Functional Association of CD9 with the Fcγ Receptors in Macrophages Keisuke Kaji, Sunao Takeshita, Kensuke Miyake, Toshiyuki Takai and Akira Kudo This information is current as of September 29, 2021. J Immunol 2001; 166:3256-3265; ; doi: 10.4049/jimmunol.166.5.3256 http://www.jimmunol.org/content/166/5/3256 Downloaded from References This article cites 40 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/166/5/3256.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Functional Association of CD9 with the Fc␥ Receptors in Macrophages1 Keisuke Kaji,* Sunao Takeshita,* Kensuke Miyake,† Toshiyuki Takai,‡ and Akira Kudo2* CD9, a member of the tetraspan family of proteins, is highly expressed on macrophages. Although a clear function for the molecule has yet to be described, we have found that the anti-CD9 mAb activates mouse macrophages. The rat anti-CD9 mAb, KMC8.8, ؅ but not the F(ab )2, induced tyrosine phosphorylation of proteins including syk and cbl and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and Fc␥R was required for the signal. Co-cross-linking of ,CD9-Fc␥R with KMC8.8 on macrophages from three different FcR-deficient mice, FcR ␥-chain؊/؊,Fc␥RIIB؊/؊, and Fc␥RIII؊/؊ revealed that Fc␥RIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-Fc␥RIIB/III mAb, 2.4G2, evoked similar phosphorylation patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 treatment led to ␣ reduce levels of TNF- production and p42/44 extracellular signal-related kinase phosphorylation relative to 2.4G2 stimulation. Downloaded from Immunofluorescence staining showed that co-cross-linking of CD9-Fc␥R with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, Fc␥RIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of Fc␥R-deficient macrophages revealed that the colocalization of CD9, Fc␥RIII, Mac-1, and F-actin requires co-cross-linking of CD9-Fc␥RIII, whereas co-cross-linking of CD9-Fc␥RIIB induced the colocaliza- tion of only CD9 and Fc␥RIIB. Our results demonstrate that co-cross-linking of CD9 and Fc␥Rs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages. The Journal of Immunology, 2001, http://www.jimmunol.org/ 166: 3256–3265. he most important accessory cells in immune responses Fc␥RI and Fc␥RIII receptors, nonreceptor tyrosine kinases includ- are phagocytes of the monocytic and myelocytic lineage, ing members of the Src and Syk/ZAP70 families are activated, T represented by the macrophage. In phagocytosis by mac- resulting in the phosphorylation of the ␥-chain ITAM. This is fol- rophages, Fc␥Rs (Fc receptor for IgG) are one of the most impor- lowed by tyrosine phosphorylation of downstream effectors, such tant receptor families. Fc␥Rs recognize IgGs, which are produced as phospholipase C␥, phosphatidylinositol 3-kinase, mitogen-acti- in response to pathogen invasions, and mediate phagocytosis of the vated protein kinase (MAPK), and cbl (2, 5, 6). Although both IgG-opsonized pathogens. During this process, cross-linking of Fc␥RI and Fc␥RIII function as phagocytotic receptors and trans- by guest on September 29, 2021 Fc␥Rs by the immune complex induces a wide variety of immune duce similar signals, the binding activity to IgGs is different; responses: Ab-dependent cellular cytotoxity, release of numerous Fc␥RI specifically binds to IgG2a with a high affinity, whereas inflammatory mediators, and expression changes of cell surface Fc␥RIII binds to IgG1, IgG2a, and IgG2b with a low affinity (2, 3). proteins involved in cell-cell adhesion and Ag presentation (1–3). In contrast, Fc␥RIIB is a monomeric receptor containing the im- Three classes of Fc␥R (Fc␥RI, Fc␥IIB, and Fc␥III) are ex- munoreceptor tyrosine-based inhibitory motif (ITIM), which re- pressed on mouse macrophages and share a highly homologous cruits the phosphatases Src homology 2 protein-1 and Src homol- extracellular portion for the IgG binding domain (1, 2). However, ogy 2 domain-containing inositol phosphatase, and it is unknown there are structural and functional differences in the various recep- how Fc␥RIIB contributes to phagocytosis in macrophages (2, 7). tor family members. Fc␥RI and Fc␥RIII exist as oligomeric com- CD9, which is highly expressed in macrophages, is a cell sur- plexes in which the ␣-chain, bearing the IgG binding domain, as- face glycoprotein belonging to the transmembrane 4 superfamily sociates with ␥-chain dimers that bear an immunoreceptor (TM4SF). The TM4SF is a group of cell surface proteins, includ- tyrosine-based activation motif (ITAM)3; these receptors do not ing at least 16 members such as CD37, CD53, CD63, CD81, and contain intrinsic tyrosine kinase activity (2, 4). On cross-linking of CD82. The structure of these proteins is typified by four hydro- phobic domains spanning the cell membrane and short N- and *Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan; †De- C-terminal cytoplasmic domains (around 5–14 aa in length) (8, 9). partment of Immunology, Saga Medical School, Saga, Japan; and ‡Department of CD9 was reported to be associated with various integrin family Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku molecules, CD5, CD19, and other TM4SF proteins on the cell University, Sendai, Japan surface and has been postulated to participate in the regulation of Received for publication July 18, 2000. Accepted for publication December 19, 2000. cell growth, motility, and signaling (8–16). Besides monocytes/ The costs of publication of this article were defrayed in part by the payment of page macrophages, CD9 is expressed on certain hematopoietic lineage charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells such as platelets, subpopulations of lymphocytes, eosino- 1 This work was supported by a grant-in-aid for scientific research from the Japan phils, and basophils and on some other cell lineages such as en- Ministry of Education, Science, Sports and Culture. dothelial cells, myoblasts, and neuroblasts (8, 15, 16). Addition- 2 Address correspondence and reprint requests to Dr. Akira Kudo, Department of Life ally, CD9 is expressed in oocytes, and CD9-deficient female mice Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226- showed serious sterility caused by a defect in the gamete fusion 8501, Japan. 3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; TM4SF, transmem- toxin; MAPK, mitogen-activated protein kinase; M␤CD, methyl-␤-cyclodextrin; brane 4 superfamily; DIGs, detergent-insoluble glycolipid membranes; CTx, cholera BSS, buffer saline solution. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3257 Downloaded from http://www.jimmunol.org/ FIGURE 1. Tyrosine phosphorylation and cell aggregation induced by anti-CD9 mAb. A, J774 cells were incubated with 10 ␮g/ml of a control Ab, ␥ ␤ anti-CD9, anti-Fc RIIB/III, and anti- 1 integrin mAb, respectively, for 5 min at 37°C. Tyrosine-phosphorylated proteins were detected by anti- by guest on September 29, 2021 phosphotyrosine mAb PY99 in Western blotting. B, Before or after stimulation with anti-CD9 mAb (10 ␮g/ml, 37°C, 5 min), the lysates of J774 cells were precipitated with either anti-syk or anti-cbl Ab. Phosphotyrosine of immunoprecipitates was detected by PY99 in Western blotting. Blots were stripped and reprobed with anti-syk or anti-cbl Ab to reveal equal loading. Data are representative of three similar experiments. C, J774 cells were incubated with a control mAb or anti-CD9 mAb for 10, 30, 60, 120, and 180 min in 1 mM CaCl2/HCMF at 37°C under constant agitation. The ratio of aggregation was represented by the index Nt/N0, where N0 is the initial cell number before incubation and Nt is the total particle number at incubation time t. Results are presented as mean Ϯ SD of four experiments. D, J774 cells were transferred to Counter chamber after incubation for 120 min with a control mAb (left) or anti-CD9 mAb (right), and aggregated cells were demonstrated by phase contrast photographs (ϫ200). process (17–20). However, no abnormalities were detected in any duced much less TNF-␣ production and p42/44 MAPK phosphor- other tissues in CD9-deficient mice (18–20), suggesting that the ylation than Fc␥RIIB/III cross-linking. Finally, our results suggest function(s) of CD9 may be compensated by the presence of other that CD9 on mouse macrophages functionally associates with TM4SF proteins. Fc␥Rs and may modify signals for phagocytosis and inflammatory In human platelets, CD9 is thought to functionally associate responses. with Fc␥RIIA, an isoform of Fc␥RII bearing the ITAM in the cytoplasmic tail, which is not found in mice, and co-cross-linking of CD9 and Fc␥RIIA induces cell aggregation and activation.