin E2 (PGE2) levels are elevated in the cerebrospinal fluid (Griffin et al. 1994) and serum (Delemarre et al. 1995) of AIDS patients compared to uninfected subjects. PGE2 exerts many of its biological effects through AC (Coleman et al. 1994), thereby elevating cAMP levels. To investigate whether elevated PGE2 caused the observed increase in cAMP during HIV-1 infection, PGE2 levels in the supernatant of HIV-1-infected and uninfected MDM were measured on day 7 post-infection, at the same time as the C’-mediated phagocytosis assay was performed. There was no significant difference

in

PGE2

levels

between

HIV-1-infected

and

uninfected

MDM

(Figure 13). These results suggest that elevation of cAMP in HIV-1-infected MDM is not due to elevated PGE2 levels, and that PGs do not play a significant role in the inhibition of C’mediated phagocytosis during HIV-1 infection of MDM.

Figure 13

HIV-1 infection of MDM does not alter basal secretion of PGE2

12 10

[pg]

8 6

HIV+ HIV-

4 2 0

MDM cultured for 5 days were infected with HIV-1 for 4 hours. On day 7 post-infection, the culture supernatant was collected and PGE2 levels were measured from a total of 5×104 MDM (n=4).

Results

67

Delineation of mechanisms

4.7.2 Suppression of C’-mediated phagocytosis is independent of retroviral replication We next considered whether active replication of HIV-1 within MDM was required to induce inhibition. HIV-1-infected and uninfected MDM were treated with the anti-retroviral drug lamivudine (100µM) immediately post-infection, and cultured for 7 days. This treatment effectively suppressed HIV-1 replication by 99%, however C’-phagocytosis by HIV-1infected MDM was still inhibited in the presence of lamivudine, indicating that replication of HIV-1 is not required for inhibition of phagocytosis (Figure 14). This concentration of lamivudine was not toxic to MDM, as assessed by trypan blue exclusion, and did not affect C’-phagocytosis by uninfected MDM (data not shown).

100

0,75

0,5

50

0,25

0

Figure 14

RT activity (%of HIV neg. control)

phagocytosis (% of HIV neg. control)

1

0 no drug

no drug

lamivudin

HIV-

HIV pos

HIV pos

Active replication of HIV-1 is not required for inhibition of C’-mediated phagocytosis.

Lamivudine was added to MDM immediately after HIV-1 infection, and cultured for a further 7 days under adherent conditions. Phagocytosis assays were performed on day 7 post-infection using C’-opsonised sRBC as targets. Data are presented as the phagocytic index as a percentage of mock-infected and untreated controls (n=3; mean ± s.e.m).

Discussion

68

5 Discussion In recent years, the focus of interest in HIV research has started to shift from its primary target, the T-lymphocyte, towards a cell species chiefly known for its main function as a professional phagocyte, termed macrophage. Recent findings have revealed that macrophages, scavengers by nature, provide an indispensable gateway for HIV into the host shortly after exposure (Graziosi et al. 1998). Thus, extensive studies have gained priority, in an attempt to elucidate their role as essential regulators of the immune response in regard to the spreading of progression of the disease following HIV infection. Two principal considerations led us to investigate Complement-mediated phagocytosis in HIV-infected monocyte-derived macrophages: firstly, a rapid increase of the cyclic nucleotides cGMP and cAMP (4 fold after 4 days and 40 fold after 8 days) in MT-4 cells, U973, and PBMCs in the course of HIV infection, which was conserved among at least three different strains of virus (Nokta & Pollard 1991, Hofmann et al. 1993a, Thomas et al. 1997). Secondly, several lines of evidence demonstrate HIV-1 to generally impair both receptormediated particle uptake by phagocytes from infected individuals, (Wehle et al. 1993, Chaturvedi et al. 1995, Koziel et al. 1998), as well as specifical Complement-mediated phagocytosis in MDM (Biggs et al. 1995). Regarding alterations in levels of intracellular cAMP, generally implicated to interfere with several functions of professional phagocytes (Rossi et al. 1998), it was tempting to investigate this coincidence based on the assumption that HIV-induced impact on phagocytosis might be directly related to cAMPi levels. Although observations have repeatedly confirmed a defective phagocytosis of pathogens such as MAC, Candida albicans, and Toxoplasma gondii by HIV-1 infected MDM in vitro (Crowe et al. 1994, Biggs et al. 1995, Kedzierska et al. 2000), the true mechanisms of the underlying pathogenesis are still a matter of conjecture. This study shows that HIV-1 infection of MDM leads to a significant impairment of C’-mediated phagocytosis, and provides evidence that the underlying mechanism involves cAMP.

Discussion

69

Our data demonstrate that (i) agents that elevate intracellular cAMP decrease phagocytosis by uninfected MDM; (ii) infection of MDM with HIV-1 is associated with higher levels of intracellular cAMP 7; (iii) agents that block cAMP production neutralise the effect of HIV-1 infection, and restore C’-mediated phagocytosis by HIV-infected MDM; (iv) inhibition occurs post-viral-binding but pre-reverse transcription. Infection of MDM with HIV-1 in vitro did not have a consistent effect on PG secretion, suggesting that PGs do not contribute significantly to elevated cAMP levels, nor to defective phagocytosis by HIV-1-infected MDM.

5.1.1 Long-term adherent MDM as a model for tissue-resident macrophages Tissue macrophages, a cell type in discussion for its ability to cross the blood-brain barrier and to provide a sanctuary for HIV, are long-lived cells, typically found in a steady state of adherence in the extracellular matrix (Zalavary & Bengtsson 1998). It should be taken into account, however, that cells of this lineage are highly heterogeneous with respect to phenotype and function (Kreutz et al. 1992). Tissue-resident intestinal macrophages, as opposed to peripheral monocytes, have been proven to be hyporesponsive to many stimuli while not migrating from their compartment, where they fulfill their duty as scavenger cells (Fais & Pallone 1995). In particular, the differentiation of monocytes into macrophages results (a) in the upregulation of receptors such as CCR5 (receptor for monokines and for HIV-1 ligand gp120) (Cheng-Mayer et al. 1997, Fear et al. 1998) and (b) in enhanced responsiveness to the biological effect of cytokines such as type I and II IFNs (Fantuzzi et al. 2000). To circumvent problems that arise from a physiological heterogeneous population of primary macrophage cell systems, many groups preferably used models of immortalized monocytoid cell lines, some of which constitutively express HIV-1, facilitating the experimental design and optimizing inter-experimental variability (Thomas et al. 1997). However, the permissiveness of cell lines for several strains of HIV-1 appear to deviate significantly from primary monocyte isolates and their descendents (Valentin et al. 1994), further confirming conclusions made by other groups that coherent, immortalized cells do not provide an adequate surrogate for MDMs (Schuitemaker et al. 1992).

Discussion

70

Refinement of a Colourimetric Assay

Considering that the discrepancy among different HIV isolates to rapidly infect and replicate in macrophages is strongly influenced by the procedure of isolation and culture, it has yet to be determined which of the protocol or models applied is the most relevant (Valentin et al. 1994). Other features of distinction between these cell lines and primary macrophages, apart from those mentioned, are difficult to evaluate. We therefore chose primary cells for our system in order to improve extrapolation from the in vitro to the in vivo situation. Maturation and differentiation of macrophages, on the other hand, are of prime relevance to investigate pathophysiological mechanisms in vitro (Fantuzzi et al. 2000) concerning the auto-regulation within the chemokine/cytokine network during HIV-1 infection. To further approach the physiological situation, we optimized our in vitro tissue culture system and replaced hydrophobic Teflon jars for 96-well plates, allowing day 5 macrophages to adhere for 7 days before assessing phagocytic capacity. We therefore examined these phagocytes adhered to plastic tissue culture plates in preliminary experiments for varying lengths of time, observing a 4.7 fold increase in phagocytic capacity after 72 hours. This might be explained by a general increase in cell size, whereas effects of autocrine priming through cytokines (e.g. GM-CSF and M-CSF) appear minor. However, it is not unlikely for adherent macrophages to start cytokine production upon adhesion, thereby enhancing proliferation, differentiation and phagocytic response. On the basis of our data, replenishment of culture medium devoid of cytokines on day 3, 4, or 6 did not affect phagocytic indices of PMA-stimulated or unstimulated cells. Despite a general elevation in phagocytosis and high cell survival rates during long-term culture of adherent, uninfected MDMs, no further evidence was obtained for the presence of either stimulatory or inhibitory cytokines in the culture medium at levels permitting interference with our assay. Together with monocytes, LPS-stimulated macrophages proved relatively refractory to HIV infection. We thus adjusted our experimental design to find formal proof for the fraction of MDM on the plate showing active replication of viral particles. As assays merely quantifying RT activity in supernatant were not able to provide this information, we screened our culture on day 12 post-infection for the expression of viral p24 antigen. Interestingly, only 46.7% (± 9.8), or 199 out of 436 cells stained positive for HIV p24 protein. However, this value corresponds with observations by other groups (Glienke et al. 1994), and was taken into account for further interpretation of our findings.

Discussion

71

Refinement of a Colourimetric Assay

5.1.2 Adoption and refinement of a colourimetric assay for C’-mediated phagocytosis of long-term adherent, HIV-1 infected MDM For our experiments, we took advantage of a colourimetric phagocytosis assay previously developed in our laboratory (Chan et al. 2001) for a precise, expedient, and reproducible evaluation of both Fc’ and Complement-mediated phagocytosis under HIV infection conditions. Initially, a variety of agents such as phorbol esters (PMA), phosphotyrosine kinase inhibitors (Genisteine), and non-metabolizable cAMP analogues (8-Bromo cAMP) were evaluated for their effect on our model. This pharmacological manipulation of cell-signalling pathways enabled us to validate the new technique and to further characterize certain dissimilarities in Fc-receptor and Complement-mediated phagocytosis. Disparities observed between our data and some studies published by other groups, are likely to be derived from: (i) the protocols used to culture MDM, (ii) varying target to MDM ratio, (iii) the agents required for maximum stimulation of MDMs prior to the experiment, and (iv) the assays applied to quantify phagocytosis. Our results, however, were similar to those obtained by Brown and Newman, (Brown et al. 1987, Newman et al. 1991) who established a novel technique, frequently copied in the meantime, to measure engulfment of C’-opsonized targets by microscopic counting. According to conclusions from other groups (Aderem & Underhill 1999) increased surface expression of CR3 was achieved using a phorbol ester, phorbol myristate acetate, (presumably via a mechanism involving PKC and MacMARCKS (Zhou & Li 2000) resulting in high activation of otherwise constitutively inactive CRs. Due to this property unique in CRs, it was possible to specifically select mode and strength of receptor activation, which, corresponding to other investigators (Castagna et al. 1982, Wright & Meyer 1986) allowed to discriminate between Complement-mediated and alternative means of ingestion.

Discussion

72

Second messengers

5.1.3 Expression of CR in the course of HIV-infection Early studies showed that clearance of Cr-labelled, C’-opsonised, autologous erythrocytes in HIV-positive patients is impaired (Bender et al. 1988). This indicates a defect in C’-mediated phagocytosis in splenic macrophages. We have previously reported impairment of C’mediated phagocytosis by human monocytes and MDM following HIV-1 infection (Kedzierska et al. 2000, Chan et al. 2001, Kedzierska et al. 2001). In the present study MDM have been used as a model for human tissue macrophages to determine the mechanism of inhibition. Several investigators have studied the effect of HIV-1 on the surface expression of C’-receptors on monocytes and macrophages ex vivo and in vitro, with reports yielding conflicting results. Petit et al (Petit et al. 1987) reported increased CD11b expression on HIV1-infected U937 cells, while CD11b was found to be either increased (Palmer & Hamblin 1993) or unaltered (Stent et al. 1994) on monocytes from HIV-1 infected subjects, and decreased on HIV-1-infected MDM three weeks post infection in vitro (Kent et al. 1994). At the time we measured phagocytosis by MDM (P12), HIV-1 infection did not change the surface expression of CD11b or CD11c, or affect the binding of C’-opsonised targets to C’Rs. This indicates that the HIV-1 induced inhibition of phagocytosis occurs downstream of complement receptor binding. However, as the phagocytosis assay detects phagocytosis by both C’R3 and C’R4, the relative contribution of each receptor to the phagocytic index is unclear, as is whether HIV-1 inhibits phagocytosis by either or both receptors

5.1.4 Second messenger & cytokines Several macrophage functions are fundamentally influenced by a series of cytokines including IL-6, TNF-α, and IFNs (types 1 and 2), which are produced by the macrophages themselves, as well as by concomitant cell species (Fantuzzi et al. 2000). Generally, a phagocyte’s state of activity might be due to the combined net effect of several pathways, each being is explicitely governed and driven by the particular composition of the proximate cytokines. Intersecting at one or more points in their functional sequelae, these biochemical cascades finally merge in an integrated response, thereby shifting the equilibrium either towards or against phagocytosis.

Discussion

73

Second messengers

Many studies have been undertaken to elicit which pathways potentially contribute to particle internalization, of which Fc’, C’-mediated, or phagocytosis of apoptotic cells (Rossi et al. 1998) comprise only a fraction of all possible mechanisms (reviewed in (Allen & Aderem 1996a, Aderem & Underhill 1999, Chimini & Chavrier 2000)). However, research has made significant progress in the improvement of inhibitor specifity. Thus, by precisely terminating second messenger signalling, it has become feasible to further delineate the order and relevance of single stages of pathways withregard to cell performance.

Previous investigations of Complement-mediated phagocytosis in uninfected cells have demonstrated that general inhibition of phosphotyrosine kinases by Genisteine caused no significant impairment. This is in direct contrast to cAMP elevation, which strongly and specifically abrogated the process (Newman et al. 1991, Chan et al. 2001). This information implies that tyrosine kinases (such as syk, Hck, Pyk2 in MDM), as opposed to FcR pathways, do not assist CRs in conferring ingestion of opsonized targets. As to HIV-induced alterations to cell functions, it is intriguing to examine the role of Nef, a viral product implicated to interact widely in cell-signalling and cytoskeletal proteins. Athough elusive, prime targets thought to most likely confer the Nef-function have been detected among a principal group of proteins associated to cell mobility, namely Vav and the small GTPases Rac1 and Cdc42 (Fackler et al. 1999). Strikingly, evidence demonstrating that HIV devoid of Nef expression still impairs C’-mediated phagocytosis renders Nef less likely to mediate this effect (Kedzierska et al. 2001). These findings fit in a picture postulated by Caron (Caron & Hall 1998), who described the small GTPases of the Rho family to be associated with distinct types of particle uptake. Whereas Rac1 and Cdc42 are implicated in phagocytosis via Fc’ receptors, CRs appear to explicitly depend on RhoA. Based on the conclusions drawn from this data, it was tempting to further investigate the putative role of cAMP with regard to the phagocytic ability of HIV infected-cells.

Discussion

74

Second messengers

5.1.5 cAMP alterations induced by HIV impose on C’-mediated phagocytosis Due to its intrinsic versatility, the chief member of second messenger molecules, cAMP, has been the subject of extensive studies. Although well characterized in the context of several systems, its foremost physiological effect remains uncertain. In early studies using a cell model of human neutrophils, Andersson et al. reported forskolininduced elevation of cAMPi (Andersson et al. 1988) to significantly impair phagocytic capacity by Complement-receptors. We thus implemented forskolin in our experiments to trigger strong and sustained elevation of cAMP yielding a relatively low 20% inhibition in Fc-mediated phagocytosis compared to a decrease of 80% in C’-mediated (PMA stimulated) phagocytosis. In this regard our data confirms the conventional scientific opinion. Likewise, all sets of experiments exhibited an inhibtion (66% ± 0.06 of uninfected control) of C’-opsonized particle uptake by MDM infected with M-tropic HIV-1Ba-L under the condition of long-term adherence. To investigate how HIV-infected macrophages would react to artificial manipulation of cAMPi, a non-metabolizable cAMP analogue (2’5’ ddAD), was used to obtain competitive inhibition of adenylate cyclase. As demonstrated in Figure 10, C’-mediated phagocytosis of MDM on day 12 post infection with HIV-1 was elevated in both infected culture and controls with a seemingly low effect on the latter (1.57 ± 0.20 versus 1.30 ± 0.06). Treated with 2’5’ddAD, HIV-infected MDMs re-established phagocytic indices (97% ± 7%) up to levels equal to those of the uninfected and untreated group. To extend and to verify these observations, experiments were repeated by replacing 2’5’ddAD with an irreversible inhibitor (MDL-12) to gain an strongest blockage of adenylate cyclase. Taking our preliminary results into consideration, we decided to incubate with inhibitor concentrations up to 5µM, previously described in literature as totally depleting intracellular cAMP (Kanda et al. 2001). Under the effect of MDL-12, HIV-infected cultures showed a rapid restoration of C’-mediated phagocytosis (0.68 ± 0.03 to 1.02 ± 0.07 relative to control), eventually exceeding the threshold as set by the uninfected/untreated control group at a concentration level of 1µM. However, with increasing levels of MDL-12, the phagocytic index again started to decline.

Discussion

75

RhoA and PKA

5.1.6 Bridging link: Rho and PKA pathways The cytoskeleton, a regulator the for cell shape as well as for the balancing of cell motility, is a dynamic structure and intimately coupled to diverse cellular functions. It has become clear that a variety of intra and extracellular pathogens (such as Clostridia, Salmonella, and Listeria) have developed sophisticated ways of abducting eukaryotic cells by employing the complex cytoskeletal system for their own purpose.

Noteably, independent lines of evidence have lent credence to the distinct involvement of small GTPases, namely RhoA, Rac and Cdc42, in mechanisms associated with cell motility (Chimini & Chavrier 2000, Steele-Mortimer et al. 2000). Hierarchically organized, Cdc42 and Rac are thought to induce formation of filopodia (bundles of actin filaments) and lamellipodia (networks of polymerized actin in flat sheet-like structures) respectively. In detail, they use their capacity to polymerize actin and regulate cell polarity, subsequently exerting protrusive force on the leading edge (Hall & Nobes 2000). On the other hand, several lines of evidence have suggested that RhoA, a largely cytoplasmic GTPase, translocates to the plasma membrane upon activation, triggering a sequence of events including (i) stress fiber formation, (ii) focal adhesion binding, (ii) integrin clustering, and (iv) actomyosin-based contractility (Schoenwaelder & Burridge 1999). In fact, the differential involvement of Cdc42/Rac on the one hand and RhoA on the other (in regard to their requirement for FcRs and CRs) appeared to be more than a casual coincidence, leading to the description of type I and II phagocytosis. Type I, accomplished by Cdc42 and Rac, is based on active membrane protrusions that engulf the target, dragging it into the cell – as typical of FcR-mediated ingestion. Type II, however, depends on RhoA, and is characterized by particles which appear to sink more “passively” into actin-lined invaginations in the plasma membrane – commonly described as CRs. An interaction of cAMP in the intracellular signalling machinery, though, is not unlikely to occur by means of regulators that directly communicate with cytoskeleton. More precisely, this raises the question of whether or not PKA, the physiological co-player of cAMP, is potentially capable of inducing a more immediate effect on proteins that execute alterations in cell shape. Apparently, with RhoA and MLCK (myosin light chain kinase), two regulatory proteins have been suggested to be accountable for this connection between second messenger signalling and cell remodeling (Schoenwaelder & Burridge 1999).

Discussion

76

RhoA and PKA

Firstly, phosphorylation of RhoA on Ser188 by PKA results in its removal from the membrane into the cytoplasm, where it is generally presumed to be inactive. Secondly, RhoAP exhibits a decreased affinity for Rho-Kinase, usually causing the activation of MLCs by means of inhibition of myosin light chain phosphatase. Moreover, MLCK, another kinase directly targeted by PKA, usually initiates actin rearrangement and reacts to PKA phosphorylation with decreased activity. In summary, these conclusions favour the specific interaction of cAMP in cell remodeling and C’-mediated phagocytosis, leading to mechanisms involving RhoA and MLCK. Compatible with our hypothesis, the picture depicted does not necessitate the cooperation of tyrosin-kinases, which appears only to be of relevance only in FcR but not in CR-signalling. As phagocytic events are confined to minute areas of the cell surface, a deeper understanding of the underlying mechanisms ruling cAMP levels and phagocytosis in MDM was obtained with the notion of a second messenger, regulated in a focal rather than a general manner, by PDE-4 and PKA at the nascent phagosome (Pryzwansky et al. 1998). Using both FcR and CR3, this group observed the catalytic subunits of PKA after dissociation (on activation by cAMP from the holoenzyme) to co-localize with PDE-4 during the initial phase of the particle engulfment. It is intriguing to argue that PDE-4, as a potential substrate for PKA, is implemented in a negative feedback loop, lowering cAMP levels and keeping actin assembly and disassembly under control. Precise translocation of PKA, mandatory under these circumstances, can be achieved by means of specific AKAP (PKA-anchoring proteins), forming a shuttle system for compartmentalization (Scott & McCartney 1994). This would underline our findings concerning a decline in C-mediated phagocytosis, subsequent to a considerable rise with increasing levels of MDL-12. The property and physiological role of cAMP is related to its focal intracellular concentration and base levels are essential for PKA-dependent actin disassembly. Our assay, however, allowed us to measure the amount of particles protected from a hypotonical environment without differentiating on how this protection was achieved, either with particle engulfment promoted by pseudopod protrusions or by particles sinking into the cell.

Discussion

77

RhoA and PKA

We suggest here a scenario where RhoA plays a double role, present both in CR- and Fc-Rmediated phagocytosis, emphasized during the formation of the phagocytic cup, whereby it contributes to the weakening of the cytoskeletal cortex beneath the bound C-opsonized target, as active pseudopod protrusion does not occur. Even if RhoA were involved in Fc-mediated internalization, cytoskeletal rearrangements and pseudopod formation initiated by Cdc42 and Rac1 are more efficient. These are not affected by PKA and mask a potentially defective RhoA pathway, as implied by the slight inhibition of FcRs in our forskolin studies. Inhibitor studies on RhoA/Rho Kinase conducted by Worthylake demonstrated defective tail retraction in monocytes, as opposed to fibroblast cultures that occurred during chemotaxis (Worthylake et al. 2001). These findings favour RhoA/Rho Kinase to mediate myosincontractility and negatively regulate integrin adhesiveness, presumed to be mandatory for receptor recycling from the tail to the leading edge during migration. This observation further promotes our idea of puzzling similarity between a migrating cell, withdrawing membrane on the leading front, and membrane retraction as observed in C’-mediated particle ingestion. This effect might further arise from the imbalance in the physiological negative signalling loop maintained by a cAMP-induced RhoA/ROK inactivation. In this study, it was demonstrated for the first time that the impairment of C’-mediated phagocytosis during HIV infection is, in fact, closely related to intracellular cAMP concentrations and highly responsive to controlled second messenger manipulation. A gradual inhibition of the cAMP pathway in HIV-infected MDM restores C’-mediated phagocytosis, whereas total blockage yielded inhibition in both infected and control cells showing that the macrophage as an entity is still functionally intact and disbalanced rather than disrupted.

Discussion

78

Disentanglement

5.1.7 Disentanglement: HIV, cAMP and phagocytosis The impaired function of macrophages is characteristic of HIV infection. Interestingly, the total number of macrophages carrying HIV is relatively low. By taking into consideration the multiplying effect through cytokine release that commences down-regulation of the immunosystem following paracrine and autocrine activation, it is conceivable that macrophages can potentiate and contribute to the exacerbation of HIV pathogenesis.

This study addressed the involvement of cellular factors in the HIV-1 mediated inhibition of C’-phagocytosis. In addition, we have considered specific virologic factors required to induce this inhibition. Our data show that inhibition of phagocytosis probably requires viral entry but not active viral replication, suggesting that immediate viral post-entry events, prior to reverse-transcription, may play a role in the HIV-1 induced inhibition. It is possible that inhibition of phagocytosis is induced by viral proteins present within the virion which are released into the cell following viral entry. In this context, our previously published data indicate that Nef is unlikely to be necessary for HIV-1-mediated inhibition of C’ phagocytosis in MDM (Kedzierska et al. 2001). However, it remains unclear whether any inhibitory HIV-1 proteins act on infected cells directly to elevate intracellular cAMP, or whether the infected cells are stimulated to secrete factors which elevate cAMP in bystander cells. Infection with HIV-1 in vitro increases intracellular cAMP in T-lymphocytes (Hofmann et al. 1993a)the MT-4 T-cell line (Nokta & Pollard 1991), and the promonocytic U937 cell line (Thomas et al. 1997). Our study provides the first direct evidence that HIV-1 infection of MDM elevates intracellular cAMP. Elevation of cAMP levels in peripheral blood cells and macrophages may have consequences for HIV-1 replication. It has been shown that cAMP upregulates the co-receptor for T-tropic strains of HIV-1 entry, CXCR4 (Cristillo et al. 2002), but downregulates that for M-tropic strains, CCR5 (Thivierge et al. 1998). Cyclic AMP also may affect HIV-1 production at the level of transcription, although both inhibitory effects (Banas et al. 2001) and stimulatory effects (Rabbi et al. 1997) have been reported. Also, it has recently been shown that enzymatically active PKA is incorporated into HIV-1 virions and that this is required for viral infectivity (Cartier et al. 2003).

Discussion

79

Disentanglement

HIV-1-enhanced cAMP levels may also affect host cell immune function directly. Tlymphocytes from HIV-seropositive individuals demonstrate increases in intracellular cAMP levels, associated with poor T-cell proliferation and cytotoxicity (Hofmann et al. 1993b). This is in keeping with reports that cAMP inhibits the proliferation and effector functions of T cells (Murray et al. 1972, Bourne et al. 1974, Kemp et al. 1975). cAMP regulates aspects of monocyte/macrophage function, including suppression of phagocytosis (Zalavary et al. 1994, Rossi et al. 1998), apoptosis (von Knethen et al. 1999, von Knethen & Brune 2000), and chemotaxis (Stephens & Snyderman 1982, Fine et al. 2001). cAMP also regulates cytokine synthesis. Increases in intracellular cAMP have been shown to shift the immune response from a T-helper (TH) 1 to a TH2 response (Thanhauser et al. 1993, Lacour et al. 1994), similar to that postulated to occur during HIV-1 infection (reviewed in (Lucey et al. 1996) (Kedzierska & Crowe 2001)). HIV-1-enhancement of cAMP levels may therefore contribute to the defective cell-mediated immunity observed in AIDS patients.

Prostaglandins have garnered exceptional interest due their powerful effect on PKA pathways. Whilst our results demonstrate that PGE2 production by MDM is not altered during HIV-1 infection in vitro, several investigators have previously shown that HIV-1 infection of monocytes/macrophages increases PGE2 secretion in vitro (Mastino et al. 1993, Hayes et al. 2002). However, these published results differed significantly in the kinetics of PGE2 secretion, with peak levels of secretion occurring anywhere between 2 hours to 4 days post infection. Similarly, the extent of secretion differed, with differences between infected and uninfected PGE2 levels in the supernatant ranging from 1- to 4-fold. In our study of cells prepared from 4 donors, MDM infected with HIV-1 varied substantially with regard to PGE2 secretion into the culture supernatant when compared to uninfected controls. Nevertheless, whilst inhibition of phagocytosis occurred within 7 days of infection, the levels of PGE2 secretion were unaltered upon infection. Given the lack of association between defective phagocytosis and PGE2 secretion in our study, we conclude that altered PG production is unlikely to account for the observed impairment in C’-mediated phagocytosis, and hence is not the major cause of the elevation of cAMP in HIV-1-infected MDM

Discussion

80

Disentanglement

With regard to the data in literature and our own findings, we suggest cAMP, to be responsible for a specific impairment of C’-mediated phagocytosis by maintaining a temporary imbalance in the physiological negative regulator loop of PKA and PDE-4, eventually resulting in prolonged inhibition of the RhoA pathways (Picture 10). The reason for elevated cAMP levels remains unclear. Impaired C’R-mediated signalling may explain why HIV-1-infected macrophages fail to control HIV-associated opportunistic pathogens such as Mycobacterium avium complex (phagocytosed via C’Rs) and provides additional potential therapeutic strategies for the treatment of AIDS via restoration of host defence

Discussion

Picture 10 MDMs

81

Disentanglement

Hypothesis of pathogenesis ruling the impairment of C’-mediated phagocytosis in HIV infected

Abstract

v

Abstract Deficiency in Complement-mediated phagocytosis of HIV-1 infected macrophages involves a cAMP-dependent pathway D. DOISCHER1, ANTHONY JAWOROWSKI2, UWE FRANK1, SUZANNE CROWE2. 1 2

Freiburg Univ. Hosp., Germany. Burnet Institute, GPO Box 2284, Melbourne, 3001, Australia.

Background: Using cAMP analogues, evidence has been obtained that Complementmediated phagocytosis (CMP) by human monocyte-derived macrophages (MDM) is impaired by cAMP. During HIV-1 infection, increased intracellular concentrations of the nucleotide cAMP may occur which could have negative effects on CMP in macrophages. Objective: In this study we investigate the effects of HIV-1 on the mechanism of complement (C’)-mediated phagocytosis by human monocyte-derived macrophages (MDM). Materials and Methods: Using a colorimetric assay, Complement-opsonized sheeperythrocytes (E) were used as targets to quantify CMP by MDM infected with a laboratory adapted, M-tropic strain of HIV-1 for 7-10d. In selected experiments, MDM on day 7 post-infection were pretreated with forskolin (10mins, 0.01µM to 100µM), 2’-5’-dideoxyadenosine (ddAD, 30min, 0.1µM to 10µM), or MDL-12,330 A (30mins, 0.1µM to 5µ). We used lamivudine (100µM) to examine phagocytosis in the absence of retroviral replication. Results: In direct measurements, cAMP was increased (%167±60) in MDM during infection. Forskolin (100µM) strongly inhibited CMP by 80% (p<0.0016) in uninfected MDM, whereas a significant increase in phagocytic activity was observed when erythrocytes were incubated with incrementing levels of 2’5’ddAD (up to 10 µg/mL) in both HIV-1 infected cells and controls (57 ± 20%, and 30 ± 6%, respectively). In the presence of 10µg/mL 2’5’ddAD, particle ingestion by HIV-1 infected macrophages reached levels (97%) of Complementmediated phagocytosis of untreated, HIV-negative controls. In our hands, levels of PGE2 were unaltered in supernatants obtained from assays. Likewise, deficiency in particle uptake did not correlate with active viral replication, as shown by use of the anti-retroviral drug lamivudine. Conclusions: Independent of prostaglandine secretion and viral replication, in HIV-1 infected MDM, integrin-dependent phagocytosis can be restored by 2’5’ddAD to levels of uninfected macrophages suggesting that elevated cAMP levels in these cells may contribute to decreased Complement receptor function.

Zusammenfassung

vi

Zusammenfassung Durch die Anwendung von cAMP–Analoga konnte gezeigt werden, dass Komplementvermittelte Phagozytose (KVP) menschlicher, von Monozyten-abstammenden Macrophagen durch cAMP beeinträchtigt wird. Es war daher theoretisch denkbar, dass die während einer HIV-1 Infektion auftretende erhöhte intrazelluläre Konzentration dieses Nukleotids negative Effekte auf die KVP ausübt. Ziel dieser Studie war es den Einfluss von HIV-1 auf die Mechanismen von Komplementvermittelter-Phagozytose menschlicher Macrophagen offenzulegen. Mit Hilfe eines kolorimetrischen Assays und Schaferythrozyten als Phagozytose-Partikeln (opsoniert mit humanem Serum als Quelle für Komplement-Komponenten) war es möglich KVP in Makrophagen während florider Infektion (7-10d) mit einem Labor-adaptierten Mtropischen Virus zu quantifizieren. In ausgewählten Experimenten wurden Phagen 7 Tage nach Infektion mit Forskolin (10mins, 0.01µM to 100µM), 2’-5’-Dideoxyadenosin (ddAD, 30min, 0.1µM to 10µM), oder MDL12,330 A (30mins, 0.1µM to 5µM) vorbehandelt. Um das Verhalten der Zellen in Abwesenheit aktiver retroviraler Replikation zu messen, benutzten wir Lamivudin (100µM). Tatsächlich bestätigten direkte cAMP-Messungen den Anstieg von desselben unter HIV-1 in unseren Zellen. Übereinstimmend mit Resultaten aus den cAMP-Analoga-Experimenten konnte Forskolin in nicht-infizierten Phagen die KVP stark inhibieren (zu 80%, 100µM, p<0.0016). Im Gegenzug wurde eine signifikante Zunahme der Phagozytose-Aktivität unter Inkubation mit ansteigender Konzentration von ddAD (bis 10 µg/mL) sowohl in HIV-1 infizierten und Kontrollzellen (entsprechend 57 ± 20%, und 30 ± 6%) beobachtet. In Gegenwart von 10µg/mL 2’5’ddAD nahm die Partikel-Aufnahme infizierter Zellen bis auf 97% HIV-negativer, unbehandelter Kontrollen zu. In weiteren Untersuchungen konnte kein Anstieg von PGE2 in Kultur-Überstand, bekannt für seine potentierende Wirkung auf die Adenylatzyklase, bestätigt werden. Ebenso korrelierte das Defizit der KVP nicht mit aktiver viraler Replikation, wie sich unter der Behandlung mit antiviralem Agens Lamivudin zeigte. Zusammenfassend ist es uns gelungen, unabhängig von der Prostaglandin-Sekretion und viraler Replikation, in HIV-1 infizierten menschlichen Makrophagen die Integrin-vermittelte Phagozytose durch 2’5’ddAD bis auf das Niveau gesunder Zellen zu heben, was unsere Vermutung bestätigt, dass erhöhtes cAMP in diesem System zu gestörter KomplementRezeptor-Funktion beiträgt.

Note of thanks

vii

Note of thanks Es ist mir schwer bei einem Werk dieser Art und Länge, welches sich in verschiedenen Instituten und auf verschiedenen Kontinenten abgespielt hat, die grosse Anzahl von Personen zu benennen und zu würdigen, die sich im Verlauf der Jahre mit meiner Forschung und nicht zuletzt mit mir beschäftigt haben. Abgesehen von grundlegenden Fähigkeiten, die von ausgetüftelten Labortechniken bis hin zu differenzierter Bewertung wissenschaftlicher Abhandlung reichen, haben mir gerade diese Begegnungen an kritischen Punkten, und diese sind dem Charakter der Forschung entsprechend zahlreich, gezeigt und bewusst gemacht, was es bedeutet, ein Arzt zu werden und wie der Weg dorthin bestimmt ist.

Ich werde nicht dieses erste Mal vergessen, als mein Doktorvater Uwe Frank heiteren Schrittes um die Ecke bog und sich mit seiner lockeren Art meines innigsten Wunsches, eines Projektes über HIV im Ausland, annahm. Für mich gehört er zu den Menschen, die es auf verblüffend einfache Art immer wieder schaffen, Dich aufzubauen – die das Vertrauen stärken darin, dass es immer Möglichkeiten und Wege geben wird, egal was da komme. Danke Uwe. John Mills, Direktor des Macfarlane Burnet Centre, welcher mich damals in sein Reich aufnahm und von dem ich lernte wie man als „Last man standing“ für den Erhalt der Individualität

seines

Institutes,

seiner

Mitarbeiter

und

ihrer

wissenschaftlichen

Errungenschaften kämpfen kann. Hochachtung John. Suzanne Crowe, deren Labore und Budget ich benutzte und die mir meine Kritikfähigkeit im Bereich der Forschung schärfte. Vielen Dank vor allem auch an Anthony, meinen Supervisor des „Macfarlane Burnet Centre“, für all die vielen Stunden im Labor oder im Büro, in denen ich ein wahres Studium generale durch ihn erfahren durfte. Mark Chan, dessen Stelle ich übernahm, um die Kontinuität der Experimente an HIVinfizierten Macrophagen zu gewährleisten. Anne Askew, meine äusserst liebenswerte australische Bibliothekarin, die mich in die grossen Geheimnisse der Literatur-Recherche einweihte, und welche soviel Verständnis mitbrachte --für meinen inspirativen Schlaf über Stapeln von „Papers“ in ihrer Bibliothek.

Note of thanks

viii

Dem aufmerksamen Betrachter wird auffallen, dass ich mich entschied diese Arbeit in Englisch zu verfassen, was einfache Gründe hatte, aber nicht ebenso einfache Probleme mit sich trug. Nun, nach einem Jahr Forschung in Melbourne ist man ein halber Aussi und tut daher das Nächstliegende - man schreibt in der Sprache, in der man sich mit seinen Experimenten auseinandergesetzt hat und die den Goldstandard der Wissenschaft darstellt. ◊

Vielen Dank daher an Louise für Deine Korrekturen meiner Prosa, für exzellente Beitrage und einen wertvollen Gedankenaustausch in dieser Zeit.

◊

Danke Frau Lawrie-Bloom für Planung und Englisch – ohne sie hätte ich nie ein Ende gefunden.

◊

Brigid, ich werde nie unseren gemeinsamen Marathon-Nächte am Schreibtisch vergessen, in der wir im Kaffee-Delir letzten Schliff an diese Arbeit angelegt haben. Exceptional endurance….Thanks a lot

◊

Vielen Dank auch oder vielmehr insbesondere an meine Familie, weil sie mich immer unterstützt haben, in jeder Hinsicht - Hermine, Erhard, Ralf, Marianne, Elisabeth.

Curriculum vitae

ix

Curriculum vitae Daniel Doischer Kronenmattenstr.9

geboren – 29. März 1976

79100 Freiburg

in Bergneustadt, Deutschland

[email protected]

Schulische Laufbahn ƒ ƒ ƒ ƒ ƒ

1982 – 86

Gemeinschaftsgrundschule auf dem Bursten

1986 – 95

Wüllenweber-Gymnasium Bergneustadt

1995 – 96

Zivildienst beim Deutschen Roten Kreuz

Universitäre Laufbahn ƒ

1996 – 98

Studium Humanmedizin (Vorklinik) Universität Magdeburg Physikum Herbst 1998

ƒ

1998– 99

Zweiter Klinischer Abschnitt Charite der Humboldt Universität 1. Staatsexamen Herbst 1999

ƒ

1999 – 00

7. und 8. Semester Universität Freiburg

ƒ

2001 – 03

9. bis 13. Semester Universität Freiburg 3. Staatsexamen Frühjahr 2004

Beruflicher Werdegang ƒ

seit 07/2004

Wissenschaftlicher Mitarbeiter Physiologisches Institut, Universität Freiburg Projekt: Oszillation interneuronaler Netzwerke im Hippokampus

Annexes

x

Material

Praktische Tätigkeit im Ausland ƒ

April 2000

Famulatur, Department of Emergency Shaare Zedec Hospital, Jerusalem, Israel

ƒ

2000 – 01

HIV-Forschungsprojekt im Rahmen der Dissertation Macfarlane Burnet Centre, Melbourne, Australia

ƒ

September 2001

Famulatur, Department of Infectious Diseases St. Vincent Hospital, Melbourne, Australia

ƒ

April 2002

Famulatur, Service d’Urgence Centre Hospitalier de L’Ouest Guyanais “Franck Joly” Guyane française

ƒ

2003

Praktisches Jahr Chirurgie thoracique et digestive Université de Montpellier, France

Poster & Präsentationen ƒ ƒ

1-4 Mai 2004

14th ECCMID Prag, European Congress Microbiology and Infectious Diseases

ƒ ƒ

Poster-Titel:

Restoration of Complement-mediated Phagocytosis in HIV-1infected Macrophages by 2’5’dideoxyadenosine (ddAD)

ƒ ƒ

of

Clinical

D. Doischer, Anthony Jaworowski, Uwe Frank, Suzanne Crowe

Annexes

xi

Material

Annexes ◊ Material Devices (D01)

Beckman centrifuge (Fullerton, CA, USA)

(D02)

hemocytometer (Clay Adams, Becton Dickinson, MD, USA)

(D03)

FACStar plus flow cytometer

(D04)

Axiovert 100, inverted phase contrast and fluorescence microscope, (Zeiss, Germany)

(D05)

Cool Snap fx Digital Camera (Roper Scientific photometrics, Auckland, New Zealand)

(D06)

V++ (Roper Scientific Digital Optics, Auckland, New Zealand)

(D07)

multichannel pipette (Labsystem, Helsinki, Finland)

(D08)

ELISA plate reader (Labsystems, Helsinki, Finland)

(D09)

liquid scintillation counter (LKB micro betacounter; Wallax, Turku, Finland)

(D10)

Microsoft Excel®, spreadsheet programm

(D11)

inverted phase contrast microscope (Olympus, Tokyo, Japan)

Annexes

xii

Material

Supply (S01)

50ml conical tubes (Cellstar, Greiner Labortechnik, Frickenhausen, Germany)

(S02)

10mL pipettes (Becton Dickinson Labware, NJ, USA)

(S03)

plastic transfer pipette (Samco Scientific, CA, USA)

(S04)

15cm, 140cm2 plastic petri dishes (Nalge Nunc International, Denmark)

(S05)

polytetrafluorethylene (Teflon) jars (Savillex, Minnetonka, MO, USA)

(S06)

FACS tube = 5-ml round bottom polypropylene tubes (Becton Dickinson Labware, Franklin Lakes, NJ, USA)

(S07)

0.2µm filters (Sartorius, Göttingen, Germany)

(S08)

cryogenic vials (Corning Costar, Cambridge, MA, USA)

(S09)

96-well plate (Nalge Nunce International, Roskilde, Denmark)

(S10)

Eppendorf tubes

(S11)

DE81 chromatography paper (Whatman, UK)

Annexes

xiii

Material

Reagents (R01)

anti-CD14 monoclonal antibody (Mab) directly conjugated to phycoerytherin (CD14-PE) (Becton Dickinson, San Jose, CA, USA)

(R02)

ultrapure formaldehyde (Polysciences, Warrington, PA, USA)

(R03)

phytohemagglutinin (PHA, 10µg/ml, Abbott Diagnostics, Santa Clara, CA, USA)

(R04)

Alexa 568 goat anti-mouse IgG (Molecular probes, Eugene, Oregon, USA)

(R05)

CD68 mouse anti-human antibody (DAKO, Denmark)

(R06)

hybridoma, mouse anti-p24 antibody

(R07)

33P-dTTP (Amersham International, Buckinghamshire, UK)

(R08)

Meltilex scintillant (Wallax, Turku, Finland)

(R09)

Phorbol-12-myristate-13-acetate (PMA) (Sigma, St. Louis, MO, USA)

(R10)

dimethyl sulphoxide (Merck, Victoria, Australia)

(R11)

2,7-diaminofluorene (Sigma, St Louis, MO, USA)

(R12)

MDL-12, 330A, hydrochloride (Calbiochem, Kylsith, Victoria, Australia)

(R13)

2’5’ddAD, 2’,5’, dideoxyadenosin (Calbiochem, Kylsith, Victoria, Australia)

(R14)

forskolin = Coleus forskohlii, (Calbiochem, Kylsith, Victoria, Australia)

Annexes

xiv

Material

Media (M01)

Ficoll-Paque: (Amersham Pharmacia Biotech, Uppsala, Sweden)

(M02)

RF 10: RPMI medium-1640 (GibcoBRL, Life Technologies, NY, USA) supplemented with 10% fetal bovine calf serum (FCS)

(M03)

FCS: fetal bovine calf serum (P.A. Biologicals, Sydney, Australia), 2mM Lglutamine (Gibco BRL, New York, USA) and 24µg/mL gentamicin (Delta West, Bentley, West Australia)

(M04)

PBS(-): phosphate buffered saline without divalent cations magnesium and calcium, PBS-CMF (GibcoBRL, Life Technologies, NY, USA

(M05)

PBS(+): phosphate buffered saline containing magnesium and calcium

(M06)

Adherence Medium: Iscove’s modified Dulbecco medium (GibcoBRL, Life Technologies, NY, USA) supplemented with 10% heat-inactivated human AB+ serum of lower quality, 2mM L-glutamine (Gibco BRL, New York, USA), and 24µg/ml gentamycin (Delta West Western Australia, Australia)

(M07)

Heat-inactivated human serum AB(+): quality assessed by culturing monocytes in macrophage medium containg 10% of the serum and determination of cell viability with Tripan blue and cell numbers

(M08)

PFW: pyrogen free water

(M09)

RH 10: RPMI medium-1640 (GibcoBRL, Life Technologies, NY, USA) supplemented with 10% heat-inactivated human serum

Annexes (M10)

xv

Material

RT assay mixture distilled water supplemented with 50mM Tris pH 7.8, 7.5mM KCl, 5mM MgCl2, 2mM dithiothreitol

(M11)

2×SSC buffer 0.3M sodium chloride and 34mM sodium citrate (Merck, Victoria, Australia)

(M12)

Iscove’s modified Dulbecco medium (GibcoBRL, Life Technologies, NY, USA)

Annexes

xvi

Pictures, figures & tables

◊ Index of Pictures Picture 1

Cell differentiation of the macrophage lineage ---------------------------------------7

Picture 2

Molecular structure of the β2- Intgrin ----------------------------------------------- 14

Picture 3

Taxonomy of the family retroviridae ------------------------------------------------- 22

Picture 4

Retroviral phylogeny ------------------------------------------------------------------- 24

Picture 5

HIV-1 virion structure------------------------------------------------------------------ 26

Picture 6

HIV-1 replication cycle ---------------------------------------------------------------- 30

Picture 7

Phase contrast microscopy of adherent MDMs on day 5 post-isolation--------- 50

Picture 8

Verification of macrophage purity in long-term adherent cell monolayer.------ 54

Picture 9

Assessment of HIV-1 infection in long-term-adherent MDMs -------------------- 56

Picture 10 Hypothesis of pathogenesis ruling the impairment of C’-mediated phagocytosis in HIV infected MDMs ----------------------------------------------------------------- 81

Annexes

xvii

Pictures, figures & tables

◊ Index of Figures Figure 1

Monocyte purity ------------------------------------------------------------------------- 45

Figure 2

Distinct difference between preparations of SRBC using normal or heat inactivated human serum. ------------------------------------------------------------- 47

Figure 3

Standard curve for assessment of Hb concentrations per SRBC.----------------- 48

Figure 4

Adhesion improves C’-mediated phagocytosis in infected MDM. ---------------- 49

Figure 5

Effect of MDM monolayer cell density on C’-mediated phagocytosis ----------- 50

Figure 6

No effect of medium replenishing on C’-mediated ingestion following long-term adherence-------------------------------------------------------------------------------- 51

Figure 7

Effect of various DMSO concentrations (ranging from 0 to 10%) on Complement-mediated phagocytosis of SRBC. ------------------------------------- 52

Figure 8

Determination of ratio HIV-1Ba-L-infected to non-infected MDM after 7 days of infection and long-term adherence on 96well plates.------------------------------ 57

Figure 9

Effect of forskolin on C’-mediated and Fc-mediated phagocytosis -------------- 60

Figure 10

Effect of 2’5’ddAD on C’-mediated phagocytosis in HIV-infected MDM. ------ 62

Figure 11

Restoration of phagocytosis in HIV-infected MDM. ------------------------------- 63

Figure 12

Effect of irreversible and selective adenyl cyclase inihibitor MDL-12 on HIV 1infected, long-term-adherent MDM. ------------------------------------------------- 64

Figure 13

HIV-1 infection of MDM does not alter basal secretion of PGE2 ---------------- 66

Figure 14

Active replication of HIV-1 is not required for inhibition of C’-mediated phagocytosis. ---------------------------------------------------------------------------- 67

Annexes

xviii

Pictures, figures & tables

◊ Index of Tables Table 1

Designation of viral structures -------------------------------------------------------- 28

Table 2

Representative results of monoycte purity determination by flowcytometry on day of isolation. ------------------------------------------------------------------------------ 44

Table 3

Cell lysis during SRBC opsonization with normal versus heat-inactivated human serum. ------------------------------------------------------------------------------------ 47

Table 4

Data sheet for calculation of loss of SRBC during opsonization and washing steps. ------------------------------------------------------------------------------------- 48

Table 5

HIV-infection of MDM elevates intracellular cAMP ------------------------------- 58

Table 6

Inhibition of Complement receptors by forskolin ----------------------------------- 60

Table 7

Inhibition of C’-mediated phagocytosis in relation to HIV-1 infection. --------- 62

Table 8

Effect of 2’5’ddAD on C’-mediated phagocytosis in HIV-infected MDM. ------ 62

Table 9

Restoration of C’-mediated phagocytosis in HIV 1-infected MDM by competitive inhibitor 2’5’dideoxy adenosine ------------------------------------------------------ 63

Table 10

Effect of irreversible and specific adenylate cyclase inhibitor MDL-12 on C’mediated phagocytosis in HIV 1-infected MDM.----------------------------------- 65

Annexes

xix

Scientific abbreviations

◊ Scientific Abbreviations ENV envelope

#

ER endoplasmatic reticulum

µL microliter

F

µM micromol

Fc fragment cristallizable, binding immunoglobulins

A

FCS fetal calf serum FITC fluorescein-iso-thiocyanate

Ab antibody

fMLP formyl-methionyl-leucyl-phenylalanine

AC adenylate cyclase ADP adenosine diphosphate

G

AIDS acquired immunodeficiency syndrome AKAP PKA-anchoring proteins

G2 growth phase 2

APC antigen-presenting cell

GM-CSF granulocyte, macrophage colony stimulating

Arp2/3 actin-related proteins 2&3

factor

AZT Zidovudine

gp glycoprotein GTP guanosine triphosphate

B H BCG Bacille Calmette-Guerin h hour

C

HAART highly active antiretroviral therapy HBV hepatitis B virus

C3 Complement factor 3

HCl sodium chloride

CA capsid

HI HuS heat-inactivated human serum

cAMP cyclic adenosine monophosphate

HIM hematopoietic inductive microenvironment

CCP Complement control protein

HIV human immunodeficiency virus

CCR5 Chemokine receptor R5, M-Tropic

HLA human leukocyte antigen

CD Cluster of differentiation

HSC hematopoietic stem cells

Cdk9 cyclin-dependent protein kinase 9

HTLV I human T cell leukemia virus

cm centimeter CPM counts per minute

I

CR Complement receptor CXCR4 chemokine receptor, T-tropic

ICAM intercellular adhesion molecule IFN interferon

D

IL interleukin IN integrase

DAF decay accelerating factor ddAD dideoxyadenosine ddI Didanosine DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dTTP deoxy thymidin triphosphate

E

IP3 inositol-triphosphate

Annexes

xx

Scientific abbreviations PAF platelet activating factor

K

PAK p21-activated protein kinase PBMC peripheral blood mononuclear cells

kb kilo bases

PBS phosphate buffered saline, primer binding site

kD kilo Dalton

PC personal containment level

KVP Komplement-vermittelte Phagozytose

L

PDE phosphodiesterase PDGF platelet-derived growth factor PE phycoerytherin

LFA lymphocyte-function associated antigen

PFW pyrogen free water

LPS lipopolysaccharide

PGE prostaglandin

LTR long terminal repeat

PHA phytohemagglutinin PI3-K phosphoinositol-3-Kinase

M

PKA protein kinase A PKC protein kinase C

MA matrix

PM Primaer-Macrophagen

Mab mouse antibody

PMA phorbol myristate acetate

MAC membrane attack complex

PPT polypurine tract

MacMARCKS macrophage myristoylated alanine-rich C

PR protease

kinase substrate MDM monocyte-derived macrophages

R

MHC Major histocompatibility antigen MIDAS metal ion-dependent adhesion site

RANTES regulated upon activation, normal T cell

min minute

expressed and secreted

MIP macrophage inflammatory protein

RNA ribonucleic acid

mL milliliter

ROK Rho-kinase inhibitor

MLCK myosin light chain kinase

RRE rev response element

mM millimol

RT reverse transcriptase

MOI multiplicity of infection

S

mRNA messenger RNA M-tropic macrophage-tropic

N

SD standard deviation SEM standard error of means Ser serine

NC nucleocapsid

SIV simean immunodeficiency virus

NES nuclear export signal

SN supernatant

NK cells natural killer cells

snRNA small nuclear RNA

NLS nuclear localization signals

SRBC sheep red blood cells

NNRTI non-nucleoside RT inhibitor

SU surface

NO nitrogen oxyde NRTI nucleoside RT inhibitor

O OD optical density

P

Annexes

xxi

Scientific abbreviations tRNA transfer RNA

T TAR trans-activating response element

TTP thymidin triphosphate

V

TCID tissue culture infectivity dose TGF tumor growth factor

VIP vasoactive intestinal peptide

TLR toll-like receptor

Vpr viral protein regulatory

TM transmembrane TNF tumor necrosis factor TPAK terminal-protein-associated kinase

Annexes

xxii

References

◊ References (1981) Dengue type 4 infections in U.S. travelers to the Caribbean. MMWR Morb Mortal Wkly Rep 30: 249-250. (1986) Classification system for human T-lymphotropic virus type III/lymphadenopathyassociated virus infections. Centers for Disease Control, U.S. Department of Health and Human Services. Ann Intern Med 105: 234-237. (2000) AIDS epidemic update: December 2000, Joint United Nations Programme ON HIV/AIDS, UNAIDS, WHO, Geneva Aderem A, Underhill DM (1999) Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17: 593-623 Agresta

BE,

Carter

CA

(1997)

Cyclophilin

A-induced

alterations

of

human

immunodeficiency virus type 1 CA protein in vitro. J Virol 71: 6921-6927. Allen LA, Aderem A (1996a) Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J Exp Med 184: 627637 Allen LA, Aderem A (1996b) Mechanisms of phagocytosis. Curr Opin Immunol 8: 36-40 Andersson T, Fallman M, Lew DP, Stendahl O (1988) Does protein kinase C control receptormediated phagocytosis in human neutrophils? FEBS Lett 239: 371-375. Arnaout MA (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75: 1037-1050. Arthur LO, Bess JW, Jr., Sowder RC, 2nd, Benveniste RE, Mann DL, Chermann JC, Henderson LE (1992) Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 258: 1935-1938. Balter M (1996) HIV's other immune-system targets: macrophages [news]. Science 274: 1464-1465 Banas B, Eberle J, Schlondorff D, Luckow B (2001) Modulation of HIV-1 enhancer activity and virus production by cAMP. FEBS Lett 509: 207-212

Annexes

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