CRACM1 (Orai1) constitutes the pore subunit of CRAC channels that are crucial for many physiological processes 1-6 . A point mutation in CRACM1 has been associated with SCID disease in humans 2 . We have generated CRACM1 deficient mice using gene trap, where β-galactosidase (LacZ) activity identifies CRACM1 expression in tissues. We show here that the homozygous CRACM1 deficient mice are considerably smaller in size and are grossly defective in mast cell degranulation and cytokine secretion. FcεRI-mediated in vivo allergic reactions were also inhibited in CRACM1-/-mice. Other tissues expressing truncated CRACM1-LacZ fusion protein include skeletal muscles, kidney and regions in the brain and heart. Surprisingly, no CRACM1 expression was seen in the lymphoid regions of thymus. Accordingly, we found no defect in T cell development. Thus, our data reveal novel crucial roles for CRAC channels including a putative role in excitable cells.

We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM1O, all directed against the C3bi receptor of human monocytes and macrophages (M4). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on MO adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from MO lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM1O binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from '251-surface-labeled M4. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human MO is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.The third component of complement, C3, binds covalently to cell surfaces (1) yielding a species, C3b, that is recognized by receptors on leukocytes (2). Surface-bound C3b is rapidly cleaved by the serum enzyme, I, to yield an altered form, C3bi, that is also recognized by receptors on leukocytes (3, 4). There are separate receptors for C3b and C3bi on human monocytes, and it has been shown that each type of receptor can independently mediate phagocytosis of C3b-or C3bi-coated particles (5). We are particularly interested in the receptors for C3 because their ability to promote phagocytosis is regulated: human macrophages (MO) bind but do not ingest C3b-or C3bi-coated erythrocytes, but MO readily ingest both C3b-and C3bi-coated erythrocytes after a brief treatment with the tumor-priomoting compound phorbol myristate acetate (5).Fearon (6) (8). Monoclonal antibodies against the human C3b receptor (antiC3bR) have been described (9), as have monoclonal antibodies, OKM1, OKM9, OKM10, and OKM5 (10). Fab fragments of 1B4 were prepared by papain digestion (11).Cells. Human blood monocytes were purified and cultured for 5-8 days in Teflon beakers as described (5). Cultured monocytes (MO) were surface labeled with 1"I by the lactoperoxidase-glucose oxidase procedure (12).Sheep erythrocytes bearing C3b (EC3b) or C3bi (EC3bi) were prepared as described (5). The attachment of EC3b or EC3bi to monolayers of phagocytes was scored visually in duplicate wells (5). T...

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1 ␣ , which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues. ( J. Clin. Invest. 1997. 100:1137-1143.)

No abstract

T cells with immunoregulatory function have been described in human and mouse systems. In both systems these cells can be differentiated either in the thymus or from peripheral T cells. To date, more progress has been made in the study of murine regulatory T cells, because it has been very difficult to isolate human regulatory T cells of sufficient purity and in sufficient numbers to permit detailed examinations of their biochemistry. We report in this study that human T cells with regulatory function can be differentiated in vitro from naive (CD4+CD45RA+) cord blood or peripheral T cells by stimulation with anti-CD3 and anti-CD28 in the presence of TGF-β. Cells derived in this manner express a surface phenotype (CD25+, CD122+, HLA-DR+, glucocorticoid-induced TNF receptor-related gene+, CD103+, CTLA-4+) described for human and mouse regulatory T cells and express protein and message for the transcription factor forkhead/winged helix transcription factor (FOXP3). They produce primarily TGF-β and IL-10, with lesser amounts of IFN-γ and IL-13, when stimulated through their TCRs and are capable of inhibiting cytokine production and proliferation by stimulated naive T cells. Unlike Th1 and Th2 cells, these TGF-β-derived regulatory T cells do not appear to be dependent on the protein kinase Cθ pathway of NF-κB activation for Ag-induced responses.