macrophages following HIV-1 infection: underlying mechanisms and modulation by adjunctive cytokine therapy

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Defective phagocytosis by human monocyte/macrophages following HIV-1 infection: underlying mechanisms and modulation by adjunctive cytokine therapy Katherine Kedzierska a,c, Rula Azzam a,c, Philip Ellery a,b,c, Johnson Mak a,d, Anthony Jaworowski a,b,1, Suzanne M. Crowe a,b,c,1,* a

AIDS Pathogenesis Research Unit, Macfarlane Burnet Institute for Medical Research and Public Health, Cnr Punt and Commercials Rds, Prahran, Melbourne, VIC 3181, Australia b National Centre for HIV Virology Research, Melbourne, Australia c Department of Medicine, Monash University, Prahran, Melbourne, Australia d Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia

Abstract Defective immunological function of cells of the macrophage lineage contributes considerably to the pathogenesis of HIV-1 infection. Impairment of phagocytosis of opportunistic pathogens such as Mycobacterium avium complex (MAC), Pneumocystis carinii , Toxoplasma gondii or Candida albicans by peripheral blood monocytes, tissue macrophages and monocyte-derived macrophages following in vivo and in vitro HIV-1 infection is well documented. The development of opportunistic infections due to these pathogens in HIV-infected individuals at late stages of disease is attributed to defective monocyte/macrophage function. The mechanisms whereby HIV-1 impairs phagocytosis are not well known. A number of phagocytic receptors normally mediate engulfment of specific opportunistic pathogens by cells of macrophage lineage; distinct mechanisms are triggered by pathogen
/receptor binding to promote cytoskeletal rearrangements and engulfment. This review focuses on the signalling events occurring during Fcg receptor- and complement receptor-mediated phagocytosis, and considers the mechanisms by which HIV-1 inhibits those signalling events. Since macrophage function is enhanced by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-g), the use of these immunomodulators is of potential interest as adjunctive immunotherapy in immunosuppressed individuals. In this review we present examples of clinical applications of GM-

Abbreviations: MAC, Mycobacterium avium complex; MDM, monocyte-derived macrophages; HIV, human immunodeficiency virus; R, receptor; C’, complement; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-g, interferon-gamma.  This work was supported in part by funding from the Australian National Council on HIV, AIDS and Related Diseases to the National Centre in HIV Virology Research, the Macfarlane Burnet Centre Research Fund and the Monash University. * Corresponding author. Tel.: /61-3-9282-2294; fax: /61-3-9282-2142 E-mail address: [email protected] (S.M. Crowe). 1 These authors contributed equally. 1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 2 3 - 3

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CSF and IFN-g therapy for the treatment of opportunistic infections in HIV-infected individuals receiving antiretroviral drugs. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Monocytes/macrophages; HIV-1; IgG-phagocytosis; Complement-phagocytosis; GM-CSF; IFN-gamma

1. Introduction Cells of macrophage lineage contribute to the pathogenesis of HIV-1 infection throughout the course of the disease (reviewed in Crowe, 1995). They express CD4 receptor (Crowe et al., 1987) and CCR5 co-receptor (Alkhatib et al., 1996) for HIV-1 entry, and hence are targets for macrophage (M)-tropic or R5 strains of HIV-1. Both blood monocytes and tissue macrophages can be infected with HIV-1, although monocytes are less susceptible than macrophages (Sonza et al., 1996), and persist in the circulation or tissues as viral reservoirs (Crowe et al., 1987; McElrath et al., 1989). Following HIV-1 infection, cells of macrophage lineage display impaired effector functions, including phagocytosis (Kedzierska et al., 2000), intracellular killing (Biggs et al., 1995) and cytokine production (reviewed in Kedzierska and Crowe, 2001). These defects contribute to the pathogenesis of AIDS by allowing reactivation and development of opportunistic infections with their attendant morbidity and mortality. Although the treatment of HIV-infected individuals has improved with the introduction of highly active antiretroviral therapy (HAART), research into the pathogenesis of AIDS-related opportunistic infections is still important and relevant. HAART dramatically reduces the plasma viral load and increases CD4 T-cell numbers, thereby delaying progression of the disease and decreasing the occurrence of opportunistic infections (reviewed in Crowe and Sonza, 2000). Despite the clinical improvement associated with HAART, current antiviral drugs are not able to eradicate HIV-1 due to the persistence of the latent reservoirs of the virus (Ho, 1998). In patients receiving HAART who maintain undetectable viral loads

for long periods, replication-competent HIV-1 can be recovered from resting CD4 T cells (Finzi et al., 1997) and peripheral blood monocytes (Crowe and Sonza, 2000). Viral reservoirs therefore represent a potentially life long persistence of replicationcompetent forms of HIV-1 that cannot be suppressed by current antiretroviral treatment. Strains of HIV-1 that are resistant to reverse transcriptase and protease inhibitors arise in the majority of treated patients who have not adhered to treatment regimens or have low plasma drug levels for other reasons. Viral load rises as a result of the emergence of drug resistant HIV-1. Opportunistic infections are the major problem for patients failing HAART and are likely to increase, as resistance to antiretroviral drugs becomes more widespread. Therefore, an improved understanding of the mechanisms by which HIV-1 impairs cell-mediated immunity is important for the full control of HIV-1 disease progression. Development of novel adjunctive immunotherapies able to control opportunistic pathogens is also needed. Although current drugs used for treatment of opportunistic infections are usually effective, drug resistance leading to persistent clinical symptoms continues to be recognised. Furthermore, some of the antibiotics used for the treatment of opportunistic infections (e.g. rifampicin) lower the blood levels of some antiretroviral drugs, and therefore must be used cautiously in combination with HAART. The use of immunomodulators such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon-gamma (IFNg) for the treatment of opportunistic infections has been successful in a limited number of studies. This review outlines the overall impairment of phagocytic function by cells of macrophage lineage following HIV-1 infection, considers the mechanisms underlying such defective function and pre-

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Table 1 Summary of studies of phagocytosis by peripheral blood monocytes obtained from HIV-infected individuals No. of subjects and category

Mean CD4 count (cells/ml)

Viral load (copies/ml)

Phagocytic target

Result

Reference

16 AIDS 19 HIV/

ND ND

C. albicans Opsonised zymosan

Normal Impaired

Nielsen et al., 1986 Bravo-Cuellar et al., 1992

ND ND

S. aureus Opsonised zymosan

Impaired Impaired

Pos et al., 1992 Pittis et al., 1993

20 AIDS

ND Variable range: 21
/913 B/200 Variable range: 120
/2225 ND

ND

Impaired

Capsoni et al., 1994

26 12 17 30

200 591, 70 ND

ND ND

IgG-opsonised erythrocytes T. gondii Escherichia coli

Impaired Increased Impaired Impaired

Delemarre et al., 1995 Dobmeyer et al., 1995

Variable range: 138
/713

Variable range: B/400
/385,500

Impaired

Kedzierska et al., 2001a

15 AIDS 20 HIV/

AIDS HIV/, AIDS HIV/

16 HIV/

Variable

Saccharomyces species MAC

Baqui et al., 1999

HIV/, HIV-seropositive patients; AIDS, AIDS patients; ND, not determined.

sents clinical examples of the use of adjunctive cytokine therapy combined with antiretroviral drugs and specific treatments for the management of opportunistic infections.

2. Defective phagocytosis by cells of macrophage lineage following HIV-1 infection In immunocompetent individuals peripheral blood monocytes and tissue macrophages provide critical functions in the cell-mediated response to a variety of pathogens such as bacteria (Mycobacterium avium complex (MAC), Mycobacterium tuberculosis ), parasites (Toxoplasma gondii , microsporidia) and fungi (Candida albicans , Cryptococcus neoformans , Pneumocystis carinii ). Inefficient control of opportunistic pathogens contributes to the development of opportunistic infections in HIV-infected individuals at late stages of disease. As monocytes and macrophages are distinct cell populations that differ in their susceptibility to HIV-1 infection (monocytes being refractory, whereas macrophages are fully permissive to HIV-1) (Sonza et al., 1996), this review considers independently defective phagocytosis by monocytes and macrophages.

2.1. Peripheral blood monocytes from HIV-1 infected individuals Peripheral blood monocytes are infrequently infected with HIV-1 in vivo (Schuitemaker et al., 1992) and only a very small proportion of blood monocytes (0.001
/1%) harbour HIV-1 at any time throughout the course of infection (McElrath et al., 1989). Freshly isolated monocytes are much less permissive to HIV-1 infection in vitro compared to monocyte-derived macrophages (MDM) cultured for a few days prior to in vitro infection (Rich et al., 1992; Sonza et al., 1996). That peripheral blood monocytes from HIVinfected individuals have defective phagocytic capacity for opportunistic pathogens has been well documented by our group and others. Impaired phagocytosis of T. gondii , Candida pseudotropicalis , Porphyromonas gingivalis , Fusobacterium nucleatum , C. neoformans and Staphylococcus aureus by peripheral monocytes obtained from HIV-infected individuals at different stages of disease have been reported (BravoCuellar et al., 1992; Pos et al., 1992; Cameron et al., 1993; Delemarre et al., 1995; Estevez et al., 1986; Dobmeyer et al., 1995; Baqui et al., 1999) (summarised in Table 1). Using a whole blood ex

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vivo assay (Hewish et al., 1996), we have also shown impaired phagocytosis of the common AIDS-related opportunistic pathogens MAC and T. gondii by monocytes from HIV-infected individuals compared to cells from uninfected controls (Kedzierska et al., 2000, 2001a). Decreased phagocytosis has been found in monocytes from patients with both undetectable (B/400 copies/ ml) and high viral loads (/10,000 copies/ml) (Baqui et al., 1999; Kedzierska et al., 2001a) as well as over a wide range of CD4 count (Roilides et al., 1993; Kedzierska et al., 2001a). However, a correlation between defective phagocytosis and CD4 counts or disease stage has been also reported (Pittis et al., 1993; Dobmeyer et al., 1995). As only a small proportion of blood monocytes (0.001
/ 1%) is infected with HIV-1, poor phagocytosis by monocytes from HIV-infected individuals may reflect an indirect consequence of infection. Although most studies have shown defective phagocytosis by monocytes following HIV-1 infection, a few have shown normal function (Nielsen et al., 1986; Eversole et al., 1994). Our laboratory has also investigated phagocytosis of the opportunistic pathogens MAC and T. gondii by peripheral blood monocytes obtained from the Sydney Blood Bank Cohort (SBBC) members (Kedzierska et al., 2001a). This cohort consist of a blood donor and eight transfusion recipients infected with an attenuated strain of HIV-1 with deletions in the nef gene as well as duplications and rearrangements in the overlapping LTR (Deacon et al., 1995). Whilst there is evidence of recent clinical progression in two of the cohort members after 17 years of infection (Learmont et al., 1992, 1999), the virus is clearly attenuated in comparison to wild-type HIV-1 strains. We found that blood monocytes from SBBC members phagocytosed MAC and T. gondii with efficiency similar to that of monocytes from HIV-uninfected individuals, but better than monocytes from individuals infected with wild-type HIV-1 (Fig. 1). These results provide evidence for the importance of Nef/LTR region in inhibition of phagocytosis by peripheral blood monocytes following HIV-1 infection in vivo (Kedzierska et al., 2001a).

2.2. Macrophages from HIV-infected individuals Tissue macrophages are major targets for HIV-1 infection (reviewed in Crowe, 1995). Resident tissue macrophages such as alveolar macrophages (Rich et al., 1992; Lewin et al., 1996), peritoneal macrophages (Olafsson et al., 1991) and placental macrophages (Kesson et al., 1993) are highly susceptible to in vitro HIV-1 infection on the day of isolation. Other specialised tissue macrophages including Kupffer cells (Schmitt et al., 1990) and microglia (Jordan et al., 1991) can also be productively infected with HIV-1. The proportion of HIV-infected macrophages within tissues is relatively high and ranges from 1 to 50% depending on the tissue source (McElrath et al., 1989; Embretson et al., 1993; Orenstein et al., 1997). Alveolar macrophages obtained from HIVinfected individuals show defective phagocytosis of a variety of opportunistic pathogens such as P. carinii , T. gondii , Aspergillus fumigatus, Histoplasma capsulatum and S. aureus (Musher et al., 1990; Wehle et al., 1993; Koziel et al., 1998), as do MDM derived from in vitro culture of monocytes from patients with HIV-1 infection (Capsoni et al., 1992; Roilides et al., 1993; Capsoni et al., 1994; Chaturvedi et al., 1995; Delemarre et al., 1995) (summarised in Table 2). MDM infected with HIV-1 in vitro also poorly ingest the opportunistic pathogens C. albicans (Crowe et al., 1994), T. gondii (Biggs et al., 1995) and MAC (Kedzierska et al., 2000, 2001a) (Table 2). Similar to tissue macrophages, MDM are also susceptible to HIV-1 infection in vitro. They can produce HIV-1 for weeks to months, without significant cytopathic effects (Crowe et al., 1987). MDM therefore provide a useful model for the assessment of HIV1 infection of tissue macrophages. Since a high proportion of MDM (20
/70%) can be infected with HIV-1 in vitro, impaired phagocytosis by those cells may predominantly reflect a direct effect of HIV-1 on macrophage function. Such an effect may be significant in HIV-infected individuals since a high proportion of tissue macrophages is also infected with HIV-1. Despite the studies demonstrating defective phagocytosis by HIV-infected macrophages, there

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Fig. 1. Monocytes from patients with wild-type HIV-1 infection show impaired phagocytosis of opportunistic pathogens, while subjects infected with an attenuated HIV-1 strain do not. Peripheral blood was collected from HIV-seronegative individuals (white bars), a SBBC member (C64, Deacon et al., 1995) infected with an attenuated strain of HIV-1 (striped bars) or patients infected with wild-type HIV-1 (black bars). Phagocytosis was assayed using a whole blood flow cytometric system as previously described (Hewish et al., 1996). Briefly, 100 ml of blood was incubated with 1.5/107 MAC or T. gondii conjugated to FITC for 10 min at 37 8C. Phagocytosis was stopped by plunging tubes on ice. The fluorescence of adhered MAC-FITC was quenched with the Orpegen quenching agent. Monocytes within the whole blood were stained with monoclonal antibody against CD14 conjugated to PE. Following the lysis of erythrocytes, leukocytes were fixed with 1% formaldehyde. The percentage of monocytes phagocytosing MACFITC was analysed by flow cytometry. The values have been corrected for the background fluorescence. (a) MAC phagocytosis; (b) T. gondii phagocytosis.

have been reports showing normal macrophage function (Nottet et al., 1993; Gordon et al., 2001). These discordant results may be partially explained by substantial variation in methods used by investigators, including the maturation stage of

macrophages at the time of functional assessment, the intracellular pathogens used as targets, type of phagocytosis assay and in particular the failure of some groups to control for endotoxin contamination.

Table 2 Summary of studies of phagocytosis by macrophages following HIV-1 infection Source of cells

No. of experiments/subjects

HIV-1 infection

Phagocytic target

Result

Reference

AM MDM MDM AM MDM MDM MDM MDM MDM MDM U937 U937 AM MDM MDM MDM AM

15 25 19 213 4 9 20 6 58 26 3 3 28 10 5 3 24

in in in in in in in in in in in in in in in in in

S. aureus IgG-opsonised erythrocytes A. fumigatus P. carinii E. coli , S. aureus C. albicans IgG-opsonised erythrocytes T. gondii H. capsulatum T. gondii IgG-opsonised erythrocytes C’3-opsonised erythrocytes P. carinii MAC IgG-opsonised latex beads C’-opsonised latex beads Streptococcus pneumoniae

Impaired Impaired Impaired Impaired Normal Impaired Impaired Impaired Impaired Impaired Impaired Normal Impaired Impaired Impaired Impaired Normal

Musher et al., 1990 Capsoni et al., 1992 Roilides et al., 1993 Wehle et al., 1993 Nottet et al., 1993 Crowe et al., 1994 Capsoni et al., 1994 Biggs et al., 1995 Chaturvedi et al., 1995 Delemarre et al., 1995 Thomas et al., 1997 Thomas et al., 1997 Koziel et al., 1998 Kedzierska et al., 2000 Kedzierska et al., 2001a Chan et al., 2001 Gordon et al., 2001

vivo vivo vivo vivo vitro vitro vivo vitro vivo vivo vitro vitro vivo vitro vitro vitro vivo

MDM, monocyte-derived macrophages; PBMC, peripheral blood mononuclear cells; AM, alveolar macrophages; C’, complement; in vivo, cells from HIV-infected subjects; in vitro, cells from normal subjects infected with HIV-1 in vitro.

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3. Mechanisms underlying defective phagocytosis by cells of macrophage lineage The mechanisms whereby HIV-1 impairs phagocytosis are not well known. There are a number of phagocytic receptors, and engulfment of specific opportunistic pathogens is often receptor-specific (reviewed in Greenberg, 1995). Complement receptors (C’Rs) are generally responsible for MAC uptake (Schlesinger and Horwitz, 1991) and Fcg receptors (FcgRs) mediate phagocytosis of opsonised T. gondii (Joiner et al., 1990). Most studies have shown that HIV-1 infection results in either elevated or unchanged surface expression of FcgRs or C’Rs (Capsoni et al., 1992; Nottet et al., 1993; Capsoni et al., 1994), indicating that the inhibition of phagocytosis by HIV-1 occurs during or after receptor binding. Following the interaction of phagocytosis-promoting macrophage surface receptors with specific ligands on target particle surface, cells of macrophage lineage engulf the pathogen. This process occurs via a cascade of signalling events resulting in membrane extension, pseudopodia formation, actin polymerisation at the site of ingestion, reorganisation of the actin-based cytoskeleton, formation of the phagosome enclosing the phagocytosed particle and its engulfment into the cytoplasm. Different receptors utilise distinct mechanisms to promote cytoskeletal rearrangements and engulfment (Allen and Aderem, 1996). This review focuses on the signalling events which occur during FcgR- and C’R-mediated phagocytosis and considers the mechanisms by which HIV-1 inhibits those signalling events. 3.1. FcgR-mediated phagocytosis 3.1.1. Fcg receptors The receptors for the constant region of immunoglobulin (Ig) G (FcgRI, FcgRII, FcgRIII) are the major means by which cells of macrophage lineage recognise IgG-opsonised pathogens. Once engaged, the receptors can often trigger either phagocytosis or antibody-dependent cellular cytotoxicity. The major FcgRs expressed on monocytes are the high affinity FcgRI (CD64) and a low affinity FcgRII (CD32), while macrophages also

express FcgRIIIA (CD16) (reviewed in Daeron, 1997). FcgRs share a highly homologous extracellular IgG binding domain, but they have different cytoplasmic domains. FcgRI and FcgRIIIA have relatively short cytoplasmic domains and they signal via gg homodimers which contain the immunoreceptor tyrosine-based activation motifs (ITAMs containing tyrosine residues arranged into specific signalling motifs YxxLx5
12Yx2
3L/I), while FcgRII has a large cytoplasmic domain containing ITAMs. 3.1.2. Signalling by cells of macrophage lineage FcgR-mediated internalisation of IgG-opsonised particles requires tyrosine phosphorylation of proteins and involves activation of several kinases and their substrates (Fig. 2). Most studies delineating signalling events during FcgRmediated phagocytosis have examined these pathways in murine macrophages or cell lines transfected with FcgR (Park et al., 1993; Greenberg et al., 1994; Indik et al., 1995). Following clustering of FcgRs, tyrosine kinases from the Src family associated with g chain of FcgR (including Hck and Lyn) are activated (Fitzer-Attas et al., 2000), leading to a rapid and transient phosphorylation of ITAMs (Park et al., 1993). This phosphorylation is absolutely required for FcgR-mediated phagocytosis as replacement of tyrosine residues within ITAMs with phenylalanine abolishes the phagocytic signal and subsequent internalisation of target particles (Park et al., 1993). Phosphorylated ITAMs create docking sites for Syk at the cell membrane, allowing its subsequent activation coupled to receptor clustering (Indik et al., 1995). An absolute and specific requirement for Syk in FcgR-mediated phagocytosis has been shown by gene knockout studies (Crowley et al., 1997). This requirement does not necessarily extend to other phagocytic receptors, since macrophages derived from the fetal livers of Syk-deficient mice showed unimpaired phagocytosis of yeast (phagocytosed by mannose receptors) or latex particles (phagocytosed via non-specific receptors) (Crowley et al., 1997). Activated Syk promotes phagocytic cap formation and is associated with phosphorylation and/or activation of a number of downstream substrates

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and localised accumulation of kinases such as focal adhesion kinase (FAK) and the actin-binding proteins paxillin, vinculin, talin and a-actinin (Greenberg et al., 1994; Allen and Aderem, 1996) (Fig. 2c), leading to cytoskeletal rearrangement and phagocytosis. Recently, we have reported that the early signalling events during FcgR-mediated phagocytosis in human uninfected MDM are similar to those previously characterised in murine macrophages. In both species, FcgR-mediated phagocytosis is mediated via tyrosine kinasedependent pathways involving the key tyrosine kinases Hck, Syk and Pyk2 (a member of FAK family) and the actin-binding protein, paxillin (Kedzierska et al., 2001b).

Fig. 2. A model for FcgR-mediated phagocytosis. Schematic representation of signalling events occurring during FcgRmediated phagocytosis. Following the binding of IgG-opsonised particles, clustering of FcgR leads to activation of tyrosine kinases from Src family associated with ITAMcontaining g-chain of FcgR I, III or cytoplasmic domain of FcgR II. Activation of Src kinases results in phosphorylation of tyrosine residues within ITAMs, which subsequently create docking sites for Syk. Activated Syk promotes phosphorylation of a number of downstream effectors: (a) PI3-kinase; (b) GTPbinding proteins; (c) cytoskeletal kinases and substrates leading to rearrangement of the actin-based cytoskeleton and engulfment of the phagocytosed particle.

including phosphatidyl inositide 3-kinase (PI3kinase) (Crowley et al., 1997) (Fig. 2a), GTPbinding proteins (Massol et al., 1998) (Fig. 2b),

3.1.3. Signalling by HIV-infected macrophages FcgR-mediated phagocytosis of a range of IgGopsonised targets has been shown to be inhibited in HIV-infected MDM (Capsoni et al., 1992, 1994; Kedzierska et al., 2001a, 2002). Recently, our group has reported the first potential mechanism underlying defective FcgR-mediated phagocytosis (Kedzierska et al., 2002). We found that impairment of phagocytic capacity was associated with marked inhibition of tyrosine phosphorylation of the cellular proteins stimulated during FcgRmediated phagocytosis, suggesting dysfunction at an early stage in FcgR-mediated signalling events. Further results confirmed this and demonstrated impaired phosphorylation of the tyrosine kinases Hck and Syk during phagocytosis by HIV-infected MDM (Kedzierska et al., 2002). Impaired Syk phosphorylation would inhibit Syk-mediated activation of substrates required for actin polymerisation and cytoskeletal rearrangement essential for FcgR-mediated phagocytosis. Consistent with these data, HIV-infected MDM had reduced phosphorylation of paxillin, a downstream effector molecule of Syk (Kedzierska et al., 2002). Similarly, decreased phosphorylation of Pyk-2, the FAK-related kinase, during FcgR-mediated phagocytosis in HIV-infected MDM has also been observed in our laboratory (Jaworowski A, unpublished results). The activation status of other downstream substrates of Syk such as PI-3 kinase, Vav or Rho family GTPases during FcgRmediated phagocytosis in HIV-infected MDM

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has not yet been determined, although they are also likely to be reduced. In seeking to understand why activation of Syk and Hck is impaired when expression of FcgRs is normal, the levels of associated g-signalling subunit were investigated, since this is the only known intermediate between FcgRs and Src family kinases. Decreased expression of this protein was found in HIV-infected MDM (Kedzierska et al., 2002). As the g signalling subunit is critical for Syk activation and subsequent signalling events during FcgR-mediated phagocytosis (Park et al., 1993; Indik et al., 1995), reduced levels of the ITAMcontaining g-signalling subunit in HIV-infected MDM are likely to be responsible for the defective phagocytosis by these cells. Our study (Kedzierska et al., 2002) therefore provides the first reported mechanism underlying defective cellular activation in HIV-infected macrophages during FcgRmediated phagocytosis. Impaired FcgR-mediated signalling may explain why HIV-infected macrophages fail to control opportunistic pathogens such as T. gondii (Joiner et al., 1990). Since the HIV-1 protein Nef interacts with cellular proteins and kinases which are potentially involved in FcgR-mediated phagocytosis, Nef would be expected to affect FcgR-mediated phagocytosis. Surprisingly, it was found that in vitro electroporation of Nef into uninfected MDM did not alter tyrosine phosphorylation during FcgRmediated phagocytosis (Kedzierska et al., 2001a). Similarly, in vitro infection of MDM with nefdeleted strains of HIV-1 impaired phagocytosis to the same degree as infection with wild-type virus, indicating that the inhibitory effect of HIV-1 on phagocytosis by MDM in vitro is not due to Nef (Kedzierska et al., 2001a). 3.1.4. Signalling by monocytes obtained from HIVinfected individuals The mechanism underlying defective phagocytosis by peripheral blood monocytes from HIVinfected individuals cannot be investigated using similar biochemical approaches due to limitations in the amount of blood available from them. We have therefore developed a whole blood flow cytometric assay which uses only 100 ml of blood (Hewish et al., 1996). Opportunistic pathogens

relevant to HIV-1 pathogenesis such as MAC and T. gondii conjugated to FITC can be used as phagocytic targets. To investigate the mechanism underlying defective FcgR-mediated phagocytosis by blood monocytes from HIV-infected subjects, a whole blood phagocytosis assay using FITC-tagged phalloidin to measure the level of actin polymerisation was also developed by our group. Poorly phagocytic monocytes from HIV-infected individuals had dysregulated actin polymerisation (Kedzierska et al., 2001a). Increased basal levels of F-actin in monocytes from HIV-infected individuals when compared to uninfected subjects had been demonstrated previously (Elbim et al., 1999). Our findings confirm those by Elbim et al. and have further shown that monocytes from HIV-infected individuals display either minimal or no elevation of Factin above their basal level when presented with IgG-opsonised targets, in contrast to significant net increases from the basal F-actin level in uninfected controls (Kedzierska et al., 2001a). As actin polymerisation plays a critical role in the formation of the phagocytic cup and ingestion of phagocytosed particles, defective actin remodelling is a potential mechanism underlying impaired phagocytic function in HIV-infected individuals. To determine if this HIV-induced effect occurs specifically at the actin polymerisation level or as a consequence of altered signalling events after engagement of FcgRs, the early signalling pathways during FcgR-mediated phagocytosis need to be investigated. The whole blood assay developed in our laboratory to assess the level of actin polymerisation could be further modified to determine the level of tyrosine phosphorylation during FcgR-mediated phagocytosis. Similarly, the phosphorylation status of Hck or Syk could be determined by flow cytometric analysis. 3.2. Complement-mediated phagocytosis 3.2.1. Complement receptors Phagocytosis of complement-opsonised particles is mediated via C’R1 (CD35), C’R3 (CD11b/ CD18) and C’R4 (CD11c/CD18). C’R1 is a single chain transmembrane receptor containing an extracellular domain which binds complement pro-

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teins present in serum; C3b, C4b and C3bi (Fearon, 1980). C’R3 and C’R4 are b2 integrin heterodimers comprising a common b subunit (b2, CD18) and distinct a chains (aM, CD11b and aL, CD11c, respectively) (Wright et al., 1983). C’R3 plays also an important role as an opsoninindependent receptor by binding bacterial lipopolysaccharide (LPS) (Wright et al., 1989).

3.2.2. Signalling by cells of macrophage lineage Integrins on the surface of macrophages are linked to actin filaments by actin-binding proteins such as vinculin and talin (Allen and Aderem, 1996) which are required for cytoskeletal rearrangement. Unlike FcgRs that are constitutively active for phagocytosis, C’Rs are inactive and require additional stimuli to be activated and promote particle internalisation (Aderem and Underhill, 1999). The activation of integrins is achieved via inside-out signaling, a process

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whereby the stimulation of cytokine and chemokine receptors activate intracellular signals (Fig. 3). Those signals in turn regulate the avidity and/ or affinity of the integrins for the ligand, lateral mobility of integrins in the plasma membrane and subsequent binding of the cytoplasmic tails of integrins to cytoskeletal proteins (reviewed in Calderwood et al., 2000). Whilst the molecular details of inside-out signalling and the activation process of integrins are not precisely known, several potential elements in the mechanisms of activation have been identified. Cytokines (e.g. TNF-a and IFN-g) and phorbol esters (e.g. PMA) activate integrins via the activation of protein kinase C (PKC). PMA also induces an increase in phosphorylation of CD18 (Chatila et al., 1989; Buyon et al., 1997). Examination of the cytoplasmic domains of C’R3 and C’R4 suggests several candidate sites for phosphorylation by PKC (Chatila et al., 1989; Buyon et al., 1997). PKC

Fig. 3. A model for complement-receptor mediated phagocytosis. C’Rs are inactive at the cell surface. Cytokines released at a site of infection activate macrophages through cytokine receptors. This activates PKC and RhoA. PKC phosphorylates the b chain and activates the integrins. RhoA, under the regulation of GEFs, stimulates downstream effectors to promote reorganisation of the actinbased cytoskeleton. This results in the bundling of actin filaments and the clustering of integrins. It is also possible that RhoA activation stimulates contractility, allowing the opsonised particle to sink into the cytosol of the macrophage. RhoA signalling can be terminated by elevation of intracellular cAMP.

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activation is associated with increased diffusion of the b2 chain of integrins (Zhou and Li, 2000). This suggests that phosphorylation of the b2 chain by PKC may serve as a regulatory mechanism for C’mediated phagocytosis, and supports a role for b2 integrin phosphorylation by PKC that releases integrins from cytoskeletal constraints. Activation of integrins is known to be associated with a quantitative up-regulation of their functions (Chatila et al., 1989). Upon activation, an integrin is thought to undergo a conformational change that enhances its ability to bind ligand (Berton and Lowell, 1999). Following binding of the activated receptor to a C’-opsonised particle, C’-receptors cluster and re-establish a firm link with the cytoskeleton (Zhou and Li, 2000). The few contact sites between the macrophage and C’-opsonised particle during the phagocytic process are rich in F-actin and cytoskeletal proteins such as vinculin, paxillin and a-actinin (Allen and Aderem, 1996). C’-mediated phagocytosis results in the reorganisation of polymerised actin at the site of ingestion and internalisation of the opsonised particle. The signalling that induces actin polymerisation and particle uptake by C’R is not precisely known. C’-mediated phagocytosis activates RhoA, a member of the Rho family of small GTPases (Caron and Hall, 1998) (Fig. 3). Rho and other GTPases are central in the regulation of the actin cytoskeleton, and in particular, RhoA stimulates contractile processes (Schoenwalder and Burridge, 1999). RhoA functions as a molecular switch, cycling between an active GTP-bound form and an inactive GDP-bound state (Bishop and Hall, 2000). It is regulated by guanosine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP and enables the protein to associate with effector molecules to initiate a downstream response. Intrinsic GTPase activity returns GTP to GDP and terminates the signalling. Activation of RhoA results in the bundling of actin filaments and clustering of integrins (Schoenwalder and Burridge, 1999). Rho-mediated signalling also promotes the assembly of stress fibres and focal adhesions, which regulate myosin filament formation and contractility. RhoA activation has been proposed to mediate integrin clustering at the site of ingestion, while regulating contractility of

the actin cytoskeleton to allow the opsonised particle to sink inside the macrophage (Chimini and Chavrier, 2000). The nucleotide exchange on RhoA and subsequent RhoA-mediated signalling can be terminated by elevation of intracellular cyclic AMP (cAMP) (Schoenwalder and Burridge, 1999).

3.2.3. Signalling by HIV-infected macrophages Although impaired phagocytosis of specific C’opsonised targets by HIV-infected MDM has previously been reported (Kedzierska et al., 2001a; Chan et al., 2001), the mechanisms underlying this defect remain unknown. Preliminary experiments from our laboratory suggest that impaired C’-mediated phagocytosis is associated with elevated intracellular cAMP levels (Azzem et al.). It is therefore possible that HIV-1 impairs C’mediated phagocytosis by inhibiting RhoAmediated signalling and hence subsequent cytoskeletal rearrangements critical for the ingestion of C’-opsonised pathogens. However, HIV-1 inhibition of early signalling events such as PKC activation or b2 integrin phosphorylation cannot be excluded.

3.2.4. Signalling by monocytes obtained from HIVinfected individuals To date, there have been no studies investigating the mechanisms whereby HIV-1 impairs C’mediated phagocytosis by peripheral blood monocytes in HIV-infected individuals. However, it is probable that increased levels of actin polymerisation within those cells (Elbim et al., 1999; Kedzierska et al., 2001a) may explain not only defective FcgR-mediated phagocytosis (Kedzierska et al., 2001a), but also lead to the impairment of other actin-based functions such as C’mediated phagocytosis, migration and chemotaxis. To determine how HIV-1 alters early signalling events during C’-mediated phagocytosis, the activation levels of PKC, b2 integrin and RhoA could be investigated by the use of flow cytometric assays, provided that suitable monoclonal antibodies are available.

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4. Use of immunomodulators for the treatment of opportunistic infections in HIV-infected individuals The function of cells of macrophage lineage is influenced by their state of activation, which in turn is initiated by exposure to growth factors and cytokines. The key monocyte/macrophage immunomodulators are GM-CSF and IFN-g. Those cytokines are of potential interest as adjuvants to drug therapy to accelerate immunoreconstitution and protection against a variety of opportunistic pathogens in immunosuppressed individuals. 4.1. Granulocyte-macrophage colony-stimulating factor GM-CSF is a glycoprotein of 127 amino acids with a molecular weight of 23
/28 kDa and is produced by a variety of cell types including activated T lymphocytes, endothelial cells, macrophages and fibroblasts. It is a multipotential haematopoietic growth factor regulating cells of eosinophilic, neutrophilic, monocytic and megakaryocytic lineages (reviewed in Armitage, 1998). GM-CSF stimulates the proliferation and differentiation of cells of macrophage lineage as well as a number of their functions including antimicrobial and tumoricidal activities, synthesis of cytokines and antibody-dependent cell-mediated cytotoxicity. Low concentrations of GM-CSF (pM ranges) have been found to enhance monocyte survival (Eischen et al., 1991). After their migration into tissues, GM-CSF induces differentiation of circulating monocytes into specialised macrophages such as alveolar macrophages, Kupffer cells or microglial cells. In vitro, GM-CSF has been reported to enhance the effector function of mature macrophages including increasing their phagocytic, tumoricidal and antimicrobicidal capacities as well as antibody-dependent cellular cytotoxicity activity (reviewed in Armitage, 1998). Stimulation with GM-CSF increases FcgRmediated phagocytosis by both human (Capsoni et al., 1992) and murine macrophages (Coleman et al., 1988), augments antimicrobial activity against a number of other opportunistic pathogens including MAC (Bermudez and Young, 1990), C. albicans (Smith et al., 1990), A. fumigatus (Roi-

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lides et al., 1996), Trypanosoma cruzi (Reed et al., 1987) and H. capsulatum (Newman and Gootee, 1992). Additionally, our group has also reported that GM-CSF markedly augmented the ability of HIV-infected MDM to phagocytose MAC in vitro (Kedzierska et al., 2000). Phagocytosis of MAC is not altered by exposure to a signalling-defective mutant form of GM-CSF (E21R) which binds only to the alpha chain of the GM-CSF receptor (Lopez et al., 1992), suggesting that augmentation of phagocytosis by GM-CSF requires binding to the ab heterodimeric form of the receptor (Kedzierska et al., 2000). GM-CSF is used in numerous clinical applications including cancer treatment and bone marrow repopulation after chemotherapy or irradiationdepletion (reviewed in Armitage, 1998). This cytokine is potentially useful in combating opportunistic infections in AIDS patients by stimulating monocyte/macrophage number and function. Currently GM-CSF is rarely used for the treatment of HIV-infected patients due to concerns regarding potential activation of HIV-1 replication, with adverse effects on viral load. Early clinical trials investigating the effect of GM-CSF on plasma HIV-1 levels showed that GM-CSF treatment of HIV-infected patients in the absence of antiretroviral therapy results in increased serum p24 antigen levels (Kaplan et al., 1991). However, when used in combination with effective antiretroviral therapy, GM-CSF has been safely administered to patients without any significant increase in viral load (Krown et al., 1992). In fact GM-CSF has been found to increase the activity of antiretroviral drugs (Perno et al., 1992). Data from studies of HIV-infected patients receiving antiretroviral therapy combined with GM-CSF have shown that patients experienced a decrease in viral load and an increase in CD4 counts (Brites et al., 2000). Clinical improvement and augmented monocyte phagocytic function against mycobacteria and fungal infections, again without an increase in viral load, has also been reported in patients with advanced HIV-1 infection and drug-resistant oropharyngeal candidiasis treated with GM-CSF (Vazquez et al., 1998). Our previous study also confirmed the beneficial effect of adjunctive GMCSF therapy in a patient with AIDS and dissemi-

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nated multi-drug resistant MAC infection, in which GM-CSF therapy improved phagocytosis of MAC by the patient’s peripheral blood monocytes and reduced MAC bacteremia (Kedzierska et al., 2000). Taken together, these studies suggest that GM-CSF might be useful adjunctive therapy in augmenting macrophage function in HIV-infected patients with opportunistic infections provided that effective antiretroviral therapy can be given concurrently. 4.2. Interferon-gamma IFN-g is a homodimeric glycoprotein which is produced by activated T and NK cells and binds to a type II IFN receptor. IFN-g promotes T and B cell proliferation, activation of mononuclear phagocytes and neutrophils, augmentation of NK cell lytic function, MHC I and II expression and suppresses IL-4 responses. IFN-g displays a wide variety of antiviral, antiproliferative, immunomodulatory and apoptotic functions, and is capable of inhibiting viral infection in a non-specific manner (reviewed in Le Page et al., 2000). Several studies demonstrate a significance of IFN-g in the immune responses against a variety of fungal, bacterial and parasitic infections. IFN-g or the IFN-g receptor gene knockout mice are highly susceptible to opportunistic infections with mycobacteria, poxviruses and listeria (Dalton et al., 1993; Huang et al., 1993). Similarly, children with congenital deficiency in the IFN-g receptor develop severe atypical mycobacterial infections (Newport et al., 1996). Conversely, in vitro treatment of monocyte/macrophages with IFN-g enhances antimicrobial activities against A. fumigatus (Roilides et al., 1996), C. neoformans (Herrmann et al., 1994), Listeria monocytogenes (Peck, 1991) and T. cruzi (Reed et al., 1987), T. gondii (Nathan et al., 1983). IFN-g-mediated augmentation of monocyte/macrophage function is associated with increased oxidative metabolism and secretion of H2O2 by those cells (Nathan et al., 1983; Murray et al., 1985b). IFN-g can also restore deficient functions of HIV-infected macrophages. In vitro exposure of HIV-infected MDM to IFN-g augments their oxidative burst activity in response to intracellular

pathogens such as T. gondii , Leishmania chagasi and T. cruzi (Reed et al., 1992). This cytokine also augments intracellular killing of T. gondii in HIVinfected MDM (Biggs et al., 1995) and FcgRmediated phagocytosis in HIV-1 transfected U937 cells (Thomas et al., 1997). Furthermore, IFN-g increases the antimicrobial activity of monocytes (Murray et al., 1987) and alveolar macrophages (Murray et al., 1985a) from AIDS patients against the intracellular pathogens L. donovani and T. gondii. Similar to GM-CSF, IFN-g has received increasing attention as a potential adjunctive immunomodulatory therapy for treatment of immunosuppressed patients. Clinical trials have indicated that IFN-g immunotherapy increases mononuclear cell biological responses including increased H2O2 secretory capacity, phagocytosis, NK/LAK activation, and improves outcomes in patients with advanced cancer, chronic granulomatous disease or after the autologous bone marrow transplants (reviewed in Murray, 1996). Administration of IFN-g to patients with advanced HIV-1 infection reduces the incidence of opportunistic infections and is effective especially against candida, herpes simplex and cytomegalovirus infections (Murphy et al., 1988; Riddell et al., 2001). INF-g has also been used successfully for treatment of M. avium -intracellulare complex infection (Squires et al., 1992) and Kaposi’s sarcoma (Murray, 1996) in AIDS patients. However, further clinical trials are necessary to establish a better understanding of the role of IFN-g adjunctive therapy on the treatment of HIVinfected individuals.

5. Summary Monocytes and macrophages are distinct cell populations that differ in the expression of surface receptors, cytokine/chemokine production and their susceptibility to HIV-1 infection. We therefore propose that there are different mechanisms underlying defective phagocytosis in those two cell populations. Since only a small proportion of blood monocytes (0.001
/1%) is infected with HIV-1, the high level of inhibition of phagocytosis

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in monocytes obtained from HIV-infected individuals may reflect an indirect consequence of HIV1 infection. Conversely, the high proportion of MDM and tissue macrophages infected with HIV1 (1
/50%) suggest a predominantly direct effect of HIV-1 on phagocytosis. This hypothesis is supported by a previous report from our laboratory demonstrating defective phagocytosis of T. gondii predominantly in MDM shown by flow cytometric analysis to be positive for p24 antigen (Biggs et al., 1995), and by evidence that heat-killed HIV-1 did not impair phagocytosis of C. albicans (Crowe et al., 1994). Since different phagocytic receptors use distinct mechanisms to engulf specific pathogens, we have summarised the present state of knowledge regarding the signalling events occurring during FcgRand C’-mediated phagocytosis in normal uninfected monocyte/macrophages. HIV-1 inhibits FcgR-mediated phagocytosis in human MDM by blocking early signalling events, specifically at the g-signalling chain of FcgRs (Kedzierska et al., 2002). Defective FcgR-mediated phagocytosis by blood monocytes from HIV-infected individuals is associated with dysregulated actin polymerisation (Kedzierska et al., 2001a). The underlying mechanisms whereby HIV-1 impairs C’-mediated phagocytosis by cells of macrophage lineage remain to be elucidated. Cytokines such as GMCSF and IFN-g are the most promising immunomodulators for augmenting monocyte/macrophage-mediated immunity against opportunistic infections.

Acknowledgements We would like to thank Geza Paukovics for his flow cytometric expertise and HiuTat Chan for stimulating discussions.

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