HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaji, K. Articles by Kudo, A. Search for Related Content PubMed PubMed Citation Articles by Kaji, K. Articles by Kudo, A.

Functional Association of CD9 with the Fc Receptors in Macrophages¹

Keisuke Kaji^*, Sunao Takeshita^*, Kensuke Miyake, Toshiyuki Takai and Akira Kudo^2,*

^* Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan; Department of Immunology, Saga Medical School, Saga, Japan; and Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 FcR was required for the signal. Co-cross-linking of CD9-FcR with KMC8.8 on macrophages from three different FcR-deficient mice, FcR -chain^-/-, FcRIIB^-/-, and FcRIII^-/-, revealed that FcRIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-FcRIIB/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. Immunofluorescence staining showed that co-cross-linking of CD9-FcR with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, FcRIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of FcR-deficient macrophages revealed that the colocalization of CD9, FcRIII, Mac-1, and F-actin requires co-cross-linking of CD9-FcRIII, whereas co-cross-linking of CD9-FcRIIB induced the colocalization of only CD9 and FcRIIB. Our results demonstrate that co-cross-linking of CD9 and FcRs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The most important accessory cells in immune responses are phagocytes of the monocytic and myelocytic lineage, represented by the macrophage. In phagocytosis by macrophages, FcRs (Fc receptor for IgG) are one of the most important receptor families. FcRs recognize IgGs, which are produced in response to pathogen invasions, and mediate phagocytosis of the IgG-opsonized pathogens. During this process, cross-linking of FcRs by the immune complex induces a wide variety of immune responses: Ab-dependent cellular cytotoxity, release of numerous inflammatory mediators, and expression changes of cell surface proteins involved in cell-cell adhesion and Ag presentation (1, 2, 3).

Three classes of FcR (FcRI, FcIIB, and FcIII) are expressed on mouse macrophages and share a highly homologous extracellular portion for the IgG binding domain (1, 2). However, there are structural and functional differences in the various receptor family members. FcRI and FcRIII exist as oligomeric complexes in which the -chain, bearing the IgG binding domain, associates with -chain dimers that bear an immunoreceptor tyrosine-based activation motif (ITAM)³; these receptors do not contain intrinsic tyrosine kinase activity (2, 4). On cross-linking of FcRI and FcRIII receptors, nonreceptor tyrosine kinases including members of the Src and Syk/ZAP70 families are activated, resulting in the phosphorylation of the -chain ITAM. This is followed by tyrosine phosphorylation of downstream effectors, such as phospholipase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinase (MAPK), and cbl (2, 5, 6). Although both FcRI and FcRIII function as phagocytotic receptors and transduce similar signals, the binding activity to IgGs is different; FcRI specifically binds to IgG2a with a high affinity, whereas FcRIII binds to IgG1, IgG2a, and IgG2b with a low affinity (2, 3). In contrast, FcRIIB is a monomeric receptor containing the immunoreceptor tyrosine-based inhibitory motif (ITIM), which recruits the phosphatases Src homology 2 protein-1 and Src homology 2 domain-containing inositol phosphatase, and it is unknown how FcRIIB contributes to phagocytosis in macrophages (2, 7).

CD9, which is highly expressed in macrophages, is a cell surface glycoprotein belonging to the transmembrane 4 superfamily (TM4SF). The TM4SF is a group of cell surface proteins, including at least 16 members such as CD37, CD53, CD63, CD81, and CD82. The structure of these proteins is typified by four hydrophobic domains spanning the cell membrane and short N- and C-terminal cytoplasmic domains (around 5–14 aa in length) (8, 9). CD9 was reported to be associated with various integrin family molecules, CD5, CD19, and other TM4SF proteins on the cell surface and has been postulated to participate in the regulation of cell growth, motility, and signaling (8, 9, 10, 11, 12, 13, 14, 15, 16). Besides monocytes/macrophages, CD9 is expressed on certain hematopoietic lineage cells such as platelets, subpopulations of lymphocytes, eosinophils, and basophils and on some other cell lineages such as endothelial cells, myoblasts, and neuroblasts (8, 15, 16). Additionally, CD9 is expressed in oocytes, and CD9-deficient female mice showed serious sterility caused by a defect in the gamete fusion process (17, 18, 19, 20). However, no abnormalities were detected in any other tissues in CD9-deficient mice (18, 19, 20), suggesting that the function(s) of CD9 may be compensated by the presence of other TM4SF proteins.

In human platelets, CD9 is thought to functionally associate with FcRIIA, an isoform of FcRII bearing the ITAM in the cytoplasmic tail, which is not found in mice, and co-cross-linking of CD9 and FcRIIA induces cell aggregation and activation. Moreover, in various types of cells, CD9 is proposed to be involved in signal transduction coupled with cell activation, proliferation, and adhesion, as mAbs against CD9 can induce the activation of T cells, the homotypic aggregation of pre-B cells, the adhesion and proliferation of Schwann cells, and can inhibit the migration of leukemia cells (13, 21, 22, 23). However, in mouse macrophages, the function of CD9 has not yet been investigated, and the association of molecules with CD9 on macrophages are not known, despite their high level of CD9 expression.

In the present study, we found that an anti-CD9 mAb, KMC8.8, induced protein tyrosine phosphorylation, filopodium extension, and cell aggregation caused by FcRIIB/III-CD9 co-cross-linking on mouse macrophages. The FcRIIB/III-CD9 co-cross-linking induced much less TNF- production and p42/44 MAPK phosphorylation than FcRIIB/III cross-linking. Finally, our results suggest that CD9 on mouse macrophages functionally associates with FcRs and may modify signals for phagocytosis and inflammatory responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

The monoclonal rat anti-mouse CD9 Ab, KMC8.8, was reported previously (24), the isotype of which is IgG2a. KMC8.8 and anti-IgM mAb (MA6), as an isotype (IgG2a)-matched control mAb, were purified from ascitic fluids as described (24), and KMC8.8 was biotinylated by the manufacturer’s instruction (Pierce, Rockford, IL). F(ab')₂ of KMC8.8 were generated by Takara Shuzo (Tokyo, Japan). Anti-FcRIIB/III mAb (2.4G2), anti-₁ integrin mAb, biotinylated anti-ICAM-1 mAb, and FITC-conjugated anti-Mac-1 mAb were obtained from BD PharMingen (San Diego, CA). Anti-syk mAb, anti-cbl-b mAb, anti-phosphorylated p42/44 MAPK mAb, anti-p42 MAPK mAb, and HRP-conjugated anti-phosphotyrosine mAb (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-rat Fab polyclonal Abs, HRP-conjugated anti-rabbit IgG, anti-mouse IgG Ab, and FITC-conjugated anti-rat IgG Ab were purchased from Jackson ImmunoResearch (West Grove, PA), Southern Biotechnology Associates (Birmingham, AL), Bio-Rad (Hercules, CA), and ICN Pharmaceuticals-Cappel Products (Costa Mesa, CA), respectively. FITC-conjugated cholera toxin (CTx) and methyl--cyclodextrin (MCD) were obtained from Sigma (St. Louis, MO). Rhodamine-conjugated phalloidin and FITC-conjugated streptavidin were purchased from Molecular Probes (Eugene, OR).

Cells

The mouse macrophage cell line J774 was cultured in DMEM with 10% FBS. M-CSF-dependent bone marrow macrophages were prepared from wild-type mice and mice deficient in the FcR -chain, FcRIIB, and FcRIII as described previously (25, 26). In brief, the bone marrow cells from femurs and tibias of adult mice were cultured in -MEM containing 10% FBS and 1/10 vol of culture supernatant from the M-CSF-producing cell line CMG14-12 (final 35,000 U/ml of M-CSF) for 3 days. After removing cells in suspension, adherent M-CSF-dependent bone marrow macrophages were harvested with 0.02% EDTA/PBS treatment and refed or used for experiments.

Detection of protein tyrosine phosphorylation by Western blottimg

J774 cells and bone marrow macrophages were washed and resuspended at 1 x 10⁷ cells/ml in DMEM or -MEM containing 10 µg/ml indicated mAb. In some experiments, J774 cells were pretreated with 10 µg/ml anti-FcRIIB/III mAb, 2.4G2 on ice for 20 min before stimulation with anti-rat IgG Ab or anti-CD9 mAb, KMC8.8. After 1–20 min incubation at 37°C, cells were washed with cold PBS and lysed with TNE buffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM Na₃VO₄, 2 mM PMSF, 1 µg/ml aprotinin, 100 µg/ml leupeptin) on ice for 30 min. The cell lysate (30 µg/lane) was separated by 7.5% or 10% SDS-PAGE in reducing conditions, electrophoretically transferred to a nitrocellulose membrane, and then reacted with Abs. Bound Abs were visualized by chemiluminescence by using an ECL immunoblotting kit (Amersham, Buckinghamshire, U.K.). To strip bound Abs, the membranes were incubated in stripping buffer (100 mM 2-ME, 2% SDS, 62.5 mM Tris-HCl, pH 6.8) at 50°C for 30 min.

Immunoprecipitation

J774 cells were lysed with TNE buffer before or after stimulation with KMC8.8 for 5 min at 37°C. Four micrograms of anti-syk or cbl-b mAb plus protein G-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) were added to lysates, and they were incubated for 2 h at 4°C under constant agitation. After washing beads with TNE buffer, the immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting.

Aggregation assay

J774 cells were incubated with Abs at 37°C for the indicated time under constant agitation in 1 mM CaCl₂/HCMF (10 mM HEPES-buffered Ca²⁺, Mg²⁺-free Hank’s solution) containing 1% BSA. After incubation for t minutes, the total number of aggregated particles and cells in the cell suspension (N_t) was counted with a counter chamber. The ratio of aggregation was represented by the index N_t/N₀, where N₀ is the initial cell number before aggregation (27).

TNF- measurement

J774 cells (5 x 10⁴) were cultured with 10 µg/ml of KMC8.8 or 2.4G2 plus anti-rat IgG Ab in 100 µl DMEM containing 10% FBS for various times (0–24 h) at 37°C. The amount of TNF- in the collected supernatant was measured with an ELISA kit (Amersham) according to the manufacturer’s instructions.

Cholesterol extraction

To remove cholesterol, suspended J774 cells (5 x 10⁶ cells/ml) were incubated for 15 or 30 min at 37°C in the presence of 20 mM MCD in BSA-containing buffer saline solution (BSA/BSS: 20 mM HEPES, pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 1 mg/ml BSA; Ref. 28) and then washed with BSA/BSS before stimulation or analysis by flow cytometry. The cells stained with anti-CD9 mAb or anti-FcRIIB/III mAb followed by FITC-conjugated anti-rat IgG were analyzed by using FACSCalibur (Becton Dickinson, San Jose, CA).

Immunohistochemistry and laser-scanning confocal microscopy

J774 cells or bone marrow macrophages cultured on a coverslip were stimulated with KMC8.8 at 37°C for 0, 5 and 60 min. After washing with PBS, cells were fixed in 4% paraformaldehyde/PBS for 30 min at 25°C and then permeabilized and blocked with 0.1% Triton X-100 and 1% skim-milk in PBS. Immunofluorescence staining was conducted by using Abs to CD9, FcRIIB/III, and ₁ integrin, followed by a second Ab, FITC-conjugated goat anti-rat IgG. ICAM-1 and Mac-1 were visualized with biotinylated anti-ICAM-1 mAb, followed by FITC-conjugated streptavidin and FITC-conjugated anti-Mac1 mAb, respectively. F-actin and ganglioside GM1, a detergent-insoluble glycolipid membranes (DIGs) marker (29), were stained with rhodamine-conjugated phalloidin and FITC-conjugated CTx subunit, respectively. Microscopy was performed with a laser-scanning confocal microscope (CSU10; Yokogawa Electric, Tokyo, Japan), and captured images were processed and superimposed by using the IPLab software package (Scanalytics, Billerica, MA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein tyrosine phosphorylation and cell aggregation induced by anti-CD9 mAb in the mouse macrophage cell line J774

In human platelets, anti-CD9 mAbs induce protein tyrosine phosphorylation and cell aggregation (10, 11, 12). To investigate the function of CD9 on macrophages, the effect of an anti-CD9 mAb, KMC8.8, on the mouse macrophage cell line, J774 was examined. After incubation with the mAb for 5 min at 37°C, J774 cells were lysed, and tyrosine-phosphorylated proteins were detected by an anti-phosphotyrosine mAb, PY99, in Western blot analysis. As shown in Fig. 1A, KMC8.8 induced the tyrosine phosphorylation of 55-, 60-, 72-, and 120-kDa proteins, and the same signals were obtained by cross-linking with an anti-FcRIIB/III mAb, 2.4G2. Neither a control mAb nor a mAb against ₁ integrin, which associates with CD9 in B cells and endothelial cells (9), induced protein tyrosine phosphorylation, despite being the same isotype as KMC8.8. CD43 and CD44 are expressed on J774 cells at a similar level to CD9, and mAbs against both surface molecules did not induced tyrosine phosphorylation. Furthermore, incubation with KMC8.8 did not induce tyrosine phosphorylation in macrophages derived from CD9-deficient mice (data not shown). Immunoprecipitation followed by Western blot analysis confirmed that the phosphorylated proteins of 72 and 120 kDa were a nonreceptor tyrosine kinase, syk, and an adapter protein, cbl, respectively (Fig. 1B). Moreover, KMC8.8 induced time-dependent cell aggregation in the presence of 1 mM CaCl₂ (Fig. 1, C and D, right), and the same result was obtained in the absence of CaCl₂ (data not shown).

View larger version (56K): [in this window] [in a new window]   FIGURE 1. Tyrosine phosphorylation and cell aggregation induced by anti-CD9 mAb. A, J774 cells were incubated with 10 µg/ml of a control Ab, anti-CD9, anti-FcRIIB/III, and anti-1 integrin mAb, respectively, for 5 min at 37°C. Tyrosine-phosphorylated proteins were detected by anti-phosphotyrosine mAb PY99 in Western blotting. B, Before or after stimulation with anti-CD9 mAb (10 µg/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 ± 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 (x200).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. No effects of F(ab')2 of KMC8.8 in tyrosine phosphorylation and cell aggregation. A, J774 cells were incubated with 10 µg/ml of F(ab')2 or the whole of anti-CD9 mAb KMC8.8 for 1 or 5 min at 37°C. After lysis of cells, tyrosine-phosphorylated proteins were detected by anti-phosphotyrosine mAb PY99 in Western blotting. To reveal equal loading, blots were stripped and reprobed with anti-syk Ab. Data are representative of three similar experiments. B, J774 cells were incubated with a control mAb, F(ab')2 of KMC8.8 and KMC8.8, respectively, for 60 min in 1 mM CaCl2/HCMF at 37°C under constant agitation. The ratio of aggregation, N60/N0, was calculated. Results are presented as mean ± SD of three experiments.

Specific induction of protein tyrosine phosphorylation by co-cross-linking of FcRIII and CD9 with KMC8.8

On mouse macrophages, three types of FcR, FcRI, FcRIIB, and FcRIII, are expressed. FcRI and FcRIII, but not FcRIIB, transduce tyrosine phosphorylation signals through the ITAM of the subunit (2), whereas FcRIIB transduces dephosphorylation signals through the ITIM. We tried to identify which FcRs can transduce tyrosine phosphorylation signals by cross-linking together with CD9. We pretreated J774 cells with 2.4G2, used as the FcRIIB/III blocker, at 4°C for 20 min before treatment of KMC8.8 so as to block co-cross-linking between FcRIIB/III and CD9 in the treatment of KMC8.8. In J774 cells pretreated with 2.4G2, slight tyrosine phosphorylation of proteins, including syk, was observed after incubation at 37°C for 5 min (Fig. 3A, lane 4), and the addition of anti-rat IgG Ab enhanced the level of phosphorylation (Fig. 3A, lane 5). This indicated that 2.4G2 did bind to FcRIIB/III at 4°C. The addition of KMC8.8 to J774 cells pretreated with 2.4G2 did not enhance the level of phosphorylation (Fig. 3A, lane 6), although KMC8.8 induced strong phosphorylation in untreated J774 cells (Fig. 3A, lane 3). These results suggested that FcRIII is more important than FcRI for phosphorylation signals induced by KMC8.8. To confirm this, we prepared M-CSF-dependent bone marrow macrophages from wild-type, FcR -chain^-/-, FcRIIB^-/-, and FcRIII^-/- mice. These macrophages express functional FcRI, FcRIIB, and FcRIII (wild-type), FcRIIB (FcR -chain^-/-), FcRI and FcRIII (FcRIIB^-/-), or FcRI and FcRIIB (FcRIII^-/-), as the FcR -chain is critical in facilitating either surface expression or ligand binding of the FcRI and FcRIII (30). Stimulation with KMC8.8 for 5 min at 37°C induced tyrosine phosphorylation of proteins, including syk, in bone marrow macrophages from wild-type and FcRIIB^-/- mice (Fig. 3B, lanes 2 and 6) but not from FcR -chain^-/- and FcRIII^-/- mice (Fig. 3B, lanes 4 and 8). These results demonstrate that co-cross-linking of FcRIII and CD9 by KMC8.8 induces tyrosine phosphorylation signals. Mouse FcRI shows 10-fold higher binding affinity to Fc region of mouse IgG2a than FcRIIB and FcRIII (2). The isotype of rat anti-CD9 mAb, KMC8.8, is IgG2a; however, the binding affinity of mouse FcRs is to the Fc region of rat IgG2a is not well known. Thus, we examined whether co-cross-linking of FcRs and CD9 with KMC8.8-F(ab')₂ followed by mouse anti-rat Fab polyclonal Abs induces tyrosine phosphorylation signals. Treatment of macrophages from wild-type, FcR -chain^-/-, FcRIIB^-/-, and FcRIII^-/- mice with KMC8.8-F(ab')₂ followed by the addition of mouse anti-rat Fab Abs induced tyrosine phosphorylation except for macrophages from FcR - chain^-/- (Fig. 3C). Moreover, in macrophages lacking FcRIIB containing the ITIM, the phosphorylation level of syk was stronger than that in wild-type macrophages (Fig. 3, B and C). These results may suggest that co-cross-linking of any combination of FcR and CD9 transduces some signals or enhances FcR signals.

View larger version (75K): [in this window] [in a new window]   FIGURE 3. Protein tyrosine phosphorylation induced by co-cross-linking of FcRIII and CD9. A, J774 cells were incubated in the presence or absence of anti-FcRIIB/III mAb, 2.4G2, for 20 min at 4°C. After washing, cells were incubated with a control Ab, anti-rat IgG Ab, or anti-CD9 mAb, KMC8.8, for 5 min at 37°C. Phosphotyrosine was detected by PY99 in Western blotting. Reblotting with anti-syk mAb was done after stripping to reveal equal loading. B and C, M-CSF-dependent bone marrow macrophages from wild-type, FcR -chain-/-, FcRIIB-/-, and FcRIII-/- mice were stimulated with KMC8.8 for 5 min at 37°C (B), or with KMC8.8-F(ab')2 for 5 min at 37°C followed by mouse anti-rat Fab Ab treatment for 15 min at 4°C (C). Western blotting was conducted as described in A. Data are representative of three (A) or two (B and C) similar experiments.

Different effects of anti-CD9 and anti-FcRIIB/III mAbs on macrophage cell aggregation and TNF- production

J774 cells were stimulated with KMC8.8, 2.4G2 alone, or 2.4G2 plus anti-rat IgG Ab, and then tyrosine-phosphorylated proteins were detected by Western blot analysis. An anti-FcRIIB/III mAb, 2.4G2, induced time-dependent protein tyrosine phosphorylation, and stronger phosphorylation was induced by super cross-linking of 2.4G2 with a secondary Ab or with KMC8.8 alone (Fig. 4A). Moreover, in an aggregation assay, unlike incubation with KMC8.8, super cross-linking of 2.4G2 with anti-rat IgG Ab did not induce cell aggregation after 60 min (Fig. 4B), despite the similar high level of tyrosine phosphorylation (Fig. 4A). The cross-linking of FcRs by IgG immune complexes triggers a wide variety of effector functions including phagocytosis, Ab-dependent cellular cytotoxity, and release of inflammatory mediators like IL-1, IL-10, and TNF- (6, 31, 32). We stimulated J774 cells with KMC8.8 or 2.4G2 plus anti-rat IgG Ab for 0, 12, 18, and 24 h and measured the TNF- production in the supernatant with an ELISA kit. FcRIIB/III cross-linking by 2.4G2 plus anti-rat IgG Ab induced the production of TNF- from J774 cells rapidly, whereas FcR-CD9 co-cross-linking by KMC8.8 induced less TNF- (Fig. 5A). An increase in TNF- mRNA expression was observed in J774 cells cultured with 2.4G2 plus anti-rat IgG Ab for 12 h but not with KMC8.8 (data not shown). It has been reported that the activation of p42 MAPK is necessary for TNF- synthesis induced by FcR cross-linking (6); therefore, the phosphorylation of p42/44 MAPK was examined. As shown in Fig. 5B, 2.4G2 plus anti-rat IgG Ab induced the phosphorylation of p42/44 MAPK after stimulation for 5 min. However, KMC8.8 did not induce but rather reduced it, although other major tyrosine-phosphorylated proteins were much the same in both treatments (Fig. 5, B and C). These results suggest that cross-linking of FcR and CD9 with KMC8.8 induces different signals from that of FcRIIB/III with 2.4G2.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Different effect of anti-CD9 mAb from that of anti-FcRIIB/III mAb in cell aggregation. A, J774 cells were incubated with or without 10 µg/ml anti-FcRIIB/III mAb 2.4G2 for 20 min at 4°C. After washing, either none or10 µg/ml anti-rat IgG Ab or anti-CD9 mAb KMC8.8 were reacted to cells for 1–20 min at 37°C. Phosphotyrosine was detected by PY99 in Western blotting. To reveal equal loading, blots were stripped and reprobed with anti-syk Ab. Data are representative of three similar experiments. B, J774 cells were incubated with a control mAb, anti-FcRIIB/III mAb 2.4G2, plus anti-rat IgG Ab, anti-CD9 mAb KMC8.8, for 60 min in 1 mM CaCl2/HCMF at 37°C under constant agitation. The ratio of aggregation, N60/N0, was calculated. Results are presented as mean ± SD of three experiments.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. TNF- production or p42/44 MAPK phosphorylation by cross-linking by anti-CD9 mAb or anti-FcRIIB/III mAb. A, J774 cells (5 x 104) were cultured with 10 µg/ml of anti-FcRIIB/III mAb 2.4G2 plus anti-rat IgG Ab (solid line) or anti-CD9 mAb KMC8.8 (dotted line) for 0, 12, 18, and 24 h (37°C, 5% CO2). The amount of TNF- in the collected supernatant was measured with an ELISA kit. Results are presented as mean ± SD of two experiments. When error bars were not seen, their sizes were smaller than the symbols. B, J774 cells were incubated with 10 µg/ml of a control mAb, 2.4G2, plus anti-rat IgG Ab or KMC8.8 for 5 min at 37°C. Phosphotyrosine was detected by PY99 in Western blotting. Phosphorylated p42/44 MAPK and p42 MAPK were detected with the corresponding Ab on the same membrane after stripping. Data are representative of three similar experiments. C, After western blotting described above, levels of phosphorylated p42 MAPK and p42 MAPK were quantified by densitometric analysis, and the specific phosphorylation was calculated by dividing the densitometric values for phosphorylated p42 MAPK by those for p42 MAPK. Data are the mean (±SD) of values relative to the specific phosphorylation in cells treated with control mAb from three independent experiments.

Induction of filopodium extension and colocalization of CD9, FcR, Mac-1, ICAM-1, and F-actin after CD9 cross-linking

KMC8.8 induced aggregation of J774 cells in a suspension culture. To observe the process of cell aggregation, J774 cells adhered on a coverslip were stimulated with KMC8.8. After fixing the cells, localization of CD9 or F-actin was examined by staining with KMC8.8, followed by FITC-conjugated anti-rat IgG Ab or rhodamine-conjugated phalloidin. Time-dependent filopodium extension was observed by staining of F-actin in J774 cells (Fig. 6). Additionally, localization of CD9 at filopodia was also observed (Fig. 6). Moreover, after Ab treatment, many cells adhered to neighboring cells as if cells had moved toward the cells attached on the extended filopodia, and CD9 and F-actin were densely colocalized at the cell-cell adhesion sites (Fig. 6, arrow). CD9 has not been reported to be associated with actin filaments or to function in cell-cell or cell-matrix adhesion directly, but CD9 can make complexes with CD5, CD19, heparin-binding-epidermal growth factor, or ₁ integrin on the plasma membrane (8, 9, 13, 14, 15, 16, 33). Therefore, we examined whether other molecules are colocalized at the cell-cell adhesion site after stimulation with KMC8.8. As shown in Fig. 7, the localization of FcRIIB/III, Mac-1 (_M2 integrin), and ICAM-1 at the cell-cell adhesion site was observed after stimulation, and these molecules were colocalized with CD9 or F-actin at this site as detected by double staining (Fig. 7A, arrow). In contrast, ₁ integrin did not change localization after stimulation, while F-actin did concentrate at the cell-cell adhesion site (Fig. 7B, arrow).

View larger version (116K): [in this window] [in a new window]   FIGURE 6. Filopodia extension and colocalization of CD9 and F-actin at the cell-cell adhesion site induced by anti-CD9 mAb. J774 cells were stimulated with anti-CD9 mAb KMC8.8 at 37°C for 0, 5, and 60 min. After fixation, permeabilization, and blocking, CD9 (left) or F-actin (center) was stained with KMC8.8 followed by FITC-conjugated anti-rat IgG Ab or rhodamine-conjugated phalloidin. Right panels represent superimposed images. Upper three panels indicate the adhesive sections to a coverslip observed by using a confocal microscopy. The lowest panels show images of the middle section of the cells. After stimulation with the Ab, CD9 and F-actin were colocalized at the cell-cell adhesion site (arrows). Data are representative of four similar experiments.

View larger version (100K): [in this window] [in a new window]   FIGURE 7. Localization of FcRIIB/III, Mac-1, ICAM-1, and GM1 at the cell-cell adhesion site. A and B, Before or after stimulation with anti-CD9 mAb KMC8.8 for 60 min, FcRIIB/III, Mac-1, ICAM-1, 1 integrin, and F-actin were stained with Abs and reagents as described in Materials and Methods. In A, ganglioside GM1 was stained with FITC-conjugated CTx subunit as a DIGs marker. Right panels present the superimposed images of the two left panels, respectively. After stimulation with the Ab, CD9, F-actin, FcRIIB/III, Mac-1, ICAM-1, and GM1 were localized at the cell-cell adhesion site (A, arrows). In B, after stimulation with the Ab, 1 integrin was not localized at the cell-cell adhesion site, although F-actin was localized at the site (arrow). Data are representative of three similar experiments.

Diminution of the signal from FcRIIB/III or FcR-CD9 cross-linking after extraction of cholesterol from DIGs

DIGs are postulated to represent plasma membrane domains that may function as centers for signal transduction and membrane trafficking (28, 29). However, FcR has not been reported to be involved in DIGs, whereas Src family kinases, which have been implicated in the FcR signals, exist in DIGs (28, 29). Therefore, we tested the effects of disrupting DIGs on FcR-CD9 cross-linking by extracting cholesterol with MCD (28). The MCD treatment (20 mM for 30 min at 37°C) did not change the amount of FcRIIB/III and CD9 on the cell surface (Fig. 8A). However, tyrosine phosphorylation signals induced by both 2.4G2 plus anti-rat IgG or KMC8.8 were diminished in a time-dependent manner (Fig. 8B). This result suggests that DIGs may function in FcR signaling in J774 cells. The result that KMC8.8 induces strong signals of tyrosine phosphorylation, filopodium extension and cell aggregation without super cross-linking by a second Ab may be explained by localization of CD9 to a specific membrane structure like DIGs as reported in T cell activation induced by anti-CD9 mAb (13).

View larger version (34K): [in this window] [in a new window]   FIGURE 8. Diminution of tyrosine phosphorylation signal induced by cross-linking of FcRIIB/III or FcR-CD9 under cholesterol extraction. A, Before (upper) or after (lower) incubation of J774 cells in the presence of 20 mM MCD in BSA/BSS for 30 min at 37°C, surface expression of FcRIIB/III (left) and CD9 (right) were analyzed by flow cytometry. The broken line shows the FACS profile from the negative control, streptavidin FITC for CD9, or FITC-conjugated anti-rat IgG for FcRIIB/III. B, After treatment with 20 mM MCD in BSA/BSS for 0, 15, and 30 min at 37°C, J774 cells were incubated with a control Ab, anti-FcRIIB/III mAb 2.4G2, plus anti-rat IgG Ab, anti-CD9 mAb KMC8.8, for 5 min at 37°C. Phosphotyrosine was detected by PY99 in Western blotting. Reblotting with anti-syk mAb was done after stripping to reveal equal loading. Data are representative of two (A) or three (B) similar experiments.

The localization of Mac-1 and F-actin at the cell-cell adhesion site induced by co-cross-linking of FcRIII and CD9

To investigate the contribution of FcR to the colocalization of CD9, F-actin, FcRIIB/III, and Mac-1 at the cell-cell adhesion site (Figs. 6 and 7), we examined the localization of these proteins by immunostaining before or after stimulation with KMC8.8. In both wild-type and FcRIIB^-/- bone marrow macrophages, CD9, F-actin, FcRIIB/III (in wild type), or FcRIII (in FcRIIB^-/-) and Mac-1 were colocalized at the cell-cell adhesion site after stimulation (Fig. 9, A and C, arrow). However, in FcR -chain^-/- and FcRIII^-/- bone marrow macrophages, CD9 and FcRIIB were colocalized at the cell-cell adhesion site, but F-actin and Mac-1 were not (Fig. 9, B and D, arrow). These results suggest that FcRIII is crucial for the localization of F-actin and Mac-1 at the cell-cell adhesion site after stimulation with KMC8.8. In addition, it is possible that FcRIIB can transduce some signal by cross-linking with CD9, because FcRIIB and CD9 are colocalized at the cell-cell adhesion site in FcRIII-deficient bone marrow macrophages.

View larger version (64K): [in this window] [in a new window]   FIGURE 9. Localization of Mac-1 and F-actin at the cell-cell adhesion site induced by co-cross-linking of FcRIII and CD9. A–D, Bone marrow macrophages from wild-type (A), FcR -chain-/- (B), FcRIIB-/- (C), and FcRIII-/- (D) mice were stimulated with anti-CD9 mAb KMC8.8 for 5 min at 37°C. Before or after stimulation with the Ab, CD9 and F-actin (A--D), FcRIIB/III and F-actin (A), FcRIIB and F-actin (B and D), FcRIII and F-actin (C), Mac-1, and CD9 (A--D) were stained with Abs and reagents described as in Materials and Methods. Right panels present the superimposed images of the two left panels, respectively. Arrows indicate localization of each molecule detected with each Ab or the reagent at the cell-cell adhesion site. Data are representative of three similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although syk was not phosphorylated after incubation with KMC8.8 in FcRIII-deficient macrophages, colocalization of FcRIIB and CD9 was detected in these cells, suggesting that KMC8.8 can co-cross-link both FcRIIB with CD9 as well as FcRIII. However, we could not detect the physical interaction of these molecules with CD9 in immunoprecipitation experiments using each detergent, Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, or Briji98 (data not shown).

Besides the association of FcRIIB/III and CD9, it seems that there is a functional association between FcRI and CD9, as co-cross-linking with an anti-CD9 F(ab')₂ followed by mouse anti-rat Fab Abs induced phosphorylation of syk in macrophages from FcRIII^-/- but not from FcR -chain^-/- mice. These results suggest that CD9 functionally associates with all FcRs and modifies the signals.

Our data suggest that CD9 may be involved in FcR-mediated phagocytosis and enhance or modify it like Mac-1/CR-3, a major receptor in the phagocytosis of complement-opsonized targets (36, 37). Moreover, in our preliminary experiments, colocalization of FcR and CD9 was observed in the phagosome when IgG-opsonized particles were engulfed (data not shown). Recently, it has been demonstrated that the small G proteins Cdc42/Rac and Rho are necessary for FcR- and Mac-1-dependent phagocytosis, respectively (38), and the cross-talk of signal transduction among the small G proteins has been described (39). The excessive filopodium extension and concentration of F-actin at the cell-cell adhesion site induced by the CD9-FcR co-cross-linking are thought to be a result of the activation of the small G proteins, which regulates the actin cytoskeleton. In fact, CD9 has been reported to be associated with small G proteins (40). The CD9 and FcR co-cross-linking may specifically enhance the signals of small G proteins downstream of FcR, and the machinery could be involved in the FcR-Mac-1-dependent phagocytosis. We demonstrated the possibility that CD9 could work functionally in associating with FcR in DIGs in J774 cells like CD9 in T cells behaving as a coreceptor of TCR in DIGs (13). In some cells, co-cross-linking between surface molecules is constitutively present in DIGs and ITAM-containing immunoreceptors, which induces recruitment to lipid raft and activation of the receptors. However, these phenomena may include some artifacts and do not reflect the biological function of these molecules. Concerning CD9 on bone marrow macrophages, MCD does not cancel the activation with KMC8.8 out of accord with the experiment carried with J774 cells under the same condition (data not shown), so it is obscure whether DIGs contribute to the activation induced with KMC8.8. Therefore, further investigation is required.

We and others have shown that CD9-deficient mice exhibit defective cell fusion between sperm and eggs, but no significant immune system defects were observed (18, 19, 20). We revealed that the structure of microvilli on the egg plasma membrane seems to capture the sperm before fertilization evokes phagocytosis and that CD9 is localized at the sperm-egg adhesion site (20). Sperm-egg fusion must require tight adherence of the egg plasma membrane to the sperm, likely being mediated by the reorganization of the actin cytoskeleton. We suggest that CD9 functions in this process.

In vivo, the reorganization of the actin cytoskeleton after recognition of IgG immune complexes by FcR is extremely important, not only for internalization of the complex, but also for activation or stabilization of adhesion molecules, which lead to chemotaxis and invasion of the inflammatory site by monocytes/macrophages. CD9 has been reported to be involved in cell adhesion and motility in various types of cells, both of which require the reorganization of the cytoskeleton. Our findings demonstrate that co-cross-linking of CD9 and FcR activates macrophages; therefore, CD9 may play a role when FcRs function in infection and inflammation on macrophages. The detailed function of CD9 on macrophages in vivo will be investigated in Ag-stimulated CD9-deficient mice.

	Acknowledgments

 
We thank Drs. Christopher Paige and Heather Fleming for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by a grant-in-aid for scientific research from the Japan Ministry of Education, Science, Sports and Culture.

² Address correspondence and reprint requests to Dr. Akira Kudo, Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.

³ Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; TM4SF, transmembrane 4 superfamily; DIGs, detergent-insoluble glycolipid membranes; CTx, cholera toxin; MAPK, mitogen-activated protein kinase; MCD, methyl--cyclodextrin; BSS, buffer saline solution.

Received for publication July 18, 2000. Accepted for publication December 19, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ravetch, J., J.-P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[Medline]
2. Gessner, J., H. Hiken, A. Tamm, R. E. Schmidt. 1998. The IgG Fc receptor family. Ann. Hematol. 76:231.[Medline]
3. Deo, Y., F. M., R. F. Robert, R. Graziano, R. Repp, G. J. van de Winkel. Jan. 1997. Clinical significance of IgG Fc receptors and FcR-directed immunotherapies. Immunol. Today 18:127.[Medline]
4. Indik, Z., J. G. K., J. G. Park, Hunter S, A. D. Schreiber. 1995. The molecular dissection of Fc receptor mediated phagocytosis. Blood 86:4389.[Abstract/Free Full Text]
5. Strzelecka, A., K. Kwiatkowska, A. Sobota. 1997. Tyrosine phosphorylation and Fc receptor-mediated phagocytosis. FEBS Lett. 400:11.[Medline]
6. Rose, D., B. W. M., E. D. Winston, D. W. Chan, P. Riches, G. L. Gerwins, G. L. Johnson, P. M. Henson. 1997. Fc receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc receptor-stimulated TNF- synthesis. J. Immunol. 158:3433.[Abstract]
7. Daeron, M.. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203.[Medline]
8. Wright, M. D., M. G. Tomlinson. 1994. The ins and outs of the transmembrane 4 superfamily. Immunol. Today 15:588.[Medline]
9. Hemler, M., B. A. E., B. A. Mannion, F. Berditchevski. 1996. Association of TM4SF proteins with integrins: relevance to cancer. Biochim. Biophys. Acta 1287:67.[Medline]
10. Gachet, C., A. Astier, H. de la Salle, C. de la Salle, W. H. Fridman, J. P. Cazenave, D. Hanau, J. L. Teillaud. 1995. Release of FcRIIa2 by activated platelets and inhibition of anti-CD9-mediated platelet aggregation by recombinant FcRIIa2. Blood 85:698.[Abstract/Free Full Text]
11. Slupsky, J., J. C. R., C. Cawley, C. Kaplan, M. Zuzel. 1997. Analysis of CD9, CD32 and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation. Br. J. Haematol. 96:275.[Medline]
12. Kuroda, K., Y. Ozaki, R. Qi, N. Asazuma, Y. Yatomi, K. Satoh, S. Nomura, M. Suzuki, S. Kume. 1995. Fc II receptor-mediated platelet activation induced by anti-CD9 monoclonal antibody opens Ca²⁺ channels which are distinct from those associated with Ca²⁺ store depletion. J. Immunol. 155:4427.[Abstract]
13. Yashiro-Ohtani, Y., X. Y. Zhou, K. Toyo-Oka, X. G. Tai, C. S. Park, T. Hamaoka, R. Abe, K. Miyake, H. Fujiwara. 2000. Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts. J. Immunol. 164:1251.[Abstract/Free Full Text]
14. Horvath, G., V. Serru, D. Clay, M. Billard, C. Boucheix, E. Rubinstein. 1998. CD19 is linked to the integrin-associated tetraspans CD9, CD81, and CD82. J. Biol. Chem. 273:30537.[Abstract/Free Full Text]
15. Tachibana, I., M. E. Hemler. 1999. Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance. J. Cell Biol. 146:893.[Abstract/Free Full Text]
16. Banerjee, S., M. A., M. Hadjiargyrou, P. H. Patterson. 1997. An antibody to the tetraspan membrane protein CD9 promotes neurite formation in a partially ₃₁ integrin-dependent manner. J. Neurosci. 17:2756.[Abstract/Free Full Text]
17. Chen, M. S., K. S. Tung, S. A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P. W. Kincade, J. C. Herr, J. M. White. 1999. Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin ₆₁: implications for murine fertilization. Proc. Natl. Acad. Sci. USA 96:11830.[Abstract/Free Full Text]
18. Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, C. Boucheix. 2000. Severely reduced female fertility in CD9-deficient mice. Science 287:319.[Abstract/Free Full Text]
19. Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Suzuki, K. Kosai, K. Inoue, A. Ogura, et al 2000. Requirement of CD9 on the egg plasma membrane for fertilization. Science 287:321.[Abstract/Free Full Text]
20. Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, A. Kudo. 2000. The gamete fusion process is defective in eggs of CD9-deficient mice. Nat. Genet. 24:279.[Medline]
21. Masellis-Smith, A., G. S. Jensen, J. G. Seehafer, J. R. Slupsky, A. R. Shaw. 1990. Anti-CD9 monoclonal antibodies induce homotypic adhesion of pre-B cell lines by a novel mechanism. J. Immunol. 144:1607.[Abstract]
22. Hadjiargyrou, M., P. H. Patterson. 1995. An anti-CD9 monoclonal antibody promotes adhesion and induces proliferation of Schwann cells in vitro. J. Neurosci. 15:574.[Abstract]
23. Ikeyama, S., M. Koyama, M. Yamaoko, R. Sasada, M. Miyake. 1993. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. J. Exp. Med. 177:1231.[Abstract/Free Full Text]
24. Oritani, K., X. Wu, K. Medina, J. Hudson, K. Miyake, J. M. Gimble, S. A. Burstein, P. W. Kincade. 1996. Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures. Blood 87:2252.[Abstract/Free Full Text]
25. Azuma, Y., K. Kaji, R. Katogi, S. Takeshita, A. Kudo. 2000. Tumor necrosis factor- induces differentiation of and bone resorption by osteoclasts. J. Biol. Chem. 275:4858.[Abstract/Free Full Text]
26. Takeshita, S., K. Kaji, A. Kudo. 2000. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J. Bone Miner. Res. 15:1477.[Medline]
27. Kawaguchi, J., S. Takeshita, T. Kashima, T. Imai, R. Machinami, A. Kudo. 1999. Expression and function of the splice variant of the human cadherin-11 gene in subordination to intact cadherin-11. J. Bone Miner. Res. 14:764.[Medline]
28. Sheets, E., D. D., D. Holowka, B. Baird. 1999. Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcRI and their association with detergent-resistant membranes. J. Cell Biol. 17:877.
29. Viola, A., S. Schroeder, Y. Sakakibara, A. Lanzavecchia. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283:680.[Abstract/Free Full Text]
30. Takai, T., M. Li, D. Sylvestre, R. Clynes, J. V. Ravetch. 1994. FcR chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
31. Sutterwala, F., G. J. S., P. Noel, P. Salgame, D. M. Mosser. 1998. Reversal of proinflammatory responses by ligating the macrophage Fc receptor type I. J. Exp. Med. 188:217.[Abstract/Free Full Text]
32. Sanchez-Mejorada, G., C. Rosales. 1998. Fc receptor-mediated mitogen-activated protein kinase activation in monocytes is independent of Ras. J. Biol. Chem. 273:27610.[Abstract/Free Full Text]
33. Sakuma, T., S. Higashiyama, S. Hosoe, S. Hayashi, N. Taniguchi. 1997. CD9 antigen interacts with heparin-binding EGF-like growth factor through its heparin-binding domain. J. Biochem. 122:474.[Abstract/Free Full Text]
34. Lebel-Binay, S., C. Lagaudriere, D. Fradelizi, H. Conjeaud. 1995. CD82, tetra-span-transmembrane protein, is a regulated transducing molecule on U937 monocytic cell line. J. Leukocyte Biol. 57:956.[Abstract]
35. Ozaki, Y., K. Satoh, K. Kuroda, R. Qi, Y. Yatomi, S. Yanagi, K. Sada, H. Yamamura, M. Yanabu, S. Nomura, et al 1995. Anti-CD9 monoclonal antibody activates p72syk in human platelets. J. Biol. Chem. 270:15119.[Abstract/Free Full Text]
36. Brown, E., J. F. J., J. F. Bohnsack, H. D. Gresham. 1988. Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen. J. Clin. Invest. 81:365.
37. Graham, I. L., H. D. Gresham, E. J. Brown. 1989. An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors. J. Immunol. 142:2352.[Abstract]
38. Caron, E., A. Hall. 1998. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 282:1717.[Abstract/Free Full Text]
39. Nobes, C. D., A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53.[Medline]
40. Seehafer, J. G., A. R. Shaw. 1991. Evidence that the signal-initiating membrane protein CD9 is associated with small GTP-binding proteins. Biochem. Biophys. Res. Commun. 179:401.[Medline]

This article has been cited by other articles:

				W M Liu, Y J Cao, Y J Yang, J Li, Z Hu, and E-K Duan Tetraspanin CD9 regulates invasion during mouse embryo implantation J. Mol. Endocrinol., February 1, 2006; 36(1): 121 - 130. [Abstract] [Full Text] [PDF]

				P. Hewins, J. M. Williams, M. J.O. Wakelam, and C. O.S. Savage Activation of Syk in Neutrophils by Antineutrophil Cytoplasm Antibodies Occurs via Fc{gamma} Receptors and CD18 J. Am. Soc. Nephrol., March 1, 2004; 15(3): 796 - 808. [Abstract] [Full Text] [PDF]

				B. N. Gantner, R. M. Simmons, S. J. Canavera, S. Akira, and D. M. Underhill Collaborative Induction of Inflammatory Responses by Dectin-1 and Toll-like Receptor 2 J. Exp. Med., May 5, 2003; 197(9): 1107 - 1117. [Abstract] [Full Text] [PDF]

				R. Waterhouse, C. Ha, and G. S. Dveksler Murine CD9 Is the Receptor for Pregnancy-specific Glycoprotein 17 J. Exp. Med., January 22, 2002; 195(2): 277 - 282. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaji, K. Articles by Kudo, A. Search for Related Content PubMed PubMed Citation Articles by Kaji, K. Articles by Kudo, A.