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Glucocorticoid-induced TNFR (GITR) has been implicated as an essential regulator of immune responses to self tissues and pathogens. We have recently shown that GITR-induced cellular events promote survival of naive T cells, but are insufficient to protect against activation-induced cell death. However, the molecular mechanisms of GITR-induced signal transduction that influence physiologic and pathologic immune responses are not well understood. TNFR-associated factors (TRAFs) are pivotal adapter proteins involved in signal transduction pathways of TNFR-related proteins. Yeast two-hybrid assays and studies in HEK293 cells and primary lymphocytes indicated interactions between TRAF2 and GITR mediated by acidic residues in the cytoplasmic domain of the receptor. GITR-induced activation of NF-kappaB is blocked by A20, an NF-kappaB-inducible protein that interacts with TRAFs and functions in a negative feedback mechanism downstream of other TNFRs. Interestingly, in contrast with its effects on signaling triggered by other TNFRs, our functional studies revealed that TRAF2 plays a novel inhibitory role in GITR-triggered NF-kappaB activation.

Glucocorticoid-induced TNFR (GITR) is a member of the TNFR family that can inhibit the suppressive function of regulatory T cells and promote the survival and activation of T cells. However, little is known about the molecular mechanisms regulating T cell survival and activation downstream of GITR. To gain further insight into the cellular events and signaling pathways triggered by GITR, survival, proliferation, and cytokine production as well as activation of MAPKs and NF-kappaB were monitored after cross-linking of the receptor on naive and activated T cells. GITR cross-linking provided costimulation of naive and activated T cells and resulted in activation of MAPKs and NF-kappaB. Although GITR-induced signaling pathways augmented the survival of naive T cells, they were not sufficient to inhibit activation-induced cell death triggered by CD3 cross-linking of activated T cells. Differences in the contributions of GITR to cell survival between naive and activated T cells suggest that the receptor triggers specific pathways depending on the activation state of the T cell.

Tissue injury triggers inflammatory responses that may result in release of degradation products or exposure of cryptic domains of extracellular matrix components. Previously, we have shown that a cryptic peptide (AQARSAASKVKVSMKF) in the alpha-chain of laminin-10 (alpha5beta1gamma1), a prominent basement membrane component, is chemotactic for both neutrophils (PMNs) and macrophages (Mphis) and induces matrix metalloproteinase-9 (MMP-9) production. To determine whether AQARSAASKVKVSMKF has additional effects on inflammatory cells, we performed microarray analysis of RNA from RAW264.7 Mphis stimulated with AQARSAASKVKVSMKF. Several cytokines and cytokine receptors were increased >3-fold in response to the laminin alpha5 peptide. Among these were TNF-alpha and one of its receptors, the p75 TNFR (TNFR-II), increasing 3.5- and 5.7-fold, respectively. However, the peptide had no effect on p55 TNFR (TNFR-I) expression. Corroborating the microarray data, the protein levels of TNF-alpha and TNFR-II were increased following stimulation of RAW264.7 cells with AQARSAASKVKVSMKF. In addition, we determined that the production of TNF-alpha and TNFR-II in response to AQARSAASKVKVSMKF preceded the production of MMP-9. Furthermore, using primary Mphis from mice deficient in TNFR-I, TNFR-II, or both TNF-alpha receptors (TNFRs), we determined that AQARSAASKVKVSMKF induces MMP-9 expression by Mphis through a pathway triggered by TNFR-II. However, TNF-alpha signaling is not required for AQARSAASKVKVSMKF-induced PMN release of MMP-9 or PMN emigration. These data suggest that interactions of inflammatory cells with basement membrane components may orchestrate immune responses by inducing expression of cytokines, recruitment of inflammatory cells, and release of proteinases.