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 References This article cites 63 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/168/6/2895.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 infec- tions 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-phosphory- lated 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. Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 2896 HIV-1 INHIBITION OF Fc␥R PHAGOCYTOSIS 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