HIV-1 Down-Modulates γ Signaling Chain of FcγR in Human Macrophages: A Possible Mechanism for Inhibition of Phagocytosis

Katherine Kedzierska, Philip Ellery, Johnson Mak, Sharon R. Lewin, Suzanne M. Crowe and Anthony Jaworowski
J Immunol March 15, 2002, 168 (6) 2895-2903; DOI: https://doi.org/10.4049/jimmunol.168.6.2895

Abstract

HIV-1 infection impairs a number of macrophage effector functions, thereby contributing to development of opportunistic infections and the pathogenesis of AIDS. FcγR-mediated phagocytosis by human monocyte-derived macrophages (MDM) is inhibited by HIV-1 infection in vitro, and the underlying mechanism was investigated in this study. Inhibition of phagocytosis directly correlated with the multiplicity of HIV-1 infection. Expression of surface FcγRs was unaffected by HIV-1 infection, suggesting that inhibition of phagocytosis occurred during or after receptor binding. HIV-1 infection of MDM markedly inhibited tyrosine phosphorylation of the cellular proteins, which occurs following engagement of FcγRs, suggesting a defect downstream of initial receptor activation. FcγR-mediated phagocytosis in HIV-infected MDM was associated with inhibition of phosphorylation of tyrosine kinases from two different families, Hck and Syk, defective formation of Syk complexes with other tyrosine-phosphorylated proteins, and inhibition of paxillin activation. Down-modulation of protein expression but not mRNA of the γ signaling subunit of FcγR (a docking site for Syk) was observed in HIV-infected MDM. Infection of MDM with a construct of HIV-1 in which nef was replaced with the gene for the γ signaling subunit augmented FcγR-mediated phagocytosis, suggesting that down-modulation of γ-chain protein expression in HIV-infected MDM caused the defective FcγR-mediated signaling and impairment of phagocytosis. This study is the first to demonstrate a specific alteration in phagocytosis signal transduction pathway, which provides a mechanism for the observed impaired FcγR-mediated phagocytosis in HIV-infected macrophages and contributes to the understanding of how HIV-1 impairs cell-mediated immunity leading to HIV-1 disease progression.

Cells of macrophage lineage, including peripheral blood monocytes and tissue macrophages, provide critical functions in the cell-mediated response to a variety of opportunistic pathogens such as Mycobacterium avium complex, Toxoplasma gondii, and Candida albicans. A number of monocyte/macrophage functions are impaired following HIV-1 infection in vivo and in vitro, including chemotaxis (1, 2), phagocytosis (3, 4, 5), intracellular killing (3), and cytokine production (reviewed in Ref. 6). These defects contribute to the pathogenesis of AIDS by allowing reactivation and development of opportunistic infections (reviewed in Ref. 7). The mechanism by which HIV-1 impairs effector functions of cells of macrophage lineage is unknown.

The HIV-1-encoded proteins Nef, Vif, Vpr, and Rev have been shown to modulate a number of signaling pathways via interactions with cytoskeletal (8, 9, 10, 11) and cytoplasmic proteins (12, 13, 14, 15, 16). These interactions include cellular proteins and kinases that are also involved in FcγR-mediated phagocytosis, e.g., the Src kinases, Hck and Lyn (12, 13, 17, 18), p21-activated kinase (14, 15), the guanine nucleotide-exchange factor, Vav (19), and actin (10). HIV-1 impairs FcγR-mediated phagocytosis (5), and the mechanism is unknown, although studies using the promonocytic U937 cell line suggest that inhibition occurs via a cAMP-dependent mechanism (20).

The receptors for the constant region of IgG (FcγRI, FcγRII, and FcγRIII) are the major means by which cells of macrophage lineage recognize IgG-opsonized pathogens, thereby triggering phagocytosis and Ab-dependent cellular cytotoxicity. Peripheral blood monocytes express mainly the high-affinity FcγRI (CD64) and a low-affinity FcγRII, whereas macrophages also express FcγRIIIA (CD16A; reviewed in Ref. 21). FcγR-mediated internalization of IgG-opsonized particles requires tyrosine phosphorylation of proteins and involves activation of several kinases and their substrates. Most studies to date have examined these pathways in murine macrophages or cell lines transfected with FcγR (22, 23, 24, 25, 26, 27). Following clustering of FcγRs, tyrosine kinases from the Src family associated with γ-chain of FcγR (including Hck and Lyn) are activated (28, 29), leading to a rapid and transient phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs)4 present on the γ signaling subunits associated with FcγRI and FcγRIII or on the cytoplasmic domain of FcγRII (30). Phosphorylation of ITAMs create docking sites for Syk, which is subsequently activated by phosphorylation (27, 31). An absolute and specific requirement for Syk in FcγR-mediated phagocytosis has been shown by gene knockout studies (32). Activation of Syk results in phosphorylation of phosphatidylinositol 3-kinase (33) and localized accumulation of kinases such as focal adhesion kinase (FAK) and cytoskeletal substrates, including actin-binding proteins paxillin, vinculin, talin, and α-actinin (23, 26, 34), leading to cytoskeletal rearrangement and phagocytosis of the opsonized particles. Recently, we have reported that the early signaling events during FcγR-mediated phagocytosis by human monocyte-derived macrophages (MDM) also involve tyrosine phosphorylation of cellular proteins, including Hck, Syk, Pyk-2 (a member of FAK family), and paxillin (35).

This study examines the mechanism by which HIV-1 inhibits FcγR-mediated phagocytosis in human MDM. Our results show that defective phagocytosis by HIV-infected MDM is due, at least in part, to decreased expression of the γ signaling subunit of the FcγR, which leads to specific signaling defects downstream of FcγRs.

Materials and Methods

Isolation and culture of monocytes

Human monocytes were isolated from buffy coats of HIV-, hepatitis B virus-, and human T cell leukemia virus-seronegative blood donors (supplied by the Red Cross Blood Bank, Melbourne, Australia) by Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation and plastic adherence as previously described (36). Cell viability was >95% and the purity of monocytes was >90% as determined by immunofluorescent staining with anti-CD14 mAb (BD Biosciences, Mountain View, CA) and analysis by flow cytometry (FACStarPlus; BD Biosciences). Monocytes were cultured in IMDM (Cytosystem, Castle Hill, Australia) supplemented with 10% heat-inactivated human AB+ serum, 2 mM l-glutamine, and 24 μg/ml gentamicin (supplemented Iscove’s medium) in suspension in polytetrafluorethylene (Teflon) jars (Savillix, Minnetonka, MN) at an initial concentration of 1 × 106 cells/ml.

HIV-1 infection of MDM

On day 5 postisolation, MDM were infected with the M-tropic strain of HIV-1Ba-L (AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD) at a multiplicity of infection (MOI) of 0.1–1 for 2 h as described previously (37). In selected experiments, MDM were infected at different MOIs, ranging from 0.1 to 3, to assess the role of HIV-1 input on FcγR-mediated phagocytosis. Control cells were mock-infected and cultured under identical conditions. HIV-1 replication in MDM was quantified by monitoring reverse transcriptase (RT) activity using a micro RT assay on day 7 postinfection (38). Under the conditions used in our experiments, HIV-1 infection of MDM at varying MOIs was not associated with decreased viability of the cells when assessed by trypan blue exclusion, decreased MDM numbers, or morphologic changes. All the reagents, viral stocks, and culture supernatants tested in this study were negative for LPS contamination (<0.5 U/ml) using the limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).

Flow cytometric analysis of FcγRI, II, and III expression

MDM on day 7 postinfection were analyzed for surface expression of FcγRsI (CD64), FcγRII (CD32), and FcγRIII (CD16). Cells were stained with mAb directed against CD64 conjugated to PE (1 μg/ml; Serotec, Raleigh, NC), CD32 conjugated to FITC (1 μg/ml; Serotec), and CD16 conjugated to CyChrome (1 μg/ml; BD PharMingen, San Diego, CA) on ice for 30 min, followed by a wash in calcium- and magnesium-free PBS (PBS-CMF). The mean fluorescence of monocytes expressing FcγRs was quantified by flow cytometric analysis. Cells were also stained with isotype-matched controls conjugated to the appropriate fluorochrome.

Intracellular staining of γ-chain of FcγRs

The intracellular γ-chain staining was determined in uninfected and HIV-infected MDM on day 7 postinfection. Cells were fixed in 3% ultrapure formaldehyde for 45 min, followed by two washes in 0.1 M glycine in PBS-CMF and permeabilization in 0.1% Triton X for 1 min. MDM were washed twice in PBS-CMF containing 1% FCS and stained for intracellular γ-chain using rabbit anti-γ subunit (TCR and FcR) polyclonal Ab (1 μg/ml; Upstate Biotechnology, Lake Placid, NY) or rabbit IgG control (1 μg/ml; Upstate Biotechnology), followed by two washes in cold (4°C) PBS-CMF and incubation with sheep anti-rabbit IgG conjugated to FITC (SILENUS Laboratories, Melbourne, Australia). All staining procedures were performed in the presence of 50% FCS to reduce the level of nonspecific staining. The fluorescence for intracellular γ-chain was quantified by flow cytometric analysis, converted to molecules of equivalent soluble fluorochrome units, and corrected for background fluorescence.

Opsonization of target particles

Target particles were opsonized immediately before the phagocytosis assay as previously reported (35). Briefly, sheep RBC (E; ICN-Cappel, Aurora, OH) were opsonized with rabbit anti-E Ab (ICN-Cappel), whereas latex beads (3 μm in diameter; Sigma-Aldrich, St. Louis, MO) were coated with BSA (Sigma-Aldrich), washed, and opsonized with rabbit anti-BSA antiserum (ICN-Cappel).

Phagocytosis assay using IgG-opsonized sheep RBC (E-IgG)

On day 7 postinfection, MDM were plated onto 96-well plates (Costar, Cambridge, MA) at 5 × 104 cells per well in 100 μl of supplemented Iscove’s medium and were allowed to adhere for 2 h at 37°C. IgG-opsonized or unopsonized E were added to adhered MDM at a E:MDM ratio of 10:1. The plate was centrifuged at 100 × g for 5 min at 4°C and was then placed at 37°C for phagocytosis to proceed. Phagocytosis was terminated after 10 min by washing cells with cold (4°C) PBS and was quantified by a colorimetric assay (35, 39). In selected experiments, MDM on day 7 postisolation were incubated with 8′bromo-cAMP (Sigma-Aldrich) at concentrations ranging from 0.1 μM to 1 mM at 37°C for 30 min or 48 h before phagocytosis assay.

Phagocytosis assays using IgG-opsonized beads

MDM (1 × 106 cells) were dispensed into 4-ml polypropylene tubes (BD Biosciences, Franklin Lakes, NJ), washed twice in PBS-CMF (500 × g for 5 min), and cooled on ice for 20 min in 100 μl of PBS-CMF. Cells were incubated with or without IgG-opsonized beads (target:MDM ratio of 10:1) at 37°C in a shaking water bath. At specified time points, phagocytosis was arrested by plunging the tubes into ice and washing MDM in ice-cold PBS-CMF, followed by centrifugation at 20,000 × g for 45 s. For immunoblotting and immunoprecipitation experiments, cells were lysed in 100 μl of Triton lysis buffer containing 25 mM Tris-HCl (pH 7.5), 0.14 M NaCl, 1 mM EDTA, and 1% Triton X-100, supplemented with the following phosphatase inhibitors: 50 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich), and 40 mM β-glycerophosphate (Sigma-Aldrich), and protease inhibitors: 1 mM pefabloc, 1 μM pepstatin, and 1 μM leupeptin (Boehringer Mannheim, Mannheim, Germany).

Immunoblotting and immunoprecipitation

MDM extracts containing equal amounts of proteins, as determined by detergent-compatible protein assay (Bio-Rad, Hercules, CA), were boiled in SDS sample buffer (10 mM Tris (pH 8), 2 mM EDTA, 1% SDS, 5% 2-ME, and 5% glycerol), resolved by 10% SDS-PAGE, transferred to nitrocellulose, and blocked for 2 h in 3% BSA. The blots were probed with Abs directed against phosphotyrosine (RC20; BD Transduction Laboratories, Lexington, KY), Syk (Santa Cruz Biotechnology, Santa Cruz, CA), paxillin (BD Transduction Laboratories), Hck (a gift from Dr. H.C. Cheng, Department of Biochemistry, University of Melbourne, Melbourne, Australia), and γ-chain of FcγR (Upstate Biotechnology) overnight at 4°C, followed by secondary Ab conjugated with HRP (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.), and they were then developed for ECL according to manufacturer’s instructions. To determine the phosphorylation of Hck, Syk, and paxillin during FcγR-mediated phagocytosis, cell lysates were immunoprecipitated with the appropriate Ab overnight at 4°C, collected with 15 μl of protein G-Sepharose beads (a 1-h incubation at 4°C; Pharmacia Biotech, Uppsala, Sweden), washed five times in Triton lysis buffer, boiled in SDS sample buffer, resolved by 10% SDS-PAGE, and probed with anti-phosphotyrosine Ab (RC20) conjugated to HRP.

mRNA extraction and amplification of γ signaling subunit

mRNA was extracted from MDM lysates (prepared on day 7 postinfection) using oligo(dT)25 beads (Dynabeads; Dynal Biotech, Carlton South, Australia) and was subsequently converted to cDNA as previously reported (38). To check for cDNA contamination samples prepared without avian myeloblastosis virus-RT were included within each experiment. For PCR, 2-fold dilutions were made using equal amounts of cDNA based on GAPDH levels determined by real-time PCR as previously described (40). PCR for β-actin was performed to confirm equal template levels (41). Reactions were performed in a total of 50 μl comprised of 0.2 mM each dNTP, 1.5 mM MgCl2, 0.4 μM each primer, 1.1 U of Taq polymerase (PerkinElmer/Cetus, Norwalk, CT), and 1× reaction buffer (PerkinElmer/Cetus). The γ-chain primer set amplified 172 bp of the human γ-chain cDNA sequence (GenBank accession no. NM004106) with the sequences 5′-GAGCCTCAGCTCTGCTATATCC-3′ and 5′-TCTCGTAAGTCTCCTGGTTCC-3′. Samples were first denatured at 95°C for 2 min and were then amplified for 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 7 min. PCR products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining.

Construction of [NL(AD8)Δnef-γ+]

The DNA constructs [pNL(AD8)] and [pNL(AD8)Δnef] were prepared by substituting the respective envelope coding DNA sequences from NL4.3 and NL4.3Δnef with monocytotropic AD8 envelope coding sequences, converting a T-tropic virus to M-tropic as previously described (5). The [NL(AD8)Δnef-γ+] plasmid was constructed using stitch PCR mutagenesis. HIV-1 sense f1 primer Rev 8392S 5′-GGGGACCCGACAGGCCCG-3′ and HIV-1 antisense f1 primer 5′-GAGCAAGACCACTGCTGGAATCATCTTATAGCAAAATCCTTTCCAAGC-3′ were used to amplify a 390-bp HIV-1 f1 fragment immediately upstream of Nef. γ-Chain sense f2 primer 5′-GCTTGGAAAGGATTTTGCTATAAGATGATTCCAGCAGTGGTCTTGCTC-3′ and antisense f2 primer 5′-CCCCTCGAGACGCGTCTACTGTGGTGGTTTCTCATGCTTCAG-3′ were used to amplify a 300-bp of γ-chain coding f2 fragment. HIV-1 f1 and γ-chain f2 fragments were joined by PCR extension as previously described (42). The resulting PCR-amplified fragment was cloned into the [NL(AD8)Δnef] proviral DNA via restriction sites BamHI and XhoI. DNA sequencing has been performed to ensure the presence of γ-chain in this mutant proviral DNA and the absence of spontaneous mutations. The production of [NL(AD8)], [NL(AD8)Δnef] viruses, and [NL(AD8)Δnef-γ+] particles was achieved by introducing 10 μg of protein into 293T cells by the calcium-phosphate transfection method as previously described (42).

Statistical analysis

The significance of the effects of HIV-1 and cAMP on FcγR-mediated phagocytosis, down-modulation of γ-chain in HIV-infected MDM, and the augmentation of phagocytosis by [NL(AD8)Δnef- γ+] construct were assessed using the Student’s paired t test. A value of p = 0.05 was used to reject null hypothesis.

Results

HIV-1 inhibits FcγR-mediated phagocytosis via a postreceptor-mediated mechanism

In vitro infection of MDM with HIV-1 inhibited phagocytosis of IgG-opsonized E (mean inhibition of 50.3 ± 4.3%, n = 33, p < 0.001) as assessed on day 7 postinfection (Fig. 1⇓A). The inhibition of phagocytosis was found in 31 of 33 donors and ranged from 18 to 91%. The percentage of inhibition of phagocytosis expressed as the phagocytic index in this study was similar to those previously reported by our group when phagocytosis was assessed as the percentage of MDM phagocytosing the fluorescent targets (mean of 51.5% inhibition for n = 10 (Ref. 5) or mean of 38.7% inhibition for n = 10 (Ref. 4)).

FIGURE 1.
FIGURE 1.

HIV-1 inhibits FcγR-mediated phagocytosis by MDM. Phagocytosis of specific IgG-opsonized sheep RBC (IgG-E) was assessed by colorimetric assay (see Materials and Methods). MDM on day 5 postisolation were either mock-infected (open bars) or infected with HIV-1Ba-L (filled bars). Phagocytosis assays were performed on day 7 postinfection using IgG-E at target:MDM ratio of 10:1. A, Data represent means ± SEM of the phagocytic index (number of phagocytosed particles per 100 MDM) from 33 donors used in this study. B, Representative donor infected with HIV-1 at varying MOI.

Under the conditions used in our experiments, the percentage of MDM infected with HIV-1 varies between 30 and 70% when assessed by intracellular p24 Ag staining (3, 36, 38). To assess the level of HIV-1 input on FcγR-mediated phagocytosis, MDM from the same donor were infected at increasing MOIs. Phagocytosis was inhibited progressively with increased MOI in MDM cultures, indicating a direct effect of HIV-1 replication on the phagocytic capacity of macrophages (Fig. 1⇑B). HIV-1 infection of MDM did not alter the surface expression of FcγRs: FcγRI (CD64), FcγRII (CD32), or FcγRIII (CD16; p = 0.22, 0.84, and 0.09, respectively, n = 5) at the time of phagocytosis, suggesting that inhibition of phagocytosis occurs during or after receptor binding (Fig. 2⇓).

FIGURE 2.
FIGURE 2.

Flow cytometric analysis of surface expression of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) on HIV-infected and uninfected macrophages. Expression of FcγRs on MDM on day 7 after HIV-1 infection was assessed by flow cytometric analysis using anti-CD64 mAb conjugated to PE, anti-CD32 mAb conjugated to FITC, and anti-CD16 mAb conjugated to CyChrome. A, Results were expressed as net mean fluorescence (corrected for background fluorescence) ± SEM and represent data from five experiments using uninfected (open bars) or HIV-infected MDM (filled bars). B, Results from a representative single donor are provided. Histograms are unimodal and demonstrate the staining with mAbs directed against FcγRs (solid lines) or isotype-matched controls (dotted lines) conjugated to appropriate fluorochrome.

HIV-1 inhibition of FcγR-mediated phagocytosis is not mediated via a cAMP-dependent mechanism

As a cAMP-dependent mechanism has been proposed as the cause of poor FcγR-mediated phagocytosis in the promonocytic U937 cell line latently infected with HIV-1 (20), we initially investigated whether a cAMP-dependent mechanism might be responsible for the inhibition of phagocytosis in MDM infected with HIV-1. However, incubation of MDM with a nonmetabolized cAMP analog, 8′bromo-cAMP for either 30 min (p > 0.05, n = 5) or 48 h (p > 0.05, n = 4) did not inhibit FcγR-mediated phagocytosis by human MDM (Table I⇓).

View this table:
Table I.

Effect of 8′-bromo-cAMP on FcγR-mediated phagocytosis by human MDMa

Tyrosine phosphorylation of cellular proteins is inhibited during FcγR-mediated phagocytosis by HIV-infected MDM

Because FcγR-mediated internalization of IgG-opsonized beads by human MDM requires rapid phosphorylation of tyrosine residues in a range of proteins (35), the effects of HIV-1 on tyrosine phosphorylation triggered by binding of IgG-opsonized targets was assessed. A range of cellular proteins was rapidly phosphorylated (by a 2-min exposure to IgG-particles) in MDM exposed to IgG-opsonized beads, and phosphorylation reached a peak between 2 and 5 min (Fig. 3⇓A). HIV-1 infection of MDM inhibited FcγR-triggered tyrosine phosphorylation of cellular proteins, suggesting a defect downstream to initial receptor activation. Inhibition of tyrosine phosphorylation in HIV-infected MDM correlated with markedly impaired phagocytosis (Fig. 3⇓B). In some cultures, HIV-infected MDM displayed a relatively high basal level of tyrosine phosphorylation, but an increase in tyrosine phosphorylation was never observed during FcγR-mediated phagocytosis by those cells.

FIGURE 3.
FIGURE 3.

Inhibition of protein tyrosine phosphorylation during FcγR-mediated phagocytosis in HIV-infected macrophages. A, MDM incubated with IgG-opsonized latex beads (target:MDM ratio of 10:1) for the indicated times were lysed in Triton X-100 buffer, and samples of lysate containing equal amount of protein (30 μg) were resolved by SDS-PAGE, then probed with anti-phosphotyrosine conjugated to HRP (RC20). Results shown are representative of three experiments using MDM prepared from different donors. The levels of tyrosine phosphorylation have been analyzed by laser densitometry. Results are expressed as net densitometry units corrected for background levels. B, Inhibition of tyrosine phosphorylation was associated with decreased FcγR-mediated phagocytosis by HIV-infected MDM from the same donor. Phagocytosis of IgG-opsonized E by uninfected (open bars) and HIV-infected (filled bars) MDM was measured after 15 or 30 min of phagocytosis via a colorimetric assay as described in Materials and Methods. Data represent means ± SEM of triplicate determinations. IB, immunoblotting; PY, phosphotyrosine.

FcγR-mediated phagocytosis is impaired at a step upstream of Hck, Syk, and paxillin

We then assessed the effect of HIV-1 on specific tyrosine kinases necessary for FcγR-mediated phagocytosis and their substrates. HIV-1 infection of MDM inhibited phosphorylation of tyrosine kinases from two different families, Hck (a Src family member; 58 kDa; Fig. 4⇓A) and Syk (72 kDa; Fig. 4⇓B). HIV-1 infection also impaired formation of FcγR-stimulated Syk complexes with other tyrosine-phosphorylated proteins (molecular mass of 38, 42, 48, 52, 58, 64, and 95 kDa; Fig. 4⇓B). HIV-infected MDM also displayed reduced phosphorylation of paxillin (an adapter protein that localizes to focal adhesions and is a substrate for Src and FAK family kinases; 68 kDa) during phagocytosis (Fig. 4⇓C). The input of Hck, Syk, and paxillin proteins was standardized according to protein estimations in each lysate. Protein levels are shown by reprobing with relevant Abs where possible (Fig. 4⇓, B and C, lower panels). Reprobing of the blots with anti-Hck Ab was not feasible, as a broad band of Ig H chain (55 kDa) interfered with the Hck band (58 kDa).

FIGURE 4.
FIGURE 4.

HIV-1 infection of MDM inhibits Hck (58 kDa), Syk (72 kDa), and paxillin (68 kDa) activation during FcγR-mediated phagocytosis. On day 7 postinfection, MDM (2 × 106) were incubated with IgG-opsonized latex beads (2 × 107) in 200 μl of PBS-CMF for the indicated times at 37°C, followed by lysis in Triton X-100 buffer. Sample of lysates containing equal amount of protein (50 μg) were immunoprecipitated with anti-Hck (A), anti-Syk (B), and anti-paxillin (C); the immunoprecipitates were resolved by SDS-PAGE and were immunoblotted with anti-phosphotyrosine-HRP. The data shown are representative of three experiments using MDM from different donors. The levels of Hck, Syk, and paxillin phosphorylation have been analyzed by laser densitometry. Results are expressed as net densitometry units corrected for background levels. Equivalent protein levels are shown by reprobing the blots with the relevant Abs where feasible (B and C, lower panels). IP, immunoprecipitation; IB, immunoblotting; PY, phosphotyrosine.

FcγR-mediated phagocytosis by HIV-infected MDM is associated with down-modulation of Fc γ-chain protein expression

Although the surface expression of FcγRs was not altered by HIV-1, the protein tyrosine phosphorylation data suggest a defect upstream of Hck and Syk, at the level of the γ signaling subunit of FcγR. HIV-1 infection-reduced protein levels of the ITAM-containing γ-chain of FcγR were demonstrated by immunoblot analysis of Triton X-100 soluble lysates from HIV-1-infected MDM when compared with lysates obtained from uninfected MDM and standardized according to total protein levels (Fig. 5⇓A). Intracellular staining using anti-γ-chain Ab confirmed these results and demonstrated a significant decrease in intracellular levels of the FcγR γ signaling subunit in HIV-infected MDM (mean inhibition of 57.3 ± 12.3%; p = 0.009, n = 4) compared with uninfected MDM (Fig. 5⇓, B and C).

FIGURE 5.
FIGURE 5.

HIV-1 infection of MDM down-modulates protein levels of the γ signaling subunit of FcγR. A, MDM (1 × 106) infected with HIV-1 for 7 days were washed twice in PBS-CMF, lysed in Triton X-100 buffer, and samples of lysate containing equal amount of protein (30 μg) were resolved by SDS-PAGE and probed with rabbit anti-γ-chain Ab overnight, followed by anti-rabbit Ab conjugated to HRP. The immunoblot shown is representative of six experiments using MDM from different donors. B, The intracellular staining of γ-chain of FcγRs on MDM on day 7 postinfection was assessed by flow cytometry using anti-rabbit γ-chain Ab (solid lines) or isotype-matched control (dotted lines), followed by a secondary anti-rabbit Ab conjugated to FITC. Results are representative of four experiments using MDM prepared from different donors. C, Net mean fluorescence values are shown for Fc γ-chain expression that has been converted to molecules of equivalent soluble fluorochrome units using QuickCal program and corrected for background fluorescence. Results are representative of four experiments using uninfected (open bars) or HIV-infected (filled bars) MDM.

Analysis of cell lysates from three donors showed no detectable change in γ-chain mRNA levels in MDM infected with HIV-1 when compared with uninfected controls (Fig. 6⇓), showing that HIV-1 does not down-modulate γ-chain at the mRNA level. Input of cDNA was standardized according to GADPH levels as assessed by real-time PCR (data not shown) and confirmed by conventional PCR using β-actin-specific primers (Fig. 6⇓).

FIGURE 6.
FIGURE 6.

HIV-1 does not modulate γ-chain mRNA expression. MDM were infected with HIV-1 on day 5 postisolation, and mRNA extractions were performed 7 days after HIV-1 infection. γ-Chain mRNA was reverse transcribed to cDNA by PCR using γ-chain-specific primers as described in Materials and Methods. Levels of cDNA were standardized according to GADPH by real-time PCR and were confirmed by β-actin levels. Neat, undiluted cDNA sample, which corresponds to 1 × 104 MDM. PCR on samples prepared without RT were negative.

In vitro infection of MDM by either wild-type [NL(AD8)] or nef-deleted [NL(AD8)Δnef] HIV-1 inhibited FcγR-mediated phagocytosis (p = 0.003 and 0.01, respectively; n = 5). In marked contrast, infection of MDM with a nef-deleted HIV-1 with the gene for the γ-chain of FcγR [NL(AD8)Δnef-γ+] inserted in the nef site augmented FcγR-mediated phagocytosis (p = 0.045, n = 5) and partially restored the phagocytic capacity of HIV-infected MDM (mean restoration of 35.2 ± 15.8%; Fig. 7⇓A). The observed increase in phagocytic capacity of MDM infected with [NL(AD8)Δnef-γ+] virus was associated with increased protein levels of the γ-chain as assessed by immunoblotting (Fig. 7⇓B). Replication of both of these nef-deleted strains of HIV-1 was equivalent to that of wild-type virus, [NL(AD8)Δnef] (p = 0.48, n = 5) or [NL(AD8)Δnef-γ+] (p = 0.89; n = 5; Fig. 7⇓C).

FIGURE 7.
FIGURE 7.

Expression of γ-chain during HIV-1 infection augments FcγR-mediated phagocytosis. MDM on day 5 postisolation were mock-infected (open bars), infected with WT [NL(AD8)] and nef-deleted [NL(AD8)Δnef] (filled bars), or infected with [NL(AD8)Δnef γ+] (hatched bars). A, Phagocytosis was performed on day 7 postinfection and was assessed via a colorimetric assay as described in Materials and Methods. These data represent means ± SEM of experiments using cells from five different donors. B, MDM (1 × 106) on day 7 postinfection were washed twice in PBS-CMF and lysed in Triton X-100 buffer, and samples of lysate containing equal amount of protein (20 μg) were resolved by SDS-PAGE and probed with rabbit anti-γ-chain Ab overnight, followed by anti-rabbit Ab conjugated to HRP. The immunoblot shown is representative of two experiments using MDM from different donors. The levels of γ-chain have been analyzed by laser densitometry. Results are expressed as net densitometry units (DU) corrected for background levels. C, RT activity was quantified in culture supernatant on day 7 postinfection. Results represent means ± SEM of experiments using cells from five different donors.

Discussion

This study shows that HIV-1 infection of MDM in vitro leads to a significant impairment of FcγR-mediated phagocytosis, resulting from inhibition of tyrosine phosphorylation of cellular proteins stimulated during phagocytosis. We have specifically demonstrated inhibition of activation of Hck, Syk, and paxillin. Upstream of Hck and Syk, we found decreased protein expression of the γ signaling subunit of FcγR in HIV-infected MDM, suggesting that HIV-1 inhibits phagocytosis via interference with γ-chain-specific signaling events within human MDM. Thus, our finding of reduced protein levels of the ITAM-containing γ signaling subunit in HIV-infected MDM is likely to be responsible for the defective phagocytosis by these cells. To our knowledge, this study is the first to report inhibition of tyrosine phosphorylation and signaling events underlying defective effector function in HIV-infected macrophages.

We and others (5, 43, 44, 45) have previously reported impaired FcγR-mediated phagocytosis by human monocytes and macrophages following HIV-1 infection, although the mechanism of inhibition was unknown. The inhibition of FcγR-mediated phagocytosis was not strain-specific, as consistent impairment of this function was observed when MDM were infected in vitro with either HIV-1Ba-L (Fig. 1⇑), HIV-1NL4–3(AD8) (Fig. 7⇑A), HIV-1DV (3), or a primary isolate amplified from a member of the Sydney Blood Bank Cohort (5). The majority of previous reports (44, 45, 46, 47), confirmed by our own observations, showed that HIV-1 infection did not change surface expression of FcγRs, indicating that the inhibition of phagocytosis results from a signaling defect downstream of FcγRs. Thomas et al. (20) showed that inhibition of FcγR-mediated phagocytosis is mediated via a cAMP-dependent mechanism in the promonocytic U937 cell line latently infected with HIV-1. This was established by showing that pretreatment of these cells with 8′bromo-cAMP for 48 h before phagocytosis significantly decreased their functional capacity. However, in primary human MDM, our results showed that FcγR-mediated phagocytosis is not inhibited by the presence of 8′bromo-cAMP. Similarly, dibutryl cAMP inhibited complement-mediated phagocytosis but not FcγR-mediated phagocytosis in human MDM (48), suggesting that elevated cAMP levels resulting from HIV-1 infection (20, 49) are not responsible for impaired FcγR-mediated phagocytosis. However, it is possible that cAMP inhibits FcγR-mediated phagocytosis in the promonocytic U937 cell line via affecting the differentiation of U937 cells, rather than inhibiting phagocytosis per se.

Our data showing inhibition of tyrosine phosphorylation of any cellular proteins following stimulation with IgG-opsonized targets in HIV-infected MDM suggested dysfunction at an early stage in FcγR-mediated signaling. Further results confirmed this hypothesis and showed impaired phosphorylation of the tyrosine kinases Hck and Syk during phagocytosis by HIV-infected MDM. Specific requirements for both Hck (associated with γ-chain of FcγRs) and Syk in FcγR-mediated phagocytosis have been demonstrated in numerous studies using knockout mice, monocytic cell lines, and human MDM (32, 35, 50, 51, 52). Although defective activation of either Syk or Hck in HIV-infected macrophages has not been previously reported, structural and functional defects of Lck and Fyn (members of Src kinases) as well as ZAP-70 (analog of Syk) have been observed in T cells from HIV-infected individuals (53). The interaction of Hck with HIV-1-encoded proteins such as Nef and Vif is known to modulate its kinase activity (16, 54, 55), thereby potentially interfering with signaling events involved in FcγR-mediated phagocytosis.

Syk activation is absolutely essential for phagocytosis, as it couples phagocytosis-promoting FcγRs to rearrangements in the actin-based cytoskeleton (32). Therefore, impaired Syk phosphorylation triggered by FcγR engagement would inhibit Syk-mediated activation of substrates required for actin polymerization and cytoskeletal rearrangement, thereby inhibiting phagocytosis (56). Paxillin, a potential downstream effector of Syk, has been shown to interact with proteins involved in actin reorganization, including vinculin, talin, and α-actinin (57). Consistent with our data showing inhibition of Syk phosphorylation, HIV-infected MDM displayed reduced phosphorylation of paxillin during phagocytosis compared with uninfected MDM.

Upstream of Hck, Syk, and paxillin, we found reduced levels of ITAM-containing γ signaling subunit of FcγR, not associated with an effect on surface expression of either FcγRI, FcγRII, or FcγRIIIA. The γ-chain of FcγR is not a prerequisite for transient expression of surface FcγRs, but it is important for their stable expression (58, 59). However, the cytoplasmic γ-chain of FcγRs is critical for Syk activation and subsequent signaling events resulting in FcγR-mediated phagocytosis (24, 30). Deletion of the intracellular γ-chain of FcγR markedly impairs phagocytosis, despite unchanged surface receptor level (60). Thus, our finding of reduced protein levels of the ITAM-containing γ signaling subunit in HIV-infected MDM is likely to be responsible for the defective phagocytosis by these cells. The HIV-induced down-regulation of γ-chain expression was specific for this subunit, as levels of other signaling molecules downstream FcγRs viz Syk and paxillin were not affected. As γ-chain mRNA levels were unaltered, we concluded that HIV-1 inhibited γ-chain expression at a post-transcriptional point. This contrasts with T cells where HIV-1 infection reduces the levels of mRNA for the homologous CD3-ζ chain (61) and CD3-γ chain (62). To determine the exact site of HIV-induced down-modulation of the γ-chain protein, but not mRNA levels, additional experiments investigating levels of γ-chain protein synthesis, transport, or degradation need to be performed. This work is currently ongoing in our laboratory.

Our data confirmed that a functional nef gene was not essential for inhibition of FcγR-mediated phagocytosis, as previously reported by our group (5). Infection of MDM with a nef-deleted HIV-1 expressing the γ-chain of FcγR augmented FcγR-mediated phagocytosis, supporting our claim that reduced expression of the γ signaling subunit of FcγR may be responsible for impaired FcγR-mediated phagocytosis in HIV-infected MDM. The increased phagocytic capacity in MDM infected with the HIV-1 construct expressing the γ-chain was consistent with increased protein levels of the γ-chain. As it was impossible to control the level of γ-chain protein input in our system, it was not possible to rigorously correlate phagocytic efficiency of macrophages and γ-chain levels. If HIV-1 infection impairs FcγR-mediated phagocytosis at more than one level along the signaling pathway, any HIV-induced phagocytic defect would not be completely restored by overexpression of γ-chain protein.

Taken together, these data suggest that the mechanism of inhibition of FcγR-mediated phagocytosis in HIV-infected MDM occurs upstream of Hck, Syk, and paxillin activation and is associated with decreased protein expression of the γ-chain signaling subunit of FcγR. It provides the first possible mechanism of defective cellular activation in HIV-infected macrophages not only during phagocytosis, but potentially also underlying other functions mediated via FcγR. Impaired FcγR-mediated signaling may explain why HIV-infected macrophages fail to control opportunistic pathogens such as T. gondii (63) and provides potential therapeutic targets for immunomodulatory therapies for the treatment of AIDS via restoration of host defense.

Acknowledgments

We thank Prof. John Mills for his critical review of the manuscript and Geza Paukovics and Ajantha Salomon for their assistance with flow cytometric analysis and real-time PCR, respectively. We also thank Drs. Miranda Shehu-Xhilaga, Melissa Hill, and Secondo Sonza for their constructive discussions, as well as Dr. Ian Cooke and Jane Hocking for their help with statistical analyses.

Footnotes

  • ↵1 This work was supported in part by funding from the Australian National Council on HIV, AIDS, and Related Diseases to the National Center for HIV Virology Research, and by the Macfarlane Burnet Center Research Fund. K.K. was a recipient of a National Health and Medical Research Council Postgraduate Scholarship. S.R.L. was supported by the National Health and Medical Research Council and the Ian Potter Foundation.

  • ↵2 S.M.C. and A.J. contributed equally to this paper.

  • ↵3 Address correspondence and reprint requests to Dr. Anthony Jaworowski, AIDS Pathogenesis Research Unit, Macfarlane Burnet Center, Yarra Bend Road, Fairfield, Victoria, Australia 3078. E-mail address: anthonyj{at}burnet.edu.au

  • ↵4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; FAK, focal adhesion kinase; MDM, monocyte-derived macrophage; MOI, multiplicity of infection; RT, reverse transcriptase.

  • Received October 29, 2001.
  • Accepted January 8, 2002.

References

  1. ↵
    Tas, M., H. A. Drexhage, J. Goudsmit. 1988. A monocyte chemotaxis inhibiting factor in serum of HIV-infected men shares epitopes with HIV transmembrane protein gp41. Clin. Exp. Immunol. 71: 13
    OpenUrlPubMed
  2. ↵
    Wahl, S. M., J. B. Allen, S. Gartner, J. M. Orenstein, M. Popovic, D. E. Chenoweth, L. O. Arthur, W. L. Farrar, L. M. Wahl. 1989. HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes. J. Immunol. 142: 3553
    OpenUrlAbstract
  3. ↵
    Biggs, B. A., M. Hewish, S. Kent, K. Hayes, S. M. Crowe. 1995. HIV-1 infection of human macrophages impairs phagocytosis and killing of Toxoplasma gondii. J. Immunol. 154: 6132
    OpenUrlAbstract
  4. ↵
    Kedzierska, K., J. Mak, A. Mijch, I. Cooke, M. Rainbird, S. Roberts, G. Paukovics, D. Jolley, A. Lopez, S. M. Crowe. 2000. Granulocyte-macrophage colony-stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type 1-infected monocytes/macrophages in vitro and in vivo. J. Infect. Dis. 181: 390
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Kedzierska, K., J. Mak, A. Jaworowski, A. Greenway, A. C. Violo, H. J. Hocking, D. Purcell, S. Sullivan, J. Mills, S. M. Crowe. 2001. nef-deleted HIV-1 inhibits phagocytosis by monocyte-derived macrophages in vitro but not by peripheral blood monocytes in vivo. AIDS 15: 945
    OpenUrlCrossRefPubMed
  6. ↵
    Kedzierska, K., S. M. Crowe. 2001. Cytokines and HIV-1: interactions and clinical implications. Antiviral Chem. Chemother. 12: 133
    OpenUrlPubMed
  7. ↵
    Crowe, S. M.. 1995. Role of macrophages in the pathogenesis of human immunodeficiency virus (HIV) infection. Aust. N. Z. J. Med. 25: 777
    OpenUrlCrossRefPubMed
  8. ↵
    Macreadie, I., L. Castelli, D. Hewish, A. Kirkpatrick, A. Ward, A. Azad. 1995. A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. Proc. Natl. Acad. Sci. USA 92: 2770
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Karczewski, M., K. Strebel. 1996. Cytoskeleton association and virion incorporation of the human immunodeficiency virus type 1 Vif protein. J. Virol. 70: 494
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Fackler, O., N. Kienzle, E. Kremmer, A. Boese, B. Schramm, T. Klimkait, C. Kucherer, N. Mueller-Lantzsch. 1997. Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization. Eur. J. Biochem. 247: 843
    OpenUrlPubMed
  11. ↵
    Watts, N., D. Sackett, R. Ward, M. Miller, P. Wingfield, S. Stahl, A. Steven. 2000. HIV-1 rev depolymerizes microtubules to form stable bilayered rings. J. Cell Biol. 150: 349
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Saksela, K., G. Cheng, D. Baltimore. 1995. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 14: 484
    OpenUrlPubMed
  13. ↵
    Greenway, A., A. Azad, J. Mills, D. A. McPhee. 1996. Human immunodeficiency virus type 1 nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity. J. Virol. 70: 6701
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Nunn, M. F., J. W. Marsh. 1996. Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family. J. Virol. 70: 6157
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Fackler, O. T., X. Lu, J. A. Frost, M. Geyer, B. Jiang, W. Luo, A. Abo, A. S. Alberts, B. M. Peterlin. 2000. p21-activated kinase 1 plays a critical role in cellular activation by Nef. Mol. Cell. Biol. 20: 2619
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Hassaine, G., M. Courcoul, G. Bessou, Y. Barthalay, C. Picard, D. Olive, Y. Collette, R. Vigne, E. Decroly. 2001. The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral vif protein. J. Biol. Chem. 276: 16885
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Lee, T., F. Sunzeri, L. Tobler, B. Williams, M. Busch. 1991. Quantitative assessment of HIV-1 DNA load by coamplification of HIV-1 gag and HLA-DQ-α genes. AIDS 5: 683
    OpenUrlCrossRefPubMed
  18. ↵
    Lee, C. H., K. Saksela, U. A. Mirza, B. T. Chait, J. Kuriyan. 1996. Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell 85: 931
    OpenUrlCrossRefPubMed
  19. ↵
    Fackler, O. T., W. Luo, M. Geyer, A. S. Alberts, B. M. Peterlin. 1999. Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions. Mol. Cell 3: 729
    OpenUrlCrossRefPubMed
  20. ↵
    Thomas, C. A., O. K. Weinberger, B. L. Ziegler, S. Greenberg, I. Schieren, S. C. Silverstein, J. El Khoury. 1997. Human immunodeficiency virus-1 env impairs Fc receptor-mediated phagocytosis via a cyclic adenosine monophosphate-dependent mechanism. Blood 90: 3760
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Daeron, M.. 1997. Fc receptor biology. Annu. Rev. Immunol. 15: 203
    OpenUrlCrossRefPubMed
  22. ↵
    Greenberg, S., P. Chang, S. C. Silverstein. 1993. Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 177: 529
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Greenberg, S., P. Chang, S. C. Silverstein. 1994. Tyrosine phosphorylation of the γ subunit of Fcγ receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 269: 3897
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Indik, Z. K., J. G. Park, S. Hunter, A. D. Schreiber. 1995. The molecular dissection of Fcγ receptor-mediated phagocytosis. Blood 86: 4389
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Park, J. G., A. D. Schreiber. 1995. Determinants of the phagocytic signal mediated by the type IIIA Fcγ receptor, Fcγ RIIIA: sequence requirements and interaction with protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 92: 7381
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Allen, L. A., A. Aderem. 1996. Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J. Exp. Med. 184: 627
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Greenberg, S., P. Chang, D. C. Wang, R. Xavier, B. Seed. 1996. Clustered syk tyrosine kinase domains trigger phagocytosis. Proc. Natl. Acad. Sci. USA 93: 1103
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Jouvin, M. H., M. Adamczewski, R. Numerof, O. Letourneur, A. Valle, J. P. Kinet. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. 269: 5918
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Fitzer-Attas, C. J., M. Lowry, M. T. Crowley, A. J. Finn, F. Meng, A. L. DeFranco, C. A. Lowell. 2000. Fcγ receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn. J. Exp. Med. 191: 669
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Park, J. G., R. K. Murray, P. Chien, C. Darby, A. D. Schreiber. 1993. Conserved cytoplasmic tyrosine residues of the γ subunit are required for a phagocytic signal mediated by Fcγ RIIIA. J. Clin. Invest. 92: 2073
    OpenUrlCrossRefPubMed
  31. ↵
    Indik, Z. K., J. G. Park, X. Q. Pan, A. D. Schreiber. 1995. Induction of phagocytosis by a protein tyrosine kinase. Blood 85: 1175
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Crowley, M. T., P. S. Costello, C. J. Fitzer-Attas, M. Turner, F. Meng, C. Lowell, V. L. Tybulewicz, A. L. DeFranco. 1997. A critical role for Syk in signal transduction and phagocytosis mediated by Fcγ receptors on macrophages. J. Exp. Med. 186: 1027
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Ninomiya, N., K. Hazeki, Y. Fukui, T. Seya, T. Okada, O. Hazeki, M. Ui. 1994. Involvement of phosphatidylinositol 3-kinase in Fcγ receptor signaling. J. Biol. Chem. 269: 22732
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Greenberg, S., K. Burridge, S. C. Silverstein. 1990. Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 172: 1853
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Kedzierska, K., N. J. Vardaxis, A. Jaworowski, S. M. Crowe. 2001. FcγR-mediated phagocytosis by human macrophages involves Hck, Syk, and Pyk2 and is augmented by GM-CSF. J. Leukocyte Biol. 70: 322
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Crowe, S. M., J. Mills, M. S. McGrath. 1987. Quantitative immunocytofluorographic analysis of CD4 surface antigen expression and HIV infection of human peripheral blood monocyte/macrophages. AIDS Res. Hum. Retroviruses 3: 135
    OpenUrlCrossRefPubMed
  37. ↵
    Kedzierska, K., S. M. Crowe. 2001. Culture of HIV in monocytes and macrophages. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and V. Strober, eds. In Current Protocols in Immunology Vol. II: 12.4.1 Wiley, New York.
    OpenUrl
  38. ↵
    Kedzierska, K., A. Maerz, T. Warby, A. Jaworowski, H. T. Chan, J. Mak, S. Sonza, A. Lopez, S. M. Crowe. 2000. Granulocyte-macrophage colony stimulating factor inhibits HIV-1 replication in monocyte-derived macrophages. AIDS 14: 1739
    OpenUrlCrossRefPubMed
  39. ↵
    Gebran, S. J., E. L. Romano, H. A. Pons, L. Cariani, A. N. Soyano. 1992. A modified colorimetric method for the measurement of phagocytosis and antibody-dependent cell cytotoxicity using 2,7-diaminofluorene. J. Immunol. Methods 151: 255
    OpenUrlCrossRefPubMed
  40. ↵
    Lewin, S., M. Vesanen, L. Kostrikis, A. Hurley, M. Duran, L. Zhang, D. Ho, M. Markowitz. 1999. Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J. Virol. 73: 6099
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Grassi, G., G. Pozzato, M. Moretti, M. Giacca. 1995. Quantitative analysis of hepatitis C virus RNA in liver biopsies by competitive reverse transcription and polymerase chain reaction. J. Hepatol. 23: 403
    OpenUrlCrossRefPubMed
  42. ↵
    Shehu-Xhilaga, M., S. Crowe, J. Mak. 2001. Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J. Virol. 75: 1834
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Bender, B., B. Davidson, R. Kline, C. Brown, T. Quinn. 1988. Role of the mononuclear phagocyte system in the immunopathogenesis of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. Rev. Infect. Dis. 10: 1142
    OpenUrlCrossRefPubMed
  44. ↵
    Capsoni, F., F. Minonzio, A. M. Ongari, G. P. Rizzardi, A. Lazzarin, C. Zanussi. 1992. Monocyte-derived macrophage function in HIV-infected subjects: in vitro modulation by rIFN-γ and rGM-CSF. Clin. Immunol. Immunopathol. 62: 176
    OpenUrlCrossRefPubMed
  45. ↵
    Capsoni, F., F. Minonzio, A. M. Ongari, P. Bonara, G. Pinto, V. Carbonelli, A. Lazzarin, C. Zanussi. 1994. Fc receptors expression and function in mononuclear phagocytes from AIDS patients: modulation by IFN-γ. Scand. J. Immunol. 39: 45
    OpenUrlCrossRefPubMed
  46. ↵
    Petit, A. J., M. Tersmette, F. G. Terpstra, R. E. de Goede, R. A. van Lier, F. Miedema. 1988. Decreased accessory cell function by human monocytic cells after infection with HIV. J. Immunol. 140: 1485
    OpenUrlAbstract
  47. ↵
    Dunne, J., C. Feighery, A. Whelan. 1996. β2-Microglobulin, neopterin and monocyte Fcγ receptors in opportunistic infections of HIV-positive patients. Br. J. Biomed. Sci. 53: 263
    OpenUrlPubMed
  48. ↵
    Newman, S. L., L. K. Mikus, M. A. Tucci. 1991. Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis. J. Immunol. 146: 967
    OpenUrlAbstract
  49. ↵
    Nokta, M., R. Pollard. 1991. Human immunodeficiency virus infection: association with altered intracellular levels of cAMP and cGMP in MT-4 cells. Virology 181: 211
    OpenUrlCrossRefPubMed
  50. ↵
    Lowell, C. A., P. Soriano, H. E. Varmus. 1994. Functional overlap in the src gene family: inactivation of hck and fgr impairs natural immunity. Genes Dev. 8: 387
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Ghazizadeh, S., J. Bolen, H. Fleit. 1994. Physical and functional association of Src-related protein tyrosine kinases with Fcγ RII in monocytic THP-1 cells. J. Biol. Chem. 269: 8878
    OpenUrlAbstract/FREE Full Text
  52. ↵
    Ghazizadeh, S., J. Bolen, H. Fleit. 1995. Tyrosine phosphorylation and association of Syk with Fcγ RII in monocytic THP-1 cells. Biochem. J. 305: 669
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Stefanova, I., M. Saville, C. Peters, F. Cleghorn, D. Schwartz, D. Venzon, K. Weinhold, N. Jack, C. Bartholomew, W. Blattner, et al 1996. HIV infection-induced post-translational modification of T cell signaling molecules associated with disease progression. J. Clin. Invest. 98: 1290
    OpenUrlCrossRefPubMed
  54. ↵
    Briggs, S., M. Sharkey, M. Stevenson, T. Smithgall. 1997. SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1. J. Biol. Chem. 272: 17899
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Greenway, A., H. Dutartre, K. Allen, D. McPhee, D. Olive, Y. Collette. 1999. Simian immunodeficiency virus and human immunodeficiency virus type 1 nef proteins show distinct patterns and mechanisms of Src kinase activation. J. Virol. 73: 6152
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Cox, D., P. Chang, T. Kurosaki, S. Greenberg. 1996. Syk tyrosine kinase is required for immunoreceptor tyrosine activation motif-dependent actin assembly. J. Biol. Chem. 271: 16597
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Turner, C. E.. 1998. Paxillin. Int. J. Biochem. Cell Biol. 30: 955
    OpenUrlCrossRefPubMed
  58. ↵
    Takai, T., M. Li, D. Sylvestre, R. Clynes, J. Ravetch. 1994. FcRγ chain deletion results in pleiotrophic effector cell defects. Cell 76: 519
    OpenUrlCrossRefPubMed
  59. ↵
    van Vugt, M., A. Heijnen, P. Capel, S. Park, C. Ra, T. Saito, J. Verbeek, J. van de Winkel. 1996. FcR γ-chain is essential for both surface expression and function of human FcγRI (CD64) in vivo. Blood 87: 3593
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Edberg, J., A. Yee, D. Rakshit, D. Chang, J. Gokhale, Z. Indik, A. Schreiber, R. Kimberly. 1999. The cytoplasmic domain of human FcγRIa alters the functional properties of the FcγR·γ-chain receptor complex. J. Biol. Chem. 274: 30328
    OpenUrlAbstract/FREE Full Text
  61. ↵
    Geertsma, M., A. van Wengen-Stevenhagen, E. van Dam, K. Risberg, F. Kroon, P. Groeneveld, P. Nibbering. 1999. Decreased expression of ζ molecules by T lymphocytes is correlated with disease progression in human immunodeficiency virus-infected persons. J. Infect. Dis. 180: 649
    OpenUrlAbstract/FREE Full Text
  62. ↵
    Segura, I., C. Delmelle-Wibaut, M. Janssens, Y. Cleuter, A. van den Broeke, R. Kettmann, K. Willard-Gallo. 1999. Human immunodeficiency virus type 2 produces a defect in CD3-γ gene transcripts similar to that observed for human immunodeficiency virus type 1. J. Virol. 73: 5207
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Joiner, K. A., S. A. Fuhrman, H. M. Miettinen, L. H. Kasper, I. Mellman. 1990. Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science 249: 641
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section