The cell-triggering properties of BCR, TCR and FcR depend on structurally related immunoreceptor tyrosine-based activation motifs (ITAMs). Fc gamma RIIB have no ITAM and do not trigger cell activation. When coaggregated to BCR, they inhibit B cell activation. We show here that, when coaggregated to these receptors, Fc gamma RIIB inhibit Fc epsilon RI-, Fc gamma RIIA-, and TCR-dependent cell activation. Inhibition also affected cell activation by single ITAMs, in isolated FcR or TCR subunits. The same tyrosine-based inhibitory motif (ITIM), which is highly conserved in murine and human Fc gamma RIIB and that was previously shown to inhibit BCR-dependent B cell activation, was required to regulate TCR- and FcR-dependent cell activation. Our findings endow Fc gamma RIIB, and thus IgG antibodies, with general immunoregulatory properties susceptible to act on all ITAM-containing receptors.

Invest. 1995. 95:577-585.)

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli.

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcγRIIIA and were negatively regulated by FcγRIIB. We found that a moderate FcγRIIB-dependent negative regulation, due not to a higher FcγRIIIA/FcγRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

Mast cells are effector cells of the innate immune system, but because they express Fc receptors (FcRs), they can be engaged in adaptive immunity by antibodies. Mast cell FcRs include immunoglobulin E (IgE) and IgG receptors and, among these, activating and inhibitory receptors. The engagement of mast cell IgG receptors by immune complexes may or may not trigger cell activation, depending on the type of mast cell. The coengagement of IgG and IgE receptors results in inhibition of mast cell activation. The Src homology-2 domain-containing inositol 5-phosphatase-1 is a major effector of negative regulation. Biological responses of mast cells depend on the balance between positive and negative signals that are generated in FcR complexes. The contribution of human mast cell IgG receptors in allergies remains to be clarified. Increasing evidence indicates that mast cells play critical roles in IgG-dependent tissue-specific autoimmune diseases. Convincing evidence was obtained in murine models of multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, and glomerulonephritis. In these models, the intensity of lesions depended on the relative engagement of activating and inhibitory IgG receptors. In vitro models of mature tissue-specific murine mast cells are needed to investigate the roles of mast cells in these diseases. One such model unraveled unique differentiation/maturation-dependent biological responses of serosal-type mast cells.

Immunoreceptor tyrosine-based inhibition motifs (ITIMs) 1 are present in the intracytoplasmic (IC) domains of a large group of transmembrane molecules that negatively regulate cell activation induced by receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs) (1). Fc␥RIIB, a subgroup of Fc receptors that bind IgG complexes (2), and killer cell Ig-like receptors with a long IC domain (KIRLs), which bind major histocompatibility complex class I molecules (3), are two prototypes of ITIM-bearing receptors. Fc␥RIIB exist as two (Fc␥RIIB1 and B2 in humans) or three (Fc␥RIIB1, B1Ј, and B2 in mice) alternatively spliced products of a single gene. Fc␥RIIB were shown to negatively regulate cell activation induced by all ITAM-bearing immunoreceptors (4) and to control the magnitude of both antibody responses and anaphylactic reactions (5). Fc␥RIIB were also recently found to negatively regulate cell proliferation induced by growth factor receptors with an intrinsic protein-tyrosine kinase activity (6). KIRLs are polymorphic molecules with two (KIR2DLs) or three (KIR3DLs) Ig-like extracellular (EC) domains encoded by related but distinct genes. KIRLs inhibit NK and T cell-mediated cytotoxicity (7,8). Negative regulation exerted by Fc␥RIIB (9) and KIR2DL3 (10) was shown to require their coaggregation with ITAM-bearing receptors by extracellular ligands. Fc␥RIIB possess one ITIM, while KIR2DL3 possess two ITIMs.ITIMs are constituted by a tyrosine, preceded by an isoleucine, valine, or leucine at position YϪ2, and followed by a valine or leucine at position Yϩ3 (1, 11). Upon coaggregation of ITIMbearing receptors with ITAM-bearing receptors, ITIMs are tyrosyl-phosphorylated by Src family protein-tyrosine kinases (12, 13). Phosphorylated ITIMs (pITIMs) then recruit cytoplasmic phosphatases containing Src homology 2 (SH2) domains that interfere with signals transduced by ITAM-bearing receptors (14 -16). These include the two-SH2 domain-containing protein-tyrosine phosphatases SHP-1 (14, 17) and SHP-2 (18) and the single-SH2 domain-containing inositol 5-phosphatases SHIP1 (19) and SHIP2 (20). SHP-1 is thought to dephosphorylate tyrosines in ITAMs, protein-tyrosine kinases, and/or adapter proteins whose phosphorylation is critical for activation signals, thereby stopping the initial steps of transduction. SHP-1 was recently found to inhibit the redistribution of cholesterol/sphyngolipid-rich membrane lipid microdomains following the engagement of a KIR3DL, on NK cells, by major histocompatibility complex class I molecules on target cells (21). The possible role of SHP-2 is not clear, because both positive and negative effects have been assigned to this phosphatase (22-24). SHIP1 and SHIP2 remove 5-phosphate groups in inositol phosphates and phosphatidylinositol phosphates (25). The preferred substrate of SHIP1 is phosphatidylinositol 3,4,5-trisphosphate, which enables the membrane recruitment of the Bruton's tyrosine kinase via its pleckstrin homology domain (15, 16). Bruton's tyrosine kinase is manda-

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