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Crosslinking of FceRI-bound IgE triggers the release of a large number of biologically active, potentially anaphylactic compounds by mast cells. FceRI activation ought to be well-controlled to restrict adverse activation. As mast cells are embedded in tissues, adhesion molecules may contribute to limiting premature activation. Here, we report that E-Cadherin serves that purpose. Having confirmed that cultured mast cells express E-Cadherin, a mast-cell-specific E-Cadherin deficiency, Mcpt5-Cre E-Cdhfl/fl mice, was used to analyze mast cell degranulation in vitro and in vivo. Cultured peritoneal mast cells from Mcpt5-Cre E-Cdhfl/fl mice were normal with respect to many parameters but showed much-enhanced degranulation in three independent assays. Soluble E-Cadherin reduced the degranulation of control cells. The release of some newly synthesized inflammatory cytokines was decreased by E-Cadherin deficiency. Compared to controls, Mcpt5-Cre E-Cdhfl/fl mice reacted much stronger to IgE-dependent stimuli, developing anaphylactic shock. We suggest E-Cadherin-mediated tissue interactions restrict mast cell degranulation to prevent their precocious activation.

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cell activates signaling pathways that trigger degranulation and the release of multiple pro-inflammatory mediators. Mature,immature and precursor mast cells are degranulation competent. We show here that the signaling protein SWAP-70 has a function in mast cell biology. While not found in many cell types, we find that apart from B cells, mast cells also express SWAP-70. In activated B cells, SWAP-70 shuttles between cytoplasm and nucleus, but in mast cells it is confined to the cytoplasm. SWAP-70(ko/ko) (double knockout) mice have reduced numbers of mature mast cells, and these are degranulation competent. However, although immature mast cells from SWAP-70(ko/ko) mice respond normally to SCF and IL-3 and have functional granules, their FcepsilonRI-mediated degranulation is inhibited. Thus, in mast cells SWAP-70 plays a role both in establishing the initial competence to degranulate and to develop into mature mast cells.

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.

The generation of plasma cells (PCs) is key for proper humoral immune responses. The transcription factors IRF-4 and BLIMP-1 (B-lymphocyte induce maturation protein-1) control PC commitment, but the underlying regulatory mechanisms are incompletely understood. Here we have identified SWAP-70 as being critically involved in Toll-like receptor (TLR)-triggered PC differentiation. Upon activation through various TLRs, Swap-70(-/-) B cells were activated and proliferated normally. However, expression of BLIMP-1 was markedly reduced and PC differentiation was impaired. Four hours of LPS stimulation were sufficient to drive PC differentiation, and SWAP-70 was required during this initial period. Swap-70(-/-) B cells pre-activated in vitro failed to efficiently differentiate into PCs upon adoptive transfer into recipient mice. Re-introduction of SWAP-70 into Swap-70(-/-) B cells rescued their development into PCs, and SWAP-70 over-expression in wild-type (WT) B cells increased PC generation. In the absence of SWAP-70, IRF-4 protein levels were reduced and the IRF-4(high) B220(+) CD138(-) compartment, including PC precursors, was strongly diminished. Ectopic expression of SWAP-70 increases IRF-4 protein levels and PC differentiation in WT and Swap-70(-/-) B cells, and IRF-4 over-expression in Swap-70(-/-) B cells elevates PC differentiation to WT levels. Thus, in a dose-dependent manner, SWAP-70 controls IRF-4 protein expression and thereby regulates the initiation of PC differentiation.

T1 and T2 transitional B cells are precursors for marginal zone B cells (MZB), which surround splenic follicles. MZB are essential for marginal zone formation, are central to the innate immune response, and contribute to adaptive immunity. Differentiation, migration, and homing of MZB and their precursors remain to be fully understood. We show that SWAP-70, a RhoGTPase-interacting and F-actin-binding protein with functions in cell polarization, migration, and adhesion regulates MZB development and marginal zone formation. The percentage of MZB in spleen of Swap70(-/-) mice was reduced to about one-third of that found in WT mice. Swap70(-/-) T1 cells accumulated in integrin ligand(high) regions of the splenic red pulp and failed to efficiently develop into T2 cells. Adoptive transfer and mixed BM chimera experiments demonstrated this to be a B-cell intrinsic phenotype. T-cell-independent antibody production was not impaired, however, and thus suggests that this process does not require correct homing of MZB precursors. B-cell adhesion through α(L)β(2) and α(4)β(1) integrins was hyper-activated in vitro and on tissue sections, and S1P-stimulated chemokinesis of MZB was reduced in the absence of SWAP-70. Thus, SWAP-70 acts as a regulator of the adhesion process, particularly important for differentiation control of B-cell precursors and their contribution to splenic tissue formation.

Mast cells, perhaps best known by their ability to trigger allergic reactions after stimulation through the FcepsilonRI, express the unusual phosphatidylinositol 3-kinase (PI3K)-dependent, Rac-binding protein SWAP-70. Here, we show that the IgE-mediated passive cutaneous and the systemic anaphylactic responses are strongly reduced in SWAP-70(-/-) mice. Cultured SWAP-70(-/-) immature bone marrow mast cells (BMMC) are also impaired in FcepsilonRI-mediated degranulation, which can be restored by expression of exogenous wild-type SWAP-70, but less so if a phosphatidylinositol trisphosphate (PIP(3)) binding mutant is expressed. SWAP-70 itself supports inositol-3-phosphate and PIP(3) production, the latter indicating a potential feedback from SWAP-70 towards PI3K. FcepsilonRI-stimulated transcription and release of cytokines is controlled by SWAP-70. Key FcepsilonRI signal transduction events like activation of LAT by phosphorylation, activation of Akt/PKB and of p38 MAP kinase are reduced in SWAP-70(-/-) BMMC, but ERK is strongly hyperactivated. Some requirements for SWAP-70 were apparent only under limited-strength signaling conditions. We suggest that SWAP-70 defines a new element of efficient mast cell activation upon FcepsilonRI signaling, important for the control of mast cell-dependent anaphylaxis.

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cell activates signaling pathways that trigger degranulation and the release of multiple pro-inflammatory mediators. Mature,immature and precursor mast cells are degranulation competent. We show here that the signaling protein SWAP-70 has a function in mast cell biology. While not found in many cell types, we find that apart from B cells, mast cells also express SWAP-70. In activated B cells, SWAP-70 shuttles between cytoplasm and nucleus, but in mast cells it is confined to the cytoplasm. SWAP-70(ko/ko) (double knockout) mice have reduced numbers of mature mast cells, and these are degranulation competent. However, although immature mast cells from SWAP-70(ko/ko) mice respond normally to SCF and IL-3 and have functional granules, their FcepsilonRI-mediated degranulation is inhibited. Thus, in mast cells SWAP-70 plays a role both in establishing the initial competence to degranulate and to develop into mature mast cells.

Protein SWAP-70 was initially isolated from nuclei of activated B cells and was implicated in the immunoglobulin class switch process. After B cell activation the protein translocates from the cytoplasm to the nucleus, and may serve to signal nuclear processes. We have generated mice deficient in SWAP-70 and found three main differences when compared to wild-type mice: (i) their B lymphocytes are two- to threefold more sensitive to gamma-irradiation than B cells of wild type; (ii) SWAP-70-deficient mice developed autoantibodies at a much higher frequency; and (iii) the CD40 signaling pathway is compromised in the mutant mice. CD40-dependent switching to the IgE isotype is reduced five- to eightfold in vitro. In SWAP-70-deficient mice, IgE levels prior to immunization were six- to sevenfold lower than in wild-type mice, and after immunization three- to fourfold lower. CD40-induced proliferation was transiently increased in the mutant. LPS-induced switching to other isotypes, however, and LPS-induced proliferation were normal. We propose that SWAP-70 serves a specific role in the CD40 signaling pathway, in particular in the IgE response.