Functional association of CD9 with the Fc gamma receptors in macrophages

CD9, a member of the tetraspan family of proteins, is highly expressed on m acrophages. Although a clear function for the molecule has Set to be descri bed, we have found that the anti-CD9 mAb activates mouse macrophages, The r at anti-CD9 mAb, KMC8.8, but not the F(ab')(2), induced tyrosine phosphoryl ation of proteins including syk and chi and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and Fc gammaR was required far the signal. Co-cross-linking of CD9-Fc gamma R with KMC8.8 on macrophages from three different FcR-deficient mice, FcR g amma -chain(-/-), Fc gamma RIIB-/-, and Fc gamma RIII-/-, revealed that Fc gamma RIII is specific and crucial for syk phosphorylation, Although both K MC8.8 and the anti-Fc gamma RIIB/III mAb, 2.4G2, evoked similar phosphoryla tion patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 t reatment led to reduce levels of TNF-alpha production and p42/44 extracellu lar signal-related kinase phosphorylation relative to 2.462 stimulation. Im munofluorescence staining showed that co-cross-linking of CD9-Fc gammaR wit h KMC8.8 induced filopodium extension before cell aggregation, which was fo llowed by simultaneous colocalization of CD9, Fc gamma RIIB/III, Mac-1, ICA M-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of Fc gammaR-deficient macrophages revealed that the colocalization of CD9 , FcyRIII, Mac-1, and F-actin requires co-cross-linking of CD9-FcyRIII, whe reas co-cross-linking of CD9-Fc gamma RIIB induced the colocalization of on ly CD9 and Fc gamma RIIB, Our results demonstrate that co-cross-linking of CD9 and Fc gamma Rs activates macrophages; therefore, CD9 mag collaborate w ith FcRs functioning in infection and inflammation on maerophages.