The Binding Site for TRAF2 and TRAF3 but Not for TRAF6 Is Essential for CD40-Mediated Immunoglobulin Class Switching

AbstractTo define the role of TRAF proteins in CD40-dependent isotype switching in B cells, we introduced wild-type (WT) and mutant CD40 transgenes that lacked the binding motifs for TRAF6 (CD40ΔTRAF6), TRAF2 and TRAF3 (CD40ΔTRAF2/3), or both (CD40ΔTRAFs) into B cells of CD40−/− mice. The in vivo isotype switch defect in CD40−/− mice was fully corrected by WT and CD40ΔTRAF6, partially by CD40ΔTRAF2/3, and not at all by CD40ΔTRAFs transgenes. CD40-mediated isotype switching, proliferation, and activation of p38, JNK, and NFκB in B cells were normal in WT and CD40ΔTRAF6 mice, severely impaired in CD40ΔTRAF2/3, and absent in CD40ΔTRAFs mice. These results suggest that binding to TRAF2 and/or TRAF3 but not TRAF6 is essential for CD40 isotype switching and activation in B cells

WIP Regulates Signaling via the High Affinity Receptor for Immunoglobulin E in Mast Cells

Wiskott-Aldrich syndrome protein–interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcεRI) in mast cells. WIP-deficient bone marrow–derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcεRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcεRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcεRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcεRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement

Structural Requirements of SLP-76 in Signaling via the High-Affinity Immunoglobulin E Receptor (FcɛRI) in Mast Cells

The adapter SLP-76 plays an essential role in FcɛRI signaling, since SLP-76(−/−) bone marrow-derived mast cells (BMMC) fail to degranulate and release interleukin-6 (IL-6) following FcɛRI ligation. To define the role of SLP-76 domains and motifs in FcɛRI signaling, SLP-76(−/−) BMMC were retrovirally transduced with SLP-76 and SLP-76 mutants. The SLP-76 N-terminal and Gads binding domains, but not the SH2 domain, were critical for FcɛRI-mediated degranulation and IL-6 secretion, whereas all three domains are essential for T-cell proliferation following T-cell receptor (TCR) ligation. Unexpectedly, the three tyrosine residues in SLP-76 critical for TCR signaling, Y112, Y128, and Y145, were not essential for IL-6 secretion, but were required for degranulation and mitogen-activated protein kinase activation. Furthermore, a Y112/128F SLP-76 mutant, but not a Y145F mutant, strongly reconstituted mast cell degranulation, suggesting a critical role for Y145 in FcɛRI-mediated exocytosis. These results point to important differences in the function of SLP-76 between T cells and mast cells

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children

BACKGROUND The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. METHODS We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. RESULTS Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response.National Institutes of HealthAugust 4, 2016. 6 Month Embargo.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]