Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcγ receptors I and II/III

The SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) adapter protein is expressed in T cells and myeloid cells, whereas its homologue BLNK (B cell linker protein) is expressed in B cells. SLP-76 and BLNK link immunoreceptor tyrosine-based activation motif-containing receptors to signaling molecules that include phospholipase C-γ, mitogen-activated protein kinases, and the GTPases Ras and Rho. SLP-76 plays a critical role in T cell receptor, FcɛRI and gpVI collagen receptor signaling, and participates in signaling via FcγR and killer cell inhibitory receptors. BLNK plays a critical role in B cell receptor signaling. We show that murine bone marrow-derived macrophages express both SLP-76 and BLNK. Selective ligation of FcγRI and FcγRII/III resulted in tyrosine phosphorylation of both SLP-76 and BLNK. SLP-76−/− bone marrow-derived macrophages display FcγR-mediated tyrosine phosphorylation of Syk, phospholipase C-γ2, and extracellular signal regulated kinases 1 and 2, and normal FcγR-dependent phagocytosis. These data suggest that both SLP-76 and BLNK are coupled to FcγR signaling in murine macrophages.

BLNK mediates Syk-dependent Btk activation

Btk is a critical molecule in B cell antigen receptor (BCR)-coupled signaling, and its activity is regulated by Lyn and Syk. Although the molecular mechanism of Lyn-dependent Btk activation has been investigated, that of Syk-dependent Btk activation has remained unidentified. We have demonstrated that BLNK mediates Syk-dependent Btk activation. In a reconstitution cell system, coexpression of BLNK allows Syk to phosphorylate Btk on its tyrosine 551, leading to the enhancement of Btk activity. This phosphorylation depends on the interaction of Btk and BLNK by means of the Btk-Src homology 2 domain. The existence of such an activation mechanism is supported by the observation that the BCR-induced Btk phosphorylation and activation are significantly reduced in BLNK-deficient B cells as well as in Syk-deficient B cells. Although previous observations have identified the function of BLNK as the linker that integrates the action of Btk and Syk into downstream effectors such as phospholipase Cγ2, our present study indicates another function of BLNK that connects the activity of Syk to that of Btk.

Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells.

Mouse bone marrow-derived mast cells (BMMCs) developed with interleukin 3 (IL-3) can be stimulated by c-kit ligand (KL) and accessory cytokines over a period of hours for direct delayed prostaglandin (PG) generation or over a period of days to prime for augmented IgE-dependent PG and leukotriene (LT) production, as previously reported. We now report that IL-4 is counterregulatory for each of these distinct KL-dependent responses. BMMCs cultured for 4 days with KL + IL-3 or with KL + IL-10 produced 5- to 7-fold more PGD2 and approximately 2-fold more LTC4 in response to IgE-dependent activation than BMMCs maintained in IL-3 alone. IL-4 inhibited the priming for increased IgE-dependent PGD2 and LTC4 production to the level obtained by activation of BMMCs maintained in IL-3 alone with an IC50 of approximately 0.2 ng/ml. IL-4 inhibited the KL-induced increase in expression of cytosolic phospholipase A2 (cPLA2) but had no effect on the incremental expression of PG endoperoxide synthase 1 (PGHS-1) and hematopoietic PGD2 synthase or on the continued baseline expression of 5-lipoxygenase, 5-lipoxygenase activating protein, and LTC4 synthase. BMMCs stimulated by KL + IL-10 for 10 h exhibited a delayed phase of PGD2 generation, which was dependent on de novo induction of PGHS-2. IL-4 inhibited the induction of PGHS-2 expression and the accompanying cytokine-initiated delayed PGD2 generation with an IC50 of approximately 6 ng/ml. IL-4 had no effect on the expression of PGHS-2 and the production of PGD2 elicited by addition of IL-1 beta to the combination of KL + IL-10. IL-4 had no effect on the immediate phase of eicosanoid synthesis elicited by KL alone or by IgE and antigen in BMMCs maintained in IL-3. Thus, the counterregulatory action of IL-4 on eicosanoid generation is highly selective for the induced incremental expression of cPLA2 and the de novo expression of PGHS-2, thereby attenuating time-dependent cytokine-regulated responses to stimulation via Fc epsilon receptor I and stimulation via c-kit, respectively.