The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-κB/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-κB/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-κB, IκBα. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-κB activity. Thus, BTK is essential for activation of NF-κB via the B cell receptor.

Treatment of small resting B cells with soluble F(ab')2 fragments of anti-IgM, an analogue of T-independent type 2 antigens, induced activation characterized by proliferation and the expression of surface CD5. In

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.

The expression of CD5 can be induced on murine B-2 cells by anti-IgM, a recognized analog of thymus-independent 2 type (TI-2) antigen. Given that cyclosporin A (CsA) sensitivity is a distinguishing feature of TI-2 type B cell activation, we asked whether the in vitro induction of CD5 on B cells by anti-mu is CsA sensitive. We report that anti-mu induced CD5 expression on B-2 cells was inhibited by CsA as well as FK-520 and rapamycin. When L-685,818, a FK-520 and rapamycin antagonist, was added to anti-mu stimulated B cell cultures containing FK-520 or rapamycin, but not CsA, suppression was abrogated and complete induction of CD5 was seen. When we used either CD4+CD8+ thymocytes or peripheral T cells activated by phorbol ester and ionomycin, the cell surface induction of CD5 was also partially blocked by CsA, FK-520 and rapamycin. Moreover, in both B and T cells, the same immunosuppressive drugs did not affect constitutive CD5 expression but only blocked de novo induction. To determine the level of CD5 regulation, we activated T cells using phorbol myristate acetate (PMA)/ionomycin and report that CD5 induction was sensitive to actinomycin D (AcD). Similarly, the induction of CD5 on anti-mu activated B cells was blocked by AcD. In addition, T cells that were activated by PMA/ionomycin expressed more abundant CD5 mRNA than CsA or FK-520 treated cells. Based on the CsA-sensitive regulation of CD5 we thought that the CsA-sensitive nuclear factor of activated T cells (NFAT) might be involved in CD5 regulation. We report evidence by Western blot analysis that NFATp is expressed by both resting and TI activated B cells but apparently not CD4+CD8+CD5+ thymocytes. We conclude that in both B and T cells the induction of CD5 requires transcriptional regulation, and that the inhibition of CD5 expression by the immunosuppressive drugs CsA, FK-520 and rapamycin requires drug-immunophilin complex formation.

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