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The Journal of Immunology, 2007, 178: 1096-1104.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Mechanism Underlying Defective Fc{gamma} Receptor-Mediated Phagocytosis by HIV-1-Infected Human Monocyte-Derived Macrophages1

Edwin Leeansyah*,{ddagger}, Bruce D. Wines{dagger}, Suzanne M. Crowe*,{ddagger} and Anthony Jaworowski2,*,{ddagger}

* AIDS Pathogenesis and Clinical Research Program, The Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia; {dagger} Helen Macpherson Smith Inflammatory Diseases Laboratory, The Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia; and {ddagger} Department of Medicine, Monash University, Melbourne, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Clearance of IgG-opsonized erythrocytes is impaired in HIV-1-infected patients, suggesting defective Fc{gamma}R-mediated phagocytosis in vivo. We have previously shown defective Fc{gamma}R-mediated phagocytosis in HIV-1-infected human monocyte-derived macrophages (MDM), establishing an in vitro model for defective tissue macrophages. Inhibition was associated with decreased protein expression of FcR {gamma}-chain, which transduces immune receptor signals via ITAM motifs. Fc{gamma}RI and Fc{gamma}RIIIa signal via {gamma}-chain, whereas Fc{gamma}RIIa does not. In this study, we showed that HIV-1 infection inhibited Fc{gamma}RI-, but not Fc{gamma}RIIa-dependent Syk activation in MDM, showing that inhibition was specific for {gamma}-chain-dependent signaling. HIV-1 infection did not impair {gamma}-chain mRNA levels measured by real-time PCR, suggesting a posttranscriptional mechanism of {gamma}-chain depletion. HIV-1 infection did not affect {gamma}-chain degradation (n = 7, p = 0.94) measured in metabolic labeling/chase experiments, whereas {gamma}-chain biosynthesis was inhibited (n = 12, p = 0.0068). Using an enhanced GFP-expressing HIV-1 strain, we showed that Fc{gamma}R-mediated phagocytosis inhibition is predominantly due to a bystander effect. Experiments in which MDM were infected in the presence of the antiretroviral drug 3TC suggest that active viral replication is required for inhibition of phagocytosis in MDM. These data suggest that HIV-1 infection may affect only {gamma}-chain-dependent Fc{gamma}R functions, but that this is not restricted to HIV-1-infected cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The primary cellular targets of HIV-1 are CD4-expressing cells (1), including cells of the macrophage lineage (2). Following HIV-1 infection, many functions of macrophages, such as chemotaxis, phagocytosis, intracellular killing, and cytokine production, are disrupted (3, 4, 5, 6). Numerous studies, both ex vivo and in vitro, have shown defective Fc{gamma}R-mediated phagocytosis by human macrophages following HIV-1 infection (4, 7, 8, 9, 10), an important immune mechanism to eliminate Ab-coated pathogens and cells. In early studies in vivo, clearance of IgG-opsonized 51Cr-labeled autologous erythrocytes was defective in AIDS patients relative to HIV-1-seronegative individuals (11), indicating defective Fc{gamma}R-mediated phagocytosis by tissue macrophages in vivo associated with HIV-1 infection. Disruption of Fc{gamma}R-mediated phagocytosis may contribute to the increased susceptibility to opportunistic infections in HIV-1-infected individuals caused by microorganisms normally controlled by macrophages.

Human macrophages express three distinct activating receptors for the Fc region of IgG (Fc{gamma}Rs): Fc{gamma}RI, Fc{gamma}RIIa, and Fc{gamma}RIIIa (12, 13, 14, 15 ; for a review see Ref. 16). Clustering of these receptors by IgG-opsonized particles induces tyrosine phosphorylation by Src tyrosine kinases of ITAM motifs either within the intracytoplasmic domain of Fc{gamma}RIIa or FcR common {gamma}-chain (17, 18, 19), a small ITAM-containing signaling protein associated with multiple receptors, including Fc{gamma}RI and Fc{gamma}RIIIa (20, 21). Phosphorylated ITAMs serve as docking sites for Syk kinase, a member of the ZAP70 family of protein tyrosine kinases, which leads to phosphorylation and activation of Syk by both phosphorylation by Src kinases (22) and subsequent autophosphorylation of the Syk activation loop (23, 24). Syk activation is absolutely required for Fc{gamma}R-mediated phagocytosis (25, 26, 27, 28), and therefore may be used as a biochemical readout for early Fc{gamma}R phagocytic signal transduction (10, 29). Activation of Syk directly or indirectly modulates a number of downstream signaling mediators, including PI3K (30); members of the Rho family of GTPases (31); Pyk-2 (29); and actin-binding proteins, including paxillin, vinculin, talin, and actinin (32, 33, 34).

We have shown in our previous study that defective Fc{gamma}R-mediated phagocytosis by HIV-1-infected human monocyte-derived macrophages (MDM)3 is associated with down-regulation of {gamma}-chain protein expression even though expression of the Fc{gamma}R is unaffected (10). Fc{gamma}RIIa signaling does not depend on ITAM-containing accessory molecules (35) due to the presence of an ITAM sequence in its intracytoplasmic domain. We therefore hypothesized that HIV-1 infection would specifically inhibit signaling via {gamma}-chain-associated Fc{gamma}Rs, such as Fc{gamma}RI, without affecting {gamma}-chain-independent signaling of Fc{gamma}RIIa. In this study, we addressed whether {gamma}-chain-dependent signaling is specifically inhibited in HIV-1-infected MDM by measuring Syk activation following cross-linking to either Fc{gamma}RI or Fc{gamma}RIIa. We also determined the mechanism of defective {gamma}-chain expression in HIV-1-infected MDM by measuring {gamma}-chain mRNA levels, protein synthesis, and protein turnover. Finally, by using an enhanced GFP (EGFP)-expressing macrophage-tropic HIV-1 strain, we investigated whether inhibition of Fc{gamma}R-mediated phagocytosis by HIV-1-infected MDM occurs only in productively infected macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines, hybridoma culture, and mAb preparation

Murine IIA1.6 cells transfected with human {gamma}-chain (IIA1.6{gamma}+) and Fc{alpha}RI (CD89) (36) were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine (both from Invitrogen Life Technologies), 10% heat-inactivated FBS (HyClone), 50 µM 2-ME, and 24 µg/ml gentamicin (supplemented RPMI 1640 medium) with 10 µg/ml puromycin (Sigma-Aldrich). Human 293T cell line and murine IV.3 hybridoma cells expressing mAb specific for human Fc{gamma}RIIa were maintained in supplemented RPMI 1640 medium. Hybridoma culture medium was collected when the hybridoma reached confluence and mAb was purified using protein G-Sepharose (Amersham Biosciences) affinity chromatography. All mAb preparations had undetectable levels of LPS contamination (<1 endotoxin unit (100 pg/ml)) using the Limulus lysate assay (BioWhittaker).

Isolation and culture of human monocytes and monocyte-derived macrophages

Human monocytes were isolated from buffy coats of HIV-, hepatitis B virus-, hepatitis C virus-, human T cell leukemia virus-, and syphilis-seronegative donors (obtained from the Australian Red Cross Blood Services) by density-gradient centrifugation (Ficoll-Paque Plus; Amersham Biosciences), followed by countercurrent elutriation (Beckman Coulter J-6M/E centrifuge JE-5.0 rotor) at 2200 rpm, 12°C. Cell viability was >95% using trypan blue dye exclusion, and the typical purity of monocytes was >90%, as determined by immunofluorescent staining with anti-CD14 mAb (BD Pharmingen) and forward and side scatter analysis (FACSCalibur; BD Biosciences). Monocytes were resuspended at a concentration of 106 cells/ml in IMDM supplemented with 2 mM L-glutamine (both supplied by Invitrogen Life Technologies), 10% heat-inactivated pooled human AB+ serum (Australian Red Cross Blood Services), and 24 µg/ml gentamicin (supplemented Iscove’s medium), and differentiated to MDM by adherent culture on plastic surfaces using 6-, 24-, or 96-well Nunclon {Delta}-surface plates (Nalge Nunc International) depending on experimental requirements.

Generation and purification of EGFP-expressing HIV-1

The HIV-1 plasmid pNL4-3 Ba-L env {Delta}nef EGFP was isolated using an endotoxin-free Maxi kit according to the manufacturer’s instructions (Qiagen). The M-tropic EGFP-expressing HIV-1 (HIV-1NL(Ba-L){Delta}nef-EGFP) was generated by introducing 12 µg of pNL4-3 Ba-L env {Delta}nef EGFP into 293T cells by polyethylenenimine transfection. Culture medium was collected 36 h posttransfection, and viral particles were concentrated by ultracentrifugation through a 20% endotoxin-free sucrose cushion (Beckman Coulter ultracentrifuge model L-90; SW 41 rotor) at 150,000 x g for 1 h at 4°C (37). Viral particles were then incubated with supplemented Iscove’s medium at 4°C for 1 h, followed by gentle resuspension before snap-freezing with liquid nitrogen. Virus stocks were stored at –80°C.

HIV-1 infection of MDM

Five days after isolation, MDM were infected with a laboratory-adapted macrophage-tropic strain, HIV-1Ba-L (AIDS Research and Reference Program, Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health) at a multiplicity of infection of 0.1–1 for 2–4 h, as previously described (10). Control cells were mock infected and cultured under identical conditions. HIV-1 replication in MDM was quantified by determining reverse transcriptase (RT) activity in culture supernatants using a micro-RT assay (38) at 7 days postinfection. In selected experiments, MDM were infected in the presence and absence of 100 µM lamivudine (3TC; GlaxoSmithKline) to inhibit HIV-1 replication. Where 3TC was added during infection, the drug was maintained in the medium throughout subsequent culturing. The 3TC concentration used was not toxic to the cells for the duration of the experiments, as previously reported (39). In selected experiments, adherent MDM cultured in 24-well plates were infected with HIV-1NL(Ba-L){Delta}nef-EGFP at indicated multiplicity of infection. Viral replication within MDM was quantified both by micro RT assay and by the proportion of EGFP-expressing MDM determined by flow cytometry.

Phagocytosis assay using IgG-opsonized SRBC

SRBC were opsonized with a subagglutinating concentration (1:714) of rabbit anti-SRBC Ab (MP Biomedicals) (10). After 7 days of infection with HIV-1, IgG-opsonized and -unopsonized SRBC were added to adherent MDM (in 96-well plates at a seeding density of 3 x 104 cells/well) at a target:MDM ratio of 10:1. The plate was centrifuged at 100 x g for 5 min at 4°C and was then incubated at 37°C for 30 min to allow phagocytosis. Phagocytosis was terminated by washing cells in cold (4°C) PBS, and then uningested SRBC were subsequently lysed using hypotonic (0.2%) NaCl for 4 min. The degree of phagocytosis was measured by a colorimetric assay, as previously described (40). To determine bystander effects of HIV-1 on Fc{gamma}R-mediated phagocytosis, MDM were cultured in 24-well plates and infected with HIV-1NL(Ba-L){Delta}nef-EGFP, and phagocytosis was performed after 7 days of infection, as described, except that MDM were fixed with 3% (v/v) formaldehyde after the lysis of uningested SRBC. Fluorescence images were visualized with a Zeiss Axiovert100 fluorescence microscope, and random fluorescence images were taken using a digital camera (CoolSnap fx; Photometrics). Fluorescence images of MDM from mock-infected and HIV-1-infected cultures were superimposed with bright-field images of the same visual field (V++ Precision Digital Imaging System; Digital Optics). Phagocytosis of SRBC was assessed microscopically by counting at least 100 MDM that had been cultured under each condition (mock-infected, green fluorescent, and nonfluorescent MDM in HIV-1-infected culture), as described (41). Background fluorescence was negligible in all experiments.

mRNA extraction and measurement of {gamma}-chain mRNA expression

MDM were mock infected or infected with HIV-1Ba-L for 7 days and lysed using RNA lysis/binding buffer (1% LiDS, 100 mM Tris-HCl (pH 7.5), 500 mM LiCl, 10 mM EDTA, 5 mM DTT). Cellular mRNA was isolated from lysates following binding to oligo(dT) magnetic beads (GenoPrep; GenoVision), and cDNA was prepared using SuperScript III RT (Invitrogen Life Technologies). Samples prepared without SuperScript III RT were included within each experiment to control for DNA contamination in the cDNA. {gamma}-Chain cDNA was amplified using real-time PCR in a total volume of 25 µl comprised of 0.8 µM each {gamma}-chain primer and iQ SYBR Green Supermix (Bio-Rad). The {gamma}-chain primer set amplified 172 bp of human {gamma}-chain cDNA sequence (GenBank accession no. NM004106) with the sequences 5'-GAGCCTCAGCTCTGCTATATCC-3' and 5'-TCTCGTAAGTCTCCTGGTTCC-3', as previously described (10). Samples were denatured at 95°C for 3 min, and were then amplified for 45 cycles of 94°C for 20 s, 57°C for 40 s, and 72°C for 40 s using a Bio-Rad iCycler (Bio-Rad). GAPDH cDNA was amplified by real-time PCR using a primer set that amplified 189 bp of human GAPDH cDNA sequence (GenBank accession no. NM002046) with the sequences 5'-TGGTATCGTGGAAGGACTCATGAC-3' and 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3' (42). The real-time PCR conditions were similar to those used for {gamma}-chain quantification, except that the hybridization temperature was 65°C. GAPDH comparative threshold (Ct) value was used to normalize {gamma}-chain Ct values in all experiments. No DNA contamination was observed in any of the SuperScript III RT-negative samples.

Cross-linking of MDM Fc{gamma}Rs

After 7 days postinfection, MDM on 6-well plates (106 cells/well) were incubated at 4°C with either 10 µg/ml mouse anti-CD64 (Fc{gamma}RI) mAb (clone 10.1; Santa Cruz Biotechnology) or 1/100 purified mouse anti-Fc{gamma}RIIa (clone IV.3) mAb preparation for 30 min. After removal of unbound mAbs, cells were incubated at 37°C with 10 µg/ml affinity-purified goat F(ab')2 against whole mouse IgG (MP Biomedicals). Cross-linking was terminated by washing cells with cold (4°C) Ca2+- and Mg2+-free PBS supplemented with 500 µM Na3VO4 and lysis with 200 µl of cold (4°C) Triton lysis buffer (TLB) (1% Triton X-100, 25 mM Tris, 140 mM NaCl, 1 mM EDTA) supplemented with phosphatase inhibitors (50 mM NaF, 1 mM Na3VO4, 40 mM beta-glycerolphosphate) and protease inhibitors (Complete; Roche). Lysates were incubated at 4°C for 10 min and clarified at 20,000 x g for 10 min at 4°C.

Metabolic labeling and chasing of {gamma}-chain and cellular proteins

Adherent MDM were cultured in 6-well plates at 106 cells/well and then labeled 7 days postinfection with 15 µCi/ml L-[U-14C]leucine (Amersham Biosciences) in leucine-free RPMI 1640 medium (MP Biomedicals). Labeling was terminated after 8 or 16 h by washing cells with cold Ca2+- and Mg2+-free PBS. MDM extracts were either immediately prepared using supplemented TLB to quantify {gamma}-chain protein synthesis or after incubation with supplemented RPMI 1640 medium containing excess nonradioactive L-leucine (500 µg/ml) for 3 h to measure {gamma}-chain stability. To quantify incorporation of label into total cellular proteins, MDM were cultured in 24-well plates (2.5 x 105 cells/well) and labeled with 5 µCi/ml L-[U-14C]leucine for 16 h. MDM extracts were prepared after labeling using 50 µl of 0.2 M NaOH or after incubation with supplemented RPMI 1640 containing excess L-leucine for 30 h before lysis.

Analysis of radioactive {gamma}-chain and total cellular proteins

Cellular extracts prepared using supplemented TLB, and which contained equal amounts of protein as determined by detergent-compatible Lowry protein assay (Bio-Rad), were immunoprecipitated using anti-{gamma}-chain antiserum (36), and then resolved using 12.5% SDS-PAGE. Gels were stained with Coomassie blue to confirm equal loading and dried. Radioactive {gamma}-chain was quantified by phosphor imaging (FujiLab) using ImageGauge software (FujiLab). To quantify incorporated L-[U-14C] leucine into total cellular proteins, MDM extracts were spotted onto filter paper (Whatman 3MM; Whatman) and allowed to air dry. Radioactivity incorporated into protein was determined following precipitation with cold 10% (w/v) TCA, followed by hydrolysis of amino acid-charged tRNA and extraction of lipids (43). Radioactivity was quantified by phosphor imaging.

Immunoprecipitation and immunoblotting

MDM extracts prepared using supplemented TLB, and which contained equal amounts of protein as determined by detergent-compatible protein assay, were immunoprecipitated with either 4 µg/ml mouse anti-Syk mAb (clone 4D10; Santa Cruz Biotechnology) or 1/200 rabbit anti-{gamma}-chain antiserum overnight at 4°C. Immune complexes were collected with 20 µl of protein G-Sepharose for 1 h at 4°C. Protein G-Sepharose-immune complexes were washed five times with cold supplemented TLB, boiled with sample buffer (10 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1% SDS, 5% 2-ME, 10% glycerol) for 10 min, resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with blocking buffer (Odyssey Blocking Buffer; LI-COR) for 1 h. Blots were probed with primary Abs directed against phosphotyrosine (RC20-HRP; Santa Cruz Biotechnology), anti-phospho-Syk (Y525/526)-specific Ab (Cell Signaling Technology), Syk (4D10), or {gamma}-chain, followed by appropriate secondary Abs (goat anti-rabbit Alexa Fluor 680 (Molecular Probes), goat anti-mouse IRDye 800 (Rockland), or goat anti-mouse HRP (DakoCytomation)). Bound Abs were visualized using either a LI-COR Odyssey infrared imager (LI-COR) or ECL (Amersham Biosciences).

Statistical analysis

Statistical analyses for pairwise comparisons were conducted using a nonparametric Sign test, adjusting for multiple comparisons where necessary using Bonferroni’s technique, and when >2 groups were compared the Friedman test was used. Friedman test was performed using SAS Institute version 9.0 software, and STATA version 9 was used for pairwise comparisons. A probability value of <0.05 was used to assess statistical significance in all cases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HIV-1 inhibition of Fc{gamma}R-mediated phagocytosis by MDM is due to decreased FcR {gamma}-chain expression

We have shown previously that HIV-1 infection of MDM inhibited Fc{gamma}R-mediated phagocytosis and was associated with down-regulation of FcR {gamma}-chain (10), an important signaling protein that transduces signals from multiple receptors, including Fc{gamma}RI and Fc{gamma}RIIIa (16). Fc{gamma}R-mediated phagocytosis of IgG-SRBC was inhibited by HIV-1 with magnitudes comparable to our previously reported studies (mean inhibition of 55.3 ± 13.4%, n = 6, p < 0.05; Fig. 1A) (9, 10). HIV-1 infection was verified in each case by measurement of RT activity in culture medium (average = 659 cpm/µl, SEM = 170 cpm/µl, range = 188–1150 cpm/µl). To better quantify {gamma}-chain protein expression in mock-infected vs HIV-1-infected MDM, we immunoprecipitated, in three of the above experiments, {gamma}-chain protein from MDM extracts and quantified the immunoprecipitated {gamma}-chain band by immunoblotting coupled with infrared fluorescence detection. With this technique, we confirmed our earlier data using semiquantitative ECL detection and showed that {gamma}-chain protein expression was decreased in HIV-1-infected MDM (average decrease = 51.7%, range = 37.8–64.2%, n = 3). A representative experiment is shown in Fig. 1B, in which HIV-1-infected MDM cultures expressed {gamma}-chain protein at a level 62% of a mock- infected control culture.


Figure 1
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  		FIGURE 1. The effect of HIV-1 infection on {gamma}-chain expression and Fc{gamma}R-mediated phagocytosis by human MDM. A, Fc{gamma}R-mediated phagocytosis by HIV-1-infected and mock-infected MDM cultures was compared for cells prepared from six independent donors (net phagocytic indices: mock infected = 172 ± 26; HIV-1 infected = 78 ± 26, n = 6, p < 0.05). B, Lysates were prepared from MDM cultured in parallel from the same donor PBMCs as analyzed in A. Samples containing equal amount of protein were analyzed for {gamma}-chain expression by immunoblotting following immunoprecipitation with rabbit anti-{gamma}-chain antiserum. Cell extracts prepared from a {gamma}-chain expressing B cell line, IIA1.6{gamma}+, were used as a positive control. Band intensities were quantified by infrared imaging and are indicated in the figure. The immunoblot shown in this figure is representative of blots from six donors used in Fc{gamma}R-mediated phagocytosis assay in A.

 
HIV-1 inhibition of Fc{gamma}R-mediated phagocytosis by MDM requires productive infection and is associated with a bystander effect of viral replication

To determine whether productive infection is essential to cause inhibition of Fc{gamma}R-mediated phagocytosis, MDM were mock infected or infected with HIV-1 in the presence or absence of 100 µM 3TC, an RT inhibitor that effectively inhibits HIV-1 replication. Our data indicate that phagocytosis of IgG-SRBC 7 days after HIV-1 infection was not inhibited when HIV-1 replication was inhibited by 3TC added at the time of infection and maintained in the culture medium (Fig. 2A). In contrast, relative phagocytosis of IgG-SRBC by HIV-1-infected MDM cultured in the absence of 3TC was inhibited by 41.3 ± 9.3% when compared with mock-infected control, consistent with our published data and the data presented in Fig. 1A. At this concentration, 3TC completely inhibited viral replication, as shown by lack of RT activity in the culture medium (Fig. 2A). Thus inhibition of viral replication in the culture correlated with loss of phagocytosis inhibition by HIV-1.


Figure 2
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  		FIGURE 2. Analysis of bystander effects of HIV-1 infection on Fc{gamma}R-mediated phagocytosis. A, MDM were mock infected or infected with HIV-1Ba-L in the presence or absence of 3TC to inhibit HIV-1 replication. Virus inoculum was removed by washing and cells cultured with maintenance of 3TC where it was added during the infection/mock infection. Phagocytosis of IgG-SRBC was determined by colorimetric assay 7 days postinfection. Data represent net phagocytic index and mean RT activity ± SEM from two independent donors. The extent of HIV-1 replication, and the effectiveness of inhibition of replication by 3TC, were determined by measuring RT activity in culture medium at day 7, as indicated. B, MDM were mock infected or infected with HIV-1NL(Ba-L){Delta}nef-EGFP using a multiplicity of infection of 2.5 infectious virions per cell, and then phagocytosis of IgG-SRBC was measured 7 days postinfection by microscopic counting of internalized SRBC. The proportion of cells within the HIV-1-infected culture phagocytosing one or more SRBC was calculated for EGFP+ and EGFP– cells separately, and this proportion compared with that of the mock-infected MDM. Data represent mean ± SEM for n = 5 independent experiments. Representative micrographs obtained from two unrelated visual fields of a single experiment are depicted in C. EGFP-positive cells (productively infected MDM) are identified by their green fluorescence. Arrows indicate examples of phagocytosed SRBC in each field.

 
To determine whether inhibition occurred only in infected MDM or also in bystander cells, we used a strain of HIV-1 expressing EGFP in place of Nef (HIV-1NL(Ba-L){Delta}nef-EGFP). This approach has been validated because we have shown previously that Nef is not required for HIV-1-induced inhibition of phagocytosis (9, 44). Ingested SRBC were counted microscopically within fluorescent MDM (EGFP-positive cells in HIV-1-infected cultures; EGFP+) and nonfluorescent MDM (EGFP-negative cells in HIV-1-infected cultures; EGFP–), as well as within mock-infected MDM (Fig. 2, B and C). The proportion of MDM that had ingested one or more SRBC was calculated to assess HIV-1 inhibition of Fc{gamma}R-mediated phagocytosis. From five independent experiments, the proportion of MDM undergoing phagocytosis in mock-infected control cultures was 44.6 ± 4.9%, whereas in HIV-1-infected cultures, the proportion was 25.7 ± 3.8%. Using Friedman’s test, a significant difference was found in phagocytosis between the groups consisting of EGFP+ or EGFP– cells within HIV-1-infected cultures and mock-infected cells (p = 0.015). In these experiments, HIV-1 infection inhibited phagocytosis by an average of 40.5% in five of five independent donors (Fig. 2B). This degree of phagocytosis inhibition is consistent with the data obtained using the colorimetric assay (Figs. 1A and 2A) (9, 10). Within HIV-1-infected cultures, both EGFP+ (productively infected) cells and EGFP– (nonproductively infected) cells (Fig. 2B) were inhibited relative to mock-infected cells (median phagocytosis = 84.6 and 49.1%, respectively; p = 0.126 after Bonferroni’s correction, for both comparisons). In the experiments depicted in Fig. 2, the EGFP+ cells represented an average of 21.5% (range = 8–40%) of total cells within the infected culture.

A similar decrease in phagocytosis was therefore observed following infection by HIV-1NL(Ba-L){Delta}nef-EGFP and by HIV-1Ba-L, although in the former case the virus was purified by ultracentrifuging through a sucrose cushion, showing that inhibition is not due to cytokines or other soluble inhibitors present in the stock HIV-1 preparation used to infect MDM. Taken together, our data indicate that inhibition of Fc{gamma}R-mediated phagocytosis is due to a bystander effect of a productive HIV-1 replication within infected MDM.

HIV-1 infection inhibits Fc{gamma}R-mediated phagocytosis by MDM and {gamma}-chain expression at a posttranscriptional level

We have shown previously in a single experiment that {gamma}-chain mRNA levels measured using semiquantitative RT-PCR were not affected by HIV-1 infection (10). Using a quantitative real-time PCR assay, we measured {gamma}-chain mRNA levels in MDM prepared from four independent donors mock infected or infected with HIV-1 for 7 days. There was no difference between {gamma}-chain mRNA expression in HIV-1-infected and mock-infected MDM (mean relative expression = 79.2 ± 9.7%, p = 0.13; Fig. 3A). Fc{gamma}R-mediated phagocytosis by HIV-1-infected MDM was inhibited in all four experiments (range = 53.3–77.9%; Fig. 3B). {gamma}-Chain protein expression was measured in three of the four experiments and was decreased by 51.7% (range = 37.8–64.2%), as shown in the representative immunoblot (Fig. 3C). Our data indicate that HIV-1-induced inhibition of {gamma}-chain expression and Fc{gamma}R-mediated phagocytosis occur at a posttranscriptional level.


Figure 3
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  		FIGURE 3. The effect of HIV-1 infection on {gamma}-chain mRNA levels in MDM. A, MDM prepared from four independent donors were mock infected or infected with HIV-1Ba-L for 7 days. {gamma}-Chain and GAPDH mRNA levels were measured by real-time PCR, and the levels of {gamma}-chain mRNA were expressed as Ct values standardized to GAPDH Ct values (mean ± SEM). B, For each donor analyzed in A, phagocytosis of IgG-SRBC was measured to determine the extent of HIV-1 inhibition of phagocytosis (net phagocytic indices ± SEM: mock infected = 194 ± 33; HIV infected = 67 ± 36). In three of the four donors, inhibition of {gamma}-chain protein expression was confirmed by quantitative immunoblotting, as shown in a representative blot (C).

 
HIV-1 infection inhibits {gamma}-chain protein biosynthesis

We next determined whether decreased {gamma}-chain expression was caused by decreased {gamma}-chain synthesis. Biosynthesis incorporating L-[U-14C]-leucine into total cellular proteins was measured for 1–24 h and was shown to be linear over this time period (Fig. 4A). In subsequent experiments, biosynthesis was measured following either 8- or 16-h incubation with 14C-leucine, which enabled sufficient incorporation of label into {gamma}-chain immunoprecipitates for accurate quantification. 14C-leucine incorporation into total TCA-precipitable protein was also measured to determine the effect of HIV-1 infection on global protein synthesis. HIV-1 infection inhibited {gamma}-chain protein biosynthesis by an average of 30.1 ± 6.6% (p = 0.0068; Fig. 4B, left panel) in MDM prepared from 12 independent donors, with a representative autoradiogram shown in Fig. 4C. HIV-1 infection also inhibited total cellular protein biosynthesis (mean inhibition = 13.5 ± 4.4%, n = 16, p = 0.024; Fig. 4B, right panel). The minimal inhibition of total cellular protein biosynthesis was not associated with cytotoxicity measured using an XTT (2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-2-H-tetrazolium-5-carboxanilide) dye reduction assay for metabolic respiratory activity (data not shown) and a similar total protein recovery measured by Lowry protein assay in HIV-1-infected MDM to that of mock-infected control (mean protein recovery = 94.0 ± 5.0%, n = 13, p = 0.15). Taken together, these observations confirm that similar numbers of adherent MDM were present in HIV-1-infected wells after 7 days of culture.


Figure 4
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  		FIGURE 4. {gamma}-Chain and cellular protein biosynthesis in HIV-1-infected MDM. A, MDM were incubated 12 days postisolation with 14C-leucine for the indicated times, and radioactivity in TCA-precipitable material was determined by phosphor imaging. Data are presented as average radioactivity (expressed as phosphor imager units, PSL) per µg protein from triplicate experiments using cells obtained from a single donor. B, Adherent 5-day MDM were mock infected or infected with HIV-1Ba-L for 7 days and then labeled with 14C-leucine, and extracts were immunoprecipitated using anti-{gamma}-chain antiserum. Immunoprecipitated {gamma}-chain was resolved by SDS-PAGE and radioactivity in the band quantified by phosphor imaging (left-hand panel). Alternatively, samples of lysate containing equivalent amounts of protein were analyzed for TCA-precipitable radioactivity as in A (right-hand panel). Data represent the rate of 14C-leucine incorporation into {gamma}-chain (PSL/h ± SEM, n = 12) and cellular proteins (PSL/µg protein/h ± SEM, n = 16). C, A representative autoradiogram from a single experiment illustrating 14C-labeled {gamma}-chain.

 
HIV-1 infection does not inhibit {gamma}-chain protein turnover

To determine whether effects on protein stability contribute to the inhibition of {gamma}-chain expression, we assessed the effect of HIV-1 infection on {gamma}-chain protein turnover. Total cellular protein turnover was also determined as a control. Initially, the t1/2 of {gamma}-chain and turnover of total cellular proteins in MDM were determined through labeling and chase experiments. MDM prepared from a single donor were labeled with [U-14C]L-leucine overnight, and then incubated with medium containing a 10-fold molar excess of unlabeled L-leucine for various lengths of time, as indicated in Fig. 5, A and B. The amount of radioactive {gamma}-chain decreased with apparent first order kinetics with a t1/2 of 2.26 h (Fig. 5A), whereas radioactivity in total cellular proteins decreased with apparent two-phase decay kinetics (Fig. 5B). Based on these measurements, we used a 3- and 30-h chase period to determine the effect of HIV-1 infection on {gamma}-chain and total cellular protein turnovers, respectively. Our results indicate that HIV-1 infection did not affect {gamma}-chain protein stability in MDM prepared from seven independent donors (mean percentage of 14C-{gamma}-chain remaining after 3-h chase = 101 ± 1.5%, p = 0.94; Fig. 5C). Similarly, we did not observe any difference between HIV-1-infected and mock-infected MDM with respect to total cellular protein turnover with mean protein-incorporated 14C-leucine 100 ± 3.1% compared with that of mock-infected control (n = 5, p = 1.0; Fig. 5D).


Figure 5
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  		FIGURE 5. The measurement of {gamma}-chain and cellular protein turnover in HIV-1-infected MDM. A and B, MDM were labeled after 12 days in culture with 14C-leucine for 16 h, and then incubated for the indicated times with culture medium containing excess nonradioactive leucine. Radioactivity in {gamma}-chain and total cellular protein were quantified, as described in Fig. 4. Data represent the mean 14C-leucine in {gamma}-chain (A) and total cellular protein (B) from two experiments using MDM prepared from a single donor. C, Adherent 5-day MDM were mock infected or infected with HIV-1Ba-L for 7 days, and then labeled overnight with 14C-leucine. {gamma}-Chain was immunoprecipitated from cell extracts containing equivalent amounts of protein 0 and 3 h after a chase with excess nonradioactive leucine. Immunoprecipitates were resolved by SDS-PAGE and {gamma}-chain quantified by phosphor imaging. Data are expressed as the proportion of counts remaining in {gamma}-chain after the 3-h chase (mean ± SEM, n = 7). D, MDM were mock infected or infected with HIV-1Ba-L and labeled with 14C leucine as in C. Extracts containing equivalent amounts of protein were precipitated with TCA 0 and 30 h after a chase with excess unlabeled leucine. Radioactivity in TCA precipitates was determined by phosphor imaging, as described. Data are expressed as the proportion of counts remaining in cellular protein after the 30-h chase (mean ± SEM, n = 5).

 
HIV-1 infection specifically abrogates {gamma}-chain-dependent Fc{gamma}RI signaling

{gamma}-Chain signaling is essential for Fc{gamma}RI signaling, but not for Fc{gamma}RIIa signaling. Because we have shown that HIV-1 infection inhibits {gamma}-chain protein expression, we hypothesized that {gamma}-chain-dependent Fc{gamma}RI signaling is inhibited by HIV-1, whereas Fc{gamma}RIIa signaling should not be inhibited. To investigate this, we activated receptor-specific signaling by incubating MDM with receptor-specific mAbs, followed by cross-linking with anti-mouse F(ab')2. After 7 days of infection with HIV-1, we observed a decrease in tyrosine phosphorylation of Syk in eight of nine infected MDM cultures relative to mock-infected controls, following Fc{gamma}RI cross-linking. In contrast, Fc{gamma}RIIa-dependent Syk tyrosine phosphorylation was not inhibited in any experiment (zero of nine). In selected experiments (n = 2), we assessed whether HIV-1 infection inhibits tyrosine phosphorylation of the Syk activation loop (Y525/526), which is essential for Syk function and a better marker for Syk activation than total Syk tyrosine phosphorylation (24). In both cases, Syk Y525/526 phosphorylation was inhibited following Fc{gamma}RI cross-linking in infected cultures, as shown by a representative immunoblot in Fig. 6A. Syk Y525/526 phosphorylation following Fc{gamma}RIIa cross-linking was not inhibited by HIV-1 infection (Fig. 6A), consistent with the data obtained using anti-proline-tyrosine Ab. The inhibition of Syk Y525/526 phosphorylation correlated with decreased {gamma}-chain protein expression, as shown by a representative immunoblot in Fig. 6B. Taken together, our results indicate that HIV-1 infection selectively inhibits the {gamma}-chain-dependent Fc{gamma}RI signaling pathway by down-modulating {gamma}-chain protein expression.


Figure 6
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  		FIGURE 6. Analysis of Fc{gamma}RI- and Fc{gamma}RIIa-dependent Syk activation following receptor cross-linking in HIV-1-infected MDM. A, Adherent 5-day MDM were mock infected or infected with HIV-1Ba-L for 7 days. MDM were incubated with anti-Fc{gamma}RI (10.1) or anti-Fc{gamma}RIIa (IV.3) mAbs, followed by cross-linking with goat anti-mouse F(ab')2. Syk was immunoprecipitated from extracts containing equivalent amounts of protein with anti-Syk mAb (4D10), resolved by SDS-PAGE, and visualized by immunoblotting with anti-phospho-Syk Y525/526, followed by goat anti-rabbit Ab conjugated to Alexa Fluor 680 (upper panels). The blots were reprobed with 4D10 and goat anti-mouse Ab conjugated to IRDye 800 (lower panels). Syk activation was determined by measuring the ratio of fluorescence at 680 nm compared with 800 nm. B, A representative immunoblot of {gamma}-chain expression in HIV-1-infected and mock-infected MDM used in A. G{alpha}M, goat anti-mouse F(ab')2; XL, cross-linking.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study shows that decreased FcR{gamma} ({gamma}-chain) expression in HIV-1-infected MDM is caused by decreased {gamma}-chain biosynthesis. Using quantitative real-time PCR, we show that {gamma}-chain mRNA levels were not affected by HIV-1 infection, indicating that HIV-1 inhibits {gamma}-chain expression at a posttranscriptional level. Measurements of incorporation of radioactive leucine into newly synthesized {gamma}-chain suggest that decreased expression by HIV-1 infection is the result of decreased protein biosynthesis, whereas {gamma}-chain degradation was not affected. Consistent with the hypothesis that decreased {gamma}-chain expression is the principal cause of defective Fc{gamma}R signaling, we have shown that Fc{gamma}RI-dependent Syk activation was inhibited in HIV-1-infected MDM, whereas Fc{gamma}RIIa-dependent Syk activation was not. Finally, measurement of Fc{gamma}R-mediated phagocytosis in MDM infected with HIV-1 expressing EGFP in place of Nef showed a strong bystander effect on cells not actively transcribing the viral genome. The significance of this latter finding is that inhibition of macrophage function in vivo may be more extensive than would be predicted from the proportion of macrophages infected with HIV-1.

We observed a small, but significant decrease in total protein biosynthesis in HIV-1-infected MDM. This is, to our knowledge, the first time that the effect of HIV-1 on global protein biosynthesis in macrophages has been carefully studied and described. The technique used in this study to measure biosynthetic incorporation of labeled amino acids into protein includes steps to hydrolyze aminoacyl tRNAs and to remove contaminating lipids, giving confidence that the small observed decrease in incorporation is due to decreased protein biosynthesis. The extent of decrease in global protein biosynthesis was not large enough, however, to account for the decrease in {gamma}-chain biosynthesis or levels, suggesting that there is in part a specific effect of HIV-1 on {gamma}-chain biosynthesis. It is possible that HIV-1 affects translational initiation factors particularly required for {gamma}-chain biosynthesis, for example, initiation factor 4GI, which is cleaved by HIV-1 protease (45, 46, 47).

Because our earlier experiments showed that Nef is not required for inhibition of phagocytosis (9, 44), we used an HIV-1 strain in which the Nef coding sequence was replaced with that of EGFP to compare phagocytosis in cells containing actively replicating HIV-1 and cells not actively transcribing the viral genome, within infected cultures. The degree of inhibition in HIV-1-infected cultures compared with mock-infected cultures using this technique was similar to that obtained with our colorimetric assay, reinforcing our previous observations that Nef expression is not required for inhibition. In addition, these experiments showed a strong bystander effect on Fc{gamma}R-mediated phagocytosis because EGFP– cells showed a similar degree of phagocytic inhibition as the whole infected MDM culture. Although we cannot determine whether the EGFP– MDM were latently infected with HIV-1 at the time of assay, these data suggest that inhibition is not due to production of inhibitory HIV-1-encoded proteins acting within the cell.

Experiments in which infection of MDM was suppressed by 3TC showed that although inhibition of phagocytosis was not confined to actively infected cells, replication within the culture was required for inhibition. These data are consistent with a model in which HIV infection stimulates MDM to secrete inhibitory factors. One potential candidate is TGF-beta1, a cytokine involved in cell growth and differentiation as well as immunosuppression (48). TGF-beta1 production, which is derived from infected macrophages, PBMCs, and astrocytes (49, 50, 51), is elevated in HIV-1-infected individuals (52). A study by Tridandapani et al. (53) showed that TGF-beta1 inhibited {gamma}-chain protein expression in uninfected human monocytes and myeloid cells, leading to defective Fc{gamma}R-mediated phagocytosis. TGF-beta1 also has been shown recently to suppress {gamma}-chain expression in human bone marrow-derived mast cells (54). In mast cells, however, in contrast to our findings with respect to Fc{gamma}Rs, decreased {gamma}-chain expression led to decreased Fc{epsilon}R expression. Unfortunately, in this study, the effect of TGF-beta1 on {gamma}-chain protein biosynthesis was not examined. Given the bystander effect of HIV-1 on Fc{gamma}R-mediated phagocytosis observed in the present study and the fact that TGF-beta1 is produced by HIV-1-infected macrophages (49), it would be of interest to determine whether HIV-1 inhibits {gamma}-chain expression and subsequent phagocytosis via a TGF-beta1-dependent mechanism. Another soluble factor that may potentially contribute to defective phagocytosis is the gp120 component of HIV-1 envelope, as it has been shown to down-regulate phagocytosis by human macrophages (55) and human monocytes (56). In this context, it is of interest that HIV-1 gp160/gp120 induces TGF-beta1 production by human PBMCs (50).

Because Fc{gamma}RI and IIIa, but not Fc{gamma}RIIa, require {gamma}-chain to transduce signals to the protein tyrosine kinase Syk, we tested whether HIV-1 infection selectively inhibits {gamma}-chain-dependent Syk activation. Our results showed that Fc{gamma}RI-, but not Fc{gamma}RIIa-mediated, tyrosine phosphorylation of Syk, and specifically phosphorylation of Y525/526, was inhibited following HIV-1 infection of MDM. These data show that HIV-1 infection of MDM does not inhibit {gamma}-chain-independent Fc{gamma}R signaling in MDM. Although it is not clear whether all Fc{gamma}RI signaling requires {gamma}-chain, both PI3K and rac/Cdc42 are downstream of {gamma}-chain, which means that HIV-1 infection will inhibit both membrane and cytoskeletal changes required for particle ingestion by this receptor. We observed a moderate enhancement of Syk tyrosine phosphorylation in HIV-1-infected cultures following Fc{gamma}RIIa cross-linking, in about one-half of our experiments. The reason that this occurred is unclear because HIV-1 infection of MDM does not alter the expression of Fc{gamma}RIIa (10), but may be due to a compensatory signaling pathway. Increased Fc{gamma}RIIa-specific Syk activation did not correlate with increased phagocytosis in our in vitro model of HIV-1 infection of human macrophages, which shows that phagocytosis of IgG-opsonized erythrocytes required {gamma}-chain-dependent signaling. These results suggest that phagocytosis of particles opsonized with Abs recognized predominantly by Fc{gamma}RIIa, such as Abs of the IgG2 subtype against encapsulated microorganisms, may not be inhibited by HIV-1 (57).

Inhibition of Fc{gamma}R-mediated phagocytosis by macrophages, predominantly via a bystander mechanism through inhibition of {gamma}-chain synthesis, may contribute to the susceptibility to specific opportunistic infections in HIV-infected individuals. It will be of interest, therefore, to determine whether {gamma}-chain expression is decreased in monocyte/macrophage populations in vivo. This has been impeded, however, by the lack of a mAb specific for {gamma}-chain, which would allow accurate quantitation by intracellular staining and flow cytometry. A bystander effect on {gamma}-chain expression also implies that {gamma}-chain could be depleted in other cell populations that are normally not infected with HIV-1, potentially leading to a broader role in HIV-1 immunopathogenesis. {gamma}-Chain is a signaling protein not only essential for transducing activation signals from Fc{gamma}RI and Fc{gamma}RIIIa, but other immunoreceptors, including Fc{alpha}R and Fc{epsilon}R (58, 59, 60), NK cell natural cytotoxicity receptor NKp46 (61, 62), and the TCR of effector CD4+ T cells (63). HIV-1 infection has been associated with decreased expression of the functionally related TCR-associated CD3{zeta} in CD4+ and CD8+ T cells and NK cells, which has been correlated with disease progression and loss of cytotoxic T cell activity and NK cell-mediated cytolysis (64, 65, 66, 67). Of interest, even though CD3{zeta} expression is normally required for cell surface expression of the CD3 complex, loss of expression was specific for CD3{zeta} and did not extend to other polypeptides in the complex, such as CD3{epsilon} (64). This observation is similar to that described in the present work and in our previous report in which infection of MDM in vitro leads to decreased expression of {gamma}-chain without a concomitant decrease in surface expression of Fc{gamma}RI and Fc{gamma}RIIIa (10). Loss of ITAM-containing signaling molecules without concomitant loss of their associated receptors appears to be a feature of chronic immune activation such as found in cancer and chronic viral diseases (68). In contrast to the observations of CD3{zeta} expression ex vivo, however, in which decreased expression was associated with decreased mRNA content (66, 67), our results showed that HIV-1 infection inhibits {gamma}-chain expression in vitro by suppressing protein biosynthesis. This suggests that HIV-1 may affect immunoreceptor signaling proteins via different mechanisms, indicating specific strategies used by HIV-1 to modulate a variety of host cellular immune defenses.


   Acknowledgments
 
We thank Dr. Paul Gorry and Lachlan Gray for the provision of the pNL4-3 Ba-L env {Delta}nef EGFP plasmid with permission from Dr. Christopher Aiken (Vanderbilt University, Nashville, TN), and Dr. Secondo Sonza and Dinushka Dowling for the GAPDH real-time PCR primer set and assistance with real-time PCR, respectively. Jenny Lewis is thanked for assistance with statistical analyses.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Health and Medical Research Council Program Grant 358399. E.L. is a recipient of an Australian postgraduate award, and S.M.C. of a National Health and Medical Research Council Principal Research Fellowship. Back

2 Address correspondence and reprint requests to Dr. Anthony Jaworowski, AIDS Pathogenesis and Clinical Research Program, The Macfarlane Burnet Institute for Medical Research and Public Health, 85 Commercial Road, Melbourne, Victoria, Australia 3004. E-mail address: anthonyj{at}burnet.edu.au Back

3 Abbreviations used in this paper: MDM, monocyte-derived macrophage; Ct, comparative threshold; EGFP, enhanced GFP; RT, reverse transcriptase; TLB, Triton lysis buffer. Back

Received for publication July 27, 2006. Accepted for publication November 2, 2006.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
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