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

This information is current as Katherine Kedzierska, Philip Ellery, Johnson Mak, Sharon R. of September 25, 2021. Lewin, Suzanne M. Crowe and Anthony Jaworowski J Immunol 2002; 168:2895-2903; ; doi: 10.4049/jimmunol.168.6.2895 http://www.jimmunol.org/content/168/6/2895 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. HIV-1 Down-Modulates ␥ Signaling Chain of Fc␥R in Human Macrophages: A Possible Mechanism for Inhibition of Phagocytosis1

Katherine Kedzierska,*‡ Philip Ellery,* Johnson Mak,*§ Sharon R. Lewin,¶ Suzanne M. Crowe,2*†‡ and Anthony Jaworowski2,3*†

HIV-1 infection impairs a number of macrophage effector functions, thereby contributing to development of opportunistic infections and the pathogenesis of AIDS. Fc␥R-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 Fc␥Rs 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 Downloaded from phosphorylation of the cellular proteins, which occurs following engagement of Fc␥Rs, suggesting a defect downstream of initial receptor activation. Fc␥R-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 ␥ signaling subunit of Fc␥R (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 ␥ signaling subunit augmented Fc␥R-mediated phagocytosis, suggesting that down- http://www.jimmunol.org/ modulation of ␥-chain protein expression in HIV-infected MDM caused the defective Fc␥R-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 Fc␥R-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. The Journal of Immu- nology, 2002, 168: 2895–2903.

ells of macrophage lineage, including peripheral blood tions with cytoskeletal (8–11) and cytoplasmic proteins (12–16).

monocytes and tissue macrophages, provide critical func- These interactions include cellular proteins and kinases that are by guest on September 25, 2021 C tions in the cell-mediated response to a variety of oppor- also involved in Fc␥R-mediated phagocytosis, e.g., the Src ki- tunistic pathogens such as Mycobacterium avium complex, Toxo- nases, Hck and Lyn (12, 13, 17, 18), p21-activated kinase (14, 15), plasma gondii, and Candida albicans. A number of monocyte/ the guanine nucleotide-exchange factor, Vav (19), and actin (10). macrophage functions are impaired following HIV-1 infection in HIV-1 impairs Fc␥R-mediated phagocytosis (5), and the mecha- vivo and in vitro, including chemotaxis (1, 2), phagocytosis (3–5), nism is unknown, although studies using the promonocytic U937 intracellular killing (3), and cytokine production (reviewed in Ref. cell line suggest that inhibition occurs via a cAMP-dependent 6). These defects contribute to the pathogenesis of AIDS by al- mechanism (20). lowing reactivation and development of opportunistic infections The receptors for the constant region of IgG (Fc␥RI, Fc␥RII, (reviewed in Ref. 7). The mechanism by which HIV-1 impairs and Fc␥RIII) are the major means by which cells of macrophage effector functions of cells of macrophage lineage is unknown. lineage recognize IgG-opsonized pathogens, thereby triggering The HIV-1-encoded proteins Nef, Vif, Vpr, and Rev have been phagocytosis and Ab-dependent cellular cytotoxicity. Peripheral shown to modulate a number of signaling pathways via interac- blood monocytes express mainly the high-affinity Fc␥RI (CD64) and a low-affinity Fc␥RII, whereas macrophages also express Fc␥RIIIA (CD16A; reviewed in Ref. 21). Fc␥R-mediated inter- *AIDS Pathogenesis Research Unit, Macfarlane Burnet Center, †National Center for HIV Virology Research; Departments of ‡Medicine and §Biochemistry, Monash Uni- nalization of IgG-opsonized particles requires tyrosine phosphor- versity, and ¶Department of Microbiology and Immunology, University of Mel- ylation of proteins and involves activation of several kinases and bourne, Melbourne, Australia their substrates. Most studies to date have examined these path- Received for publication October 29, 2001. Accepted for publication January 8, 2002. ways in murine macrophages or cell lines transfected with Fc␥R The costs of publication of this article were defrayed in part by the payment of page (22–27). Following clustering of Fc␥Rs, tyrosine kinases from the charges. This article must therefore be hereby marked advertisement in accordance Src family associated with ␥-chain of Fc␥R (including Hck and with 18 U.S.C. Section 1734 solely to indicate this fact. Lyn) are activated (28, 29), leading to a rapid and transient phos- 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 Re- phorylation of immunoreceptor tyrosine-based activation motifs search, and by the Macfarlane Burnet Center Research Fund. K.K. was a recipient of (ITAMs)4 present on the ␥ signaling subunits associated with a National Health and Medical Research Council Postgraduate Scholarship. S.R.L. Fc␥RI and Fc␥RIII or on the cytoplasmic domain of Fc␥RII (30). was supported by the National Health and Medical Research Council and the Ian Potter Foundation. Phosphorylation of ITAMs create docking sites for Syk, which is 2 S.M.C. and A.J. contributed equally to this paper. 3 Address correspondence and reprint requests to Dr. Anthony Jaworowski, AIDS 4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation Pathogenesis Research Unit, Macfarlane Burnet Center, Yarra Bend Road, Fairfield, motif; FAK, focal adhesion kinase; MDM, monocyte-derived macrophage; MOI, Victoria, Australia 3078. E-mail address: [email protected] multiplicity of infection; RT, reverse transcriptase.

subsequently activated by phosphorylation (27, 31). An absolute ␥-chain using rabbit anti-␥ subunit (TCR and FcR) polyclonal Ab (1 ␮g/ and specific requirement for Syk in Fc␥R-mediated phagocytosis ml; Upstate Biotechnology, Lake Placid, NY) or rabbit IgG control (1 ␮ has been shown by gene knockout studies (32). Activation of Syk g/ml; Upstate Biotechnology), followed by two washes in cold (4°C) PBS-CMF and incubation with sheep anti-rabbit IgG conjugated to FITC results in phosphorylation of phosphatidylinositol 3-kinase (33) (SILENUS Laboratories, Melbourne, Australia). All staining procedures and localized accumulation of kinases such as focal adhesion ki- were performed in the presence of 50% FCS to reduce the level of non- nase (FAK) and cytoskeletal substrates, including actin-binding specific staining. The fluorescence for intracellular ␥-chain was quantified proteins paxillin, vinculin, talin, and ␣-actinin (23, 26, 34), leading by flow cytometric analysis, converted to molecules of equivalent soluble fluorochrome units, and corrected for background fluorescence. to cytoskeletal rearrangement and phagocytosis of the opsonized particles. Recently, we have reported that the early signaling Opsonization of target particles ␥ events during Fc R-mediated phagocytosis by human monocyte- Target particles were opsonized immediately before the phagocytosis assay derived macrophages (MDM) also involve tyrosine phosphoryla- as previously reported (35). Briefly, sheep RBC (E; ICN-Cappel, Aurora, tion of cellular proteins, including Hck, Syk, Pyk-2 (a member of OH) were opsonized with rabbit anti-E Ab (ICN-Cappel), whereas latex FAK family), and paxillin (35). beads (3 ␮m in diameter; Sigma-Aldrich, St. Louis, MO) were coated with This study examines the mechanism by which HIV-1 inhibits BSA (Sigma-Aldrich), washed, and opsonized with rabbit anti-BSA anti- serum (ICN-Cappel). Fc␥R-mediated phagocytosis in human MDM. Our results show that defective phagocytosis by HIV-infected MDM is due, at least Phagocytosis assay using IgG-opsonized sheep RBC (E-IgG) in part, to decreased expression of the ␥ signaling subunit of the On day 7 postinfection, MDM were plated onto 96-well plates (Costar, Fc␥R, which leads to specific signaling defects downstream of Cambridge, MA) at 5 ϫ 104 cells per well in 100 ␮l of supplemented Fc␥Rs. Iscove’s medium and were allowed to adhere for2hat37°C. IgG-opso- Downloaded from nized or unopsonized E were added to adhered MDM at a E:MDM ratio of 10:1. The plate was centrifuged at 100 ϫ g for 5 min at 4°C and was then Materials and Methods placed at 37°C for phagocytosis to proceed. Phagocytosis was terminated Isolation and culture of monocytes 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 Human monocytes were isolated from buffy coats of HIV-, hepatitis B postisolation were incubated with 8Јbromo-cAMP (Sigma-Aldrich) at con- virus-, and human T cell leukemia virus-seronegative blood donors (sup- centrations ranging from 0.1 ␮Mto1mMat37°C for 30 min or 48 h http://www.jimmunol.org/ plied by the Red Cross Blood Bank, Melbourne, Australia) by Ficoll-Paque before phagocytosis assay. (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation and plastic adherence as previously described (36). Cell viabil- Phagocytosis assays using IgG-opsonized beads ity was Ͼ95% and the purity of monocytes was Ͼ90% as determined by 6 immunofluorescent staining with anti-CD14 mAb (BD Biosciences, Moun- MDM (1 ϫ 10 cells) were dispensed into 4-ml polypropylene tubes (BD tain View, CA) and analysis by flow cytometry (FACStarPlus; BD Bio- Biosciences, Franklin Lakes, NJ), washed twice in PBS-CMF (500 ϫ g for sciences). Monocytes were cultured in IMDM (Cytosystem, Castle Hill, Aus- 5 min), and cooled on ice for 20 min in 100 ␮l of PBS-CMF. Cells were tralia) supplemented with 10% heat-inactivated human ABϩ serum, 2 mM incubated with or without IgG-opsonized beads (target:MDM ratio of 10:1) L-glutamine, and 24 ␮g/ml gentamicin (supplemented Iscove’s medium) in at 37°C in a shaking water bath. At specified time points, phagocytosis was suspension in polytetrafluorethylene (Teflon) jars (Savillix, Minnetonka, MN) arrested by plunging the tubes into ice and washing MDM in ice-cold at an initial concentration of 1 ϫ 106 cells/ml. PBS-CMF, followed by centrifugation at 20,000 ϫ g for 45 s. For immunoblotting and immunoprecipitation experiments, cells were lysed in 100 by guest on September 25, 2021 HIV-1 infection of MDM ␮l 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 follow- On day 5 postisolation, MDM were infected with the M-tropic strain of ing phosphatase inhibitors: 50 mM NaF, 1 mM sodium orthovanadate (Sig- HIV-1Ba-L (AIDS Research and Reference Reagent Program, Division of ma-Aldrich), and 40 mM ␤-glycerophosphate (Sigma-Aldrich), and pro- AIDS, National Institute of Allergy and Infectious Disease, National In- tease inhibitors: 1 mM pefabloc, 1 ␮M pepstatin, and 1 ␮M leupeptin stitutes of Health, Bethesda, MD) at a multiplicity of infection (MOI) of (Boehringer Mannheim, Mannheim, Germany). 0.1–1for2hasdescribed previously (37). In selected experiments, MDM were infected at different MOIs, ranging from 0.1 to 3, to assess the role of Immunoblotting and immunoprecipitation HIV-1 input on Fc␥R-mediated phagocytosis. Control cells were mock- infected and cultured under identical conditions. HIV-1 replication in MDM extracts containing equal amounts of proteins, as determined by MDM was quantified by monitoring reverse transcriptase (RT) activity detergent-compatible protein assay (Bio-Rad, Hercules, CA), were boiled using a micro RT assay on day 7 postinfection (38). Under the conditions in SDS sample buffer (10 mM Tris (pH 8), 2 mM EDTA, 1% SDS, 5% used in our experiments, HIV-1 infection of MDM at varying MOIs was 2-ME, and 5% glycerol), resolved by 10% SDS-PAGE, transferred to ni- not associated with decreased viability of the cells when assessed by trypan trocellulose, and blocked for2hin3%BSA. The blots were probed with blue exclusion, decreased MDM numbers, or morphologic changes. All the Abs directed against phosphotyrosine (RC20; BD Transduction Laborato- reagents, viral stocks, and culture supernatants tested in this study were ries, Lexington, KY), Syk (Santa Cruz Biotechnology, Santa Cruz, CA), negative for LPS contamination (Ͻ0.5 U/ml) using the limulus amebocyte paxillin (BD Transduction Laboratories), Hck (a gift from Dr. H.C. Cheng, lysate assay (BioWhittaker, Walkersville, MD). Department of Biochemistry, University of Melbourne, Melbourne, Aus- tralia), and ␥-chain of Fc␥R (Upstate Biotechnology) overnight at 4°C, Flow cytometric analysis of Fc␥RI, II, and III expression followed by secondary Ab conjugated with HRP (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.), and they were then de- MDM on day 7 postinfection were analyzed for surface expression of veloped for ECL according to manufacturer’s instructions. To determine Fc␥RsI (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16). Cells were stained the phosphorylation of Hck, Syk, and paxillin during Fc␥R-mediated with mAb directed against CD64 conjugated to PE (1 ␮g/ml; Serotec, phagocytosis, cell lysates were immunoprecipitated with the appropriate Raleigh, NC), CD32 conjugated to FITC (1 ␮g/ml; Serotec), and CD16 Ab overnight at 4°C, collected with 15 ␮l of protein G-Sepharose beads (a conjugated to CyChrome (1 ␮g/ml; BD PharMingen, San Diego, CA) on 1-h incubation at 4°C; Pharmacia Biotech, Uppsala, Sweden), washed five ice for 30 min, followed by a wash in calcium- and magnesium-free PBS times in Triton lysis buffer, boiled in SDS sample buffer, resolved by 10% (PBS-CMF). The mean fluorescence of monocytes expressing Fc␥Rs was SDS-PAGE, and probed with anti-phosphotyrosine Ab (RC20) conjugated quantified by flow cytometric analysis. Cells were also stained with iso- to HRP. type-matched controls conjugated to the appropriate fluorochrome. mRNA extraction and amplification of ␥ signaling subunit Intracellular staining of ␥-chain of Fc␥Rs mRNA was extracted from MDM lysates (prepared on day 7 postinfection) ␥ The intracellular -chain staining was determined in uninfected and HIV- using oligo(dT)25 beads (Dynabeads; Dynal Biotech, Carlton South, Aus- infected MDM on day 7 postinfection. Cells were fixed in 3% ultrapure tralia) and was subsequently converted to cDNA as previously reported formaldehyde for 45 min, followed by two washes in 0.1 M glycine in (38). To check for cDNA contamination samples prepared without avian PBS-CMF and permeabilization in 0.1% Triton X for 1 min. MDM were myeloblastosis virus-RT were included within each experiment. For PCR, washed twice in PBS-CMF containing 1% FCS and stained for intracellular 2-fold dilutions were made using equal amounts of cDNA based on The Journal of Immunology 2897

GAPDH levels determined by real-time PCR as previously described (40). 0.001) as assessed on day 7 postinfection (Fig. 1A). The inhibition PCR for ␤-actin was performed to confirm equal template levels (41). of phagocytosis was found in 31 of 33 donors and ranged from 18 Reactions were performed in a total of 50 ␮l comprised of 0.2 mM each ␮ to 91%. The percentage of inhibition of phagocytosis expressed as dNTP, 1.5 mM MgCl2, 0.4 M each primer, 1.1 U of Taq polymerase (PerkinElmer/Cetus, Norwalk, CT), and 1ϫ reaction buffer (PerkinElmer/ the phagocytic index in this study was similar to those previously Cetus). The ␥-chain primer set amplified 172 bp of the human ␥-chain reported by our group when phagocytosis was assessed as the per- cDNA sequence (GenBank accession no. NM004106) with the sequences centage of MDM phagocytosing the fluorescent targets (mean of 5Ј-GAGCCTCAGCTCTGCTATATCC-3Ј and 5Ј-TCTCGTAAGTCTC 51.5% inhibition for n ϭ 10 (Ref. 5) or mean of 38.7% inhibition CTGGTTCC-3Ј. Samples were first denatured at 95°C for 2 min and were ϭ then amplified for 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for for n 10 (Ref. 4)). 1 min, with a final extension step at 72°C for 7 min. PCR products were Under the conditions used in our experiments, the percentage of analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. MDM infected with HIV-1 varies between 30 and 70% when assessed by intracellular p24 Ag staining (3, 36, 38). To assess the Construction of [NL(AD8)⌬nef-␥ϩ] level of HIV-1 input on Fc␥R-mediated phagocytosis, MDM from The DNA constructs [pNL(AD8)] and [pNL(AD8)⌬nef] were prepared by the same donor were infected at increasing MOIs. Phagocytosis substituting the respective envelope coding DNA sequences from NL4.3 was inhibited progressively with increased MOI in MDM cultures, and NL4.3⌬nef with monocytotropic AD8 envelope coding sequences, converting a T-tropic virus to M-tropic as previously described (5). The indicating a direct effect of HIV-1 replication on the phagocytic [NL(AD8)⌬nef-␥ϩ] plasmid was constructed using stitch PCR mutagene- capacity of macrophages (Fig. 1B). HIV-1 infection of MDM did sis. HIV-1 sense f1 primer Rev 8392S 5Ј-GGGGACCCGACAGGC not alter the surface expression of Fc␥Rs: Fc␥RI (CD64), Fc␥RII CCG-3Ј and HIV-1 antisense f1 primer 5Ј-GAGCAAGACCACTGCTG (CD32), or Fc␥RIII (CD16; p ϭ 0.22, 0.84, and 0.09, respectively,

Ј Downloaded from GAATCATCTTATAGCAAAATCCTTTCCAAGC-3 were used to amplify n ϭ 5) at the time of phagocytosis, suggesting that inhibition of a 390-bp HIV-1 f1 fragment immediately upstream of Nef. ␥-Chain sense f2 primer 5Ј-GCTTGGAAAGGATTTTGCTATAAGATGATTCCAGCAGT phagocytosis occurs during or after receptor binding (Fig. 2). GGTCTTGCTC-3Ј and antisense f2 primer 5Ј-CCCCTCGAGACGCGTC TACTGTGGTGGTTTCTCATGCTTCAG-3Ј were used to amplify a 300-bp HIV-1 inhibition of Fc␥R-mediated phagocytosis is not mediated of ␥-chain coding f2 fragment. HIV-1 f1 and ␥-chain f2 fragments were joined via a cAMP-dependent mechanism by PCR extension as previously described (42). The resulting PCR-ampliﬁed fragment was cloned into the [NL(AD8)⌬nef] proviral DNA via restriction As a cAMP-dependent mechanism has been proposed as the cause sites BamHI and XhoI. DNA sequencing has been performed to ensure the of poor Fc␥R-mediated phagocytosis in the promonocytic U937 http://www.jimmunol.org/ ␥ presence of -chain in this mutant proviral DNA and the absence of sponta- cell line latently infected with HIV-1 (20), we initially investigated neous mutations. The production of [NL(AD8)], [NL(AD8)⌬nef] viruses, and [NL(AD8)⌬nef-␥ϩ] particles was achieved by introducing 10 ␮g of protein whether a cAMP-dependent mechanism might be responsible for into 293T cells by the calcium-phosphate transfection method as previously the inhibition of phagocytosis in MDM infected with HIV-1. How- described (42). ever, incubation of MDM with a nonmetabolized cAMP analog, 8Јbromo-cAMP for either 30 min ( p Ͼ 0.05, n ϭ 5) or 48 h ( p Ͼ Statistical analysis 0.05, n ϭ 4) did not inhibit Fc␥R-mediated phagocytosis by hu- The signiﬁcance of the effects of HIV-1 and cAMP on Fc␥R-mediated man MDM (Table I). phagocytosis, down-modulation of ␥-chain in HIV-infected MDM, and the augmentation of phagocytosis by [NL(AD8)⌬nef- ␥ϩ] construct were as- by guest on September 25, 2021 sessed using the Student’s paired t test. A value of p ϭ 0.05 was used to Tyrosine phosphorylation of cellular proteins is inhibited during reject null hypothesis. Fc␥R-mediated phagocytosis by HIV-infected MDM Because Fc␥R-mediated internalization of IgG-opsonized beads Results by human MDM requires rapid phosphorylation of tyrosine resi- ␥ HIV-1 inhibits Fc R-mediated phagocytosis via a postreceptor- dues in a range of proteins (35), the effects of HIV-1 on tyrosine mediated mechanism phosphorylation triggered by binding of IgG-opsonized targets In vitro infection of MDM with HIV-1 inhibited phagocytosis of was assessed. A range of cellular proteins was rapidly phosphor- IgG-opsonized E (mean inhibition of 50.3 Ϯ 4.3%, n ϭ 33, p Ͻ ylated (by a 2-min exposure to IgG-particles) in MDM exposed to

FIGURE 1. HIV-1 inhibits Fc␥R-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 Ϯ 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. 2898 HIV-1 INHIBITION OF Fc␥R PHAGOCYTOSIS

FIGURE 2. Flow cytometric analysis of surface expression of Fc␥RI (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16) on HIV-infected and uninfected macrophages. Expression of Fc␥Rs 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) Ϯ 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. Histo- Downloaded from grams are unimodal and demonstrate the staining with mAbs directed against Fc␥Rs (solid lines) or isotype-matched controls (dotted lines) conjugated to appropriate fluorochrome. http://www.jimmunol.org/

IgG-opsonized beads, and phosphorylation reached a peak be- kDa; Fig. 4A) and Syk (72 kDa; Fig. 4B). HIV-1 infection also tween 2 and 5 min (Fig. 3A). HIV-1 infection of MDM inhibited impaired formation of Fc␥R-stimulated Syk complexes with other Fc␥R-triggered tyrosine phosphorylation of cellular proteins, sug- tyrosine-phosphorylated proteins (molecular mass of 38, 42, 48, gesting a defect downstream to initial receptor activation. Inhibi- 52, 58, 64, and 95 kDa; Fig. 4B). HIV-infected MDM also dis- tion of tyrosine phosphorylation in HIV-infected MDM correlated played reduced phosphorylation of paxillin (an adapter protein that with markedly impaired phagocytosis (Fig. 3B). In some cultures, localizes to focal adhesions and is a substrate for Src and FAK by guest on September 25, 2021 HIV-infected MDM displayed a relatively high basal level of ty- family kinases; 68 kDa) during phagocytosis (Fig. 4C). The input rosine phosphorylation, but an increase in tyrosine phosphoryla- of Hck, Syk, and paxillin proteins was standardized according to ␥ tion was never observed during Fc R-mediated phagocytosis by protein estimations in each lysate. Protein levels are shown by those cells. reprobing with relevant Abs where possible (Fig. 4, B and C, lower panels). Reprobing of the blots with anti-Hck Ab was not feasible, Fc␥R-mediated phagocytosis is impaired at a step upstream of Hck, Syk, and paxillin as a broad band of Ig H chain (55 kDa) interfered with the Hck band (58 kDa). We then assessed the effect of HIV-1 on specific tyrosine kinases necessary for Fc␥R-mediated phagocytosis and their substrates. HIV-1 infection of MDM inhibited phosphorylation of tyrosine Fc␥R-mediated phagocytosis by HIV-infected MDM is kinases from two different families, Hck (a Src family member; 58 associated with down-modulation of Fc ␥-chain protein expression ␥ Table I. Effect of 8Ј-bromo-cAMP on Fc␥R-mediated phagocytosis by Although the surface expression of Fc Rs was not altered by human MDMa HIV-1, the protein tyrosine phosphorylation data suggest a defect upstream of Hck and Syk, at the level of the ␥ signaling subunit of Phagocytic Index (% control) Fc␥R. HIV-1 infection-reduced protein levels of the ITAM-containing ␥-chain of Fc␥R were demonstrated by immunoblot anal- 8Ј-Bromo-cAMP 30-min preincubation 48-h preincubation (mM) (n ϭ 5) (n ϭ 4) ysis of Triton X-100 soluble lysates from HIV-1-infected MDM when compared with lysates obtained from uninfected MDM and 100 111.3 Ϯ 19.9 61.5 Ϯ 15.6b Ϫ standardized according to total protein levels (Fig. 5A). Intracel- 10 1 109.2 Ϯ 10.5 124.3 Ϯ 11.1 Ϫ ␥ 10 2 122.2 Ϯ 9.4 116.0 Ϯ 4.5 lular staining using anti- -chain Ab confirmed these results and 10Ϫ3 125.0 Ϯ 6.9 112.8 Ϯ 10.6 demonstrated a significant decrease in intracellular levels of the Ϫ 10 4 111.8 Ϯ 10.6 111.5 Ϯ 6.0 Fc␥R ␥ signaling subunit in HIV-infected MDM (mean inhibition a Phagocytic function of MDM from five different HIV-1-seronegative donors was of 57.3 Ϯ 12.3%; p ϭ 0.009, n ϭ 4) compared with uninfected assessed in triplicate after preincubation with 8Ј-bromo-cAMP at the indicated con- MDM (Fig. 5, B and C). centrations. The MDM to IgG-sRBC ratio was 1:10, and results were expressed as percentage of control (MDM cultured in the absence of cAMP) as mean Ϯ SEM. Analysis of cell lysates from three donors showed no detectable Values of p were Ͻ0.05 for all concentrations tested. change in ␥-chain mRNA levels in MDM infected with HIV-1 b Concentration was toxic to the cells as assessed by viable count measurements using trypan blue exclusion (viability was Ͻ75%; for all other concentrations viability when compared with uninfected controls (Fig. 6), showing that was Ͼ95%). HIV-1 does not down-modulate ␥-chain at the mRNA level. Input The Journal of Immunology 2899 Downloaded from

FIGURE 3. Inhibition of protein tyrosine phosphorylation during Fc␥R-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 ␮g) 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 http://www.jimmunol.org/ Fc␥R-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 Ϯ SEM of triplicate determinations. IB, immunoblotting; PY, phosphotyrosine. of cDNA was standardized according to GADPH levels as as- phages following HIV-1 infection, although the mechanism of in- sessed by real-time PCR (data not shown) and confirmed by con- hibition was unknown. The inhibition of Fc␥R-mediated phago- ventional PCR using ␤-actin-specific primers (Fig. 6). cytosis was not strain-specific, as consistent impairment of this In vitro infection of MDM by either wild-type [NL(AD8)] or function was observed when MDM were infected in vitro with ⌬ ␥ by guest on September 25, 2021 nef-deleted [NL(AD8) nef] HIV-1 inhibited Fc R-mediated either HIV-1Ba-L (Fig. 1), HIV-1NL4–3(AD8) (Fig. 7A), HIV-1DV phagocytosis ( p ϭ 0.003 and 0.01, respectively; n ϭ 5). In marked (3), or a primary isolate amplified from a member of the Sydney contrast, infection of MDM with a nef-deleted HIV-1 with the gene Blood Bank Cohort (5). The majority of previous reports (44–47), for the ␥-chain of Fc␥R [NL(AD8)⌬nef-␥ϩ] inserted in the nef site confirmed by our own observations, showed that HIV-1 infection augmented Fc␥R-mediated phagocytosis ( p ϭ 0.045, n ϭ 5) and did not change surface expression of Fc␥Rs, indicating that the partially restored the phagocytic capacity of HIV-infected MDM inhibition of phagocytosis results from a signaling defect down- (mean restoration of 35.2 Ϯ 15.8%; Fig. 7A). The observed in- stream of Fc␥Rs. Thomas et al. (20) showed that inhibition of crease in phagocytic capacity of MDM infected with Fc␥R-mediated phagocytosis is mediated via a cAMP-dependent [NL(AD8)⌬nef-␥ϩ] virus was associated with increased protein mechanism in the promonocytic U937 cell line latently infected levels of the ␥-chain as assessed by immunoblotting (Fig. 7B). with HIV-1. This was established by showing that pretreatment of Replication of both of these nef-deleted strains of HIV-1 was these cells with 8Јbromo-cAMP for 48 h before phagocytosis sig- equivalent to that of wild-type virus, [NL(AD8)⌬nef](p ϭ 0.48, nificantly decreased their functional capacity. However, in primary n ϭ 5) or [NL(AD8)⌬nef-␥ϩ](p ϭ 0.89; n ϭ 5; Fig. 7C). human MDM, our results showed that Fc␥R-mediated phagocytosis is not inhibited by the presence of 8Јbromo-cAMP. Similarly, Discussion dibutryl cAMP inhibited complement-mediated phagocytosis but This study shows that HIV-1 infection of MDM in vitro leads to a not Fc␥R-mediated phagocytosis in human MDM (48), suggesting significant impairment of Fc␥R-mediated phagocytosis, resulting that elevated cAMP levels resulting from HIV-1 infection (20, 49) from inhibition of tyrosine phosphorylation of cellular proteins are not responsible for impaired Fc␥R-mediated phagocytosis. stimulated during phagocytosis. We have specifically demon- However, it is possible that cAMP inhibits Fc␥R-mediated phago- strated inhibition of activation of Hck, Syk, and paxillin. Upstream cytosis in the promonocytic U937 cell line via affecting the dif- of Hck and Syk, we found decreased protein expression of the ␥ ferentiation of U937 cells, rather than inhibiting phagocytosis signaling subunit of Fc␥R in HIV-infected MDM, suggesting that per se. HIV-1 inhibits phagocytosis via interference with ␥-chain-specific Our data showing inhibition of tyrosine phosphorylation of any signaling events within human MDM. Thus, our finding of reduced cellular proteins following stimulation with IgG-opsonized targets protein levels of the ITAM-containing ␥ signaling subunit in HIV- in HIV-infected MDM suggested dysfunction at an early stage in infected MDM is likely to be responsible for the defective phago- Fc␥R-mediated signaling. Further results confirmed this hypothe- cytosis by these cells. To our knowledge, this study is the first to sis and showed impaired phosphorylation of the tyrosine kinases report inhibition of tyrosine phosphorylation and signaling events un- Hck and Syk during phagocytosis by HIV-infected MDM. Specific derlying defective effector function in HIV-infected macrophages. requirements for both Hck (associated with ␥-chain of Fc␥Rs) and We and others (5, 43–45) have previously reported impaired Syk in Fc␥R-mediated phagocytosis have been demonstrated in Fc␥R-mediated phagocytosis by human monocytes and macro- numerous studies using knockout mice, monocytic cell lines, and 2900 HIV-1 INHIBITION OF Fc␥R PHAGOCYTOSIS Downloaded from http://www.jimmunol.org/

FIGURE 4. HIV-1 infection of MDM inhibits Hck (58 kDa), Syk (72 kDa), and paxillin (68 kDa) activation during Fc␥R-mediated phagocytosis. On day 7 postinfection, MDM (2 ϫ 106) were incubated with IgG-opsonized latex beads (2 ϫ 107)in200␮l 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 ␮g) 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 by guest on September 25, 2021 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. human MDM (32, 35, 50–52). Although defective activation of (members of Src kinases) as well as ZAP-70 (analog of Syk) have either Syk or Hck in HIV-infected macrophages has not been pre- been observed in T cells from HIV-infected individuals (53). The viously reported, structural and functional defects of Lck and Fyn interaction of Hck with HIV-1-encoded proteins such as Nef and

FIGURE 5. HIV-1 infection of MDM down-modulates protein levels of the ␥ signaling subunit of Fc␥R. A, MDM (1 ϫ 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 ␮g) were resolved by SDS-PAGE and probed with rabbit anti-␥-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 ␥-chain of Fc␥Rs on MDM on day 7 postinfection was assessed by flow cytometry using anti-rabbit ␥-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 ␥-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. The Journal of Immunology 2901

FIGURE 6. HIV-1 does not modulate ␥-chain mRNA expression. MDM were infected with HIV-1 on day 5 postisolation, and mRNA ex- tractions were performed 7 days after HIV-1 infection. ␥-Chain mRNA was reverse transcribed to cDNA by PCR using ␥-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 ␤-actin levels. Neat, undiluted cDNA sample, which corresponds to 1 ϫ 104 MDM. PCR on samples prepared without RT were negative.

Vif is known to modulate its kinase activity (16, 54, 55), thereby potentially interfering with signaling events involved in Fc␥R-me- Downloaded from diated phagocytosis. Syk activation is absolutely essential for phagocytosis, as it cou- ples phagocytosis-promoting Fc␥Rs to rearrangements in the actin-based cytoskeleton (32). Therefore, impaired Syk phosphorylation triggered by Fc␥R engagement would inhibit Syk-mediated activation of substrates required for actin polymerization and cy- http://www.jimmunol.org/ toskeletal 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 ␣-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 ␥ signaling subunit of Fc␥R, not associated with by guest on September 25, 2021 an effect on surface expression of either Fc␥RI, Fc␥RII, or Fc␥RIIIA. The ␥-chain of Fc␥R is not a prerequisite for transient expression of surface Fc␥Rs, but it is important for their stable expression (58, 59). However, the cytoplasmic ␥-chain of Fc␥Rs is critical for Syk activation and subsequent signaling events resulting in Fc␥R-mediated phagocytosis (24, 30). Deletion of the intracellular ␥-chain of Fc␥R markedly impairs phagocytosis, de- spite unchanged surface receptor level (60). Thus, our finding of reduced protein levels of the ITAM-containing ␥ signaling subunit in HIV-infected MDM is likely to be responsible for the defective phagocytosis by these cells. The HIV-induced down-regulation of ␥-chain expression was specific for this subunit, as levels of other signaling molecules downstream Fc␥Rs viz Syk and paxillin were not affected. As ␥-chain mRNA levels were unaltered, we con- cluded that HIV-1 inhibited ␥-chain expression at a post-transcrip- FIGURE 7. Expression of ␥-chain during HIV-1 infection augments tional point. This contrasts with T cells where HIV-1 infection Fc␥R-mediated phagocytosis. MDM on day 5 postisolation were mock- reduces the levels of mRNA for the homologous CD3-␨ chain (61) infected (open bars), infected with WT [NL(AD8)] and nef-deleted and CD3-␥ chain (62). To determine the exact site of HIV-induced [NL(AD8)⌬nef](filled bars), or infected with [NL(AD8)⌬nef ␥ϩ] (hatched down-modulation of the ␥-chain protein, but not mRNA levels, bars). A, Phagocytosis was performed on day 7 postinfection and was as- additional experiments investigating levels of ␥-chain protein syn- sessed via a colorimetric assay as described in Materials and Methods. Ϯ thesis, transport, or degradation need to be performed. This work These data represent means SEM of experiments using cells from five ϫ 6 is currently ongoing in our laboratory. different donors. B, MDM (1 10 ) on day 7 postinfection were washed twice in PBS-CMF and lysed in Triton X-100 buffer, and samples of lysate Our data confirmed that a functional nef gene was not essential ␮ ␥ containing equal amount of protein (20 g) were resolved by SDS-PAGE for inhibition of Fc R-mediated phagocytosis, as previously re- and probed with rabbit anti-␥-chain Ab overnight, followed by anti-rabbit ported by our group (5). Infection of MDM with a nef-deleted Ab conjugated to HRP. The immunoblot shown is representative of two ␥ ␥ ␥ HIV-1 expressing the -chain of Fc R augmented Fc R-mediated experiments using MDM from different donors. The levels of ␥-chain have phagocytosis, supporting our claim that reduced expression of the been analyzed by laser densitometry. Results are expressed as net densi- ␥ signaling subunit of Fc␥R may be responsible for impaired tometry units (DU) corrected for background levels. C, RT activity was Fc␥R-mediated phagocytosis in HIV-infected MDM. The in- quantified in culture supernatant on day 7 postinfection. Results represent creased phagocytic capacity in MDM infected with the HIV-1 con- means Ϯ SEM of experiments using cells from five different donors. 2902 HIV-1 INHIBITION OF Fc␥R PHAGOCYTOSIS struct expressing the ␥-chain was consistent with increased protein 15. Fackler, O. T., X. Lu, J. A. Frost, M. Geyer, B. Jiang, W. Luo, A. Abo, levels of the ␥-chain. As it was impossible to control the level of A. S. Alberts, and B. M. Peterlin. 2000. p21-activated kinase 1 plays a critical role in cellular activation by Nef. Mol. Cell. Biol. 20:2619. ␥-chain protein input in our system, it was not possible to rigor- 16. Hassaine, G., M. Courcoul, G. Bessou, Y. Barthalay, C. Picard, D. Olive, ously correlate phagocytic efficiency of macrophages and ␥-chain Y. Collette, R. Vigne, and E. Decroly. 2001. The tyrosine kinase Hck is an ␥ inhibitor of HIV-1 replication counteracted by the viral vif protein. J. Biol. Chem. levels. If HIV-1 infection impairs Fc R-mediated phagocytosis at 276:16885. more than one level along the signaling pathway, any HIV-induced 17. Lee, T., F. Sunzeri, L. Tobler, B. Williams, and M. Busch. 1991. Quantitative phagocytic defect would not be completely restored by overex- assessment of HIV-1 DNA load by coamplification of HIV-1 gag and HLA-DQ-␣ ␥ genes. AIDS 5:683. pression of -chain protein. 18. Lee, C. H., K. Saksela, U. A. Mirza, B. T. Chait, and J. Kuriyan. 1996. Crystal Taken together, these data suggest that the mechanism of inhi- structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 bition of Fc␥R-mediated phagocytosis in HIV-infected MDM oc- domain. Cell 85:931. 19. Fackler, O. T., W. Luo, M. Geyer, A. S. Alberts, and B. M. Peterlin. 1999. curs upstream of Hck, Syk, and paxillin activation and is associ- Activation of Vav by Nef induces cytoskeletal rearrangements and downstream ated with decreased protein expression of the ␥-chain signaling effector functions. Mol. Cell 3:729. subunit of Fc␥R. It provides the first possible mechanism of de- 20. Thomas, C. A., O. K. Weinberger, B. L. Ziegler, S. Greenberg, I. Schieren, S. C. Silverstein, and J. El Khoury. 1997. Human immunodeficiency virus-1 env fective cellular activation in HIV-infected macrophages not only impairs Fc receptor-mediated phagocytosis via a cyclic adenosine monophos- during phagocytosis, but potentially also underlying other func- phate-dependent mechanism. Blood 90:3760. ␥ ␥ 21. Daeron, M. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203. tions mediated via Fc R. Impaired Fc R-mediated signaling may 22. Greenberg, S., P. Chang, and S. C. Silverstein. 1993. Tyrosine phosphorylation is explain why HIV-infected macrophages fail to control opportunis- required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. tic pathogens such as T. gondii (63) and provides potential thera- Med. 177:529.

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