Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis

Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.     

Phenylarsine oxide, a tyrosine phosphatase inhibitor, induces an increase in the intracellular concentration of calcium in rat peritoneal macrophages and human fibroblasts]

Using Fura-2 microfluorimetry, phenylarsine oxide (PAO) (10-50 microM), a potent tyrosine phosphatase inhibitor, was shown to induce a dose-dependent increase in the free Ca2+ intracellular concentration in rat peritoneal macrophages and human foreskin fibroblasts. The PAO-induced increase in [Ca2+]i is not due presumably to depletion of intracellular Ca2+ stores but to mainly a stimulation of Ca2+ entry from the extracellular medium. This PAO-activated Ca2+ entry is attenuated by the following pharmacological agents. Organic and inorganic Ca2+ channel blockers: (nifedipine, verapamil and Ni2+); nonselective cation channel blocker niflumic acid; tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate; SH-reagents dithiothreitol parachloromercuribenzoate and N-ethylmaleimide; arachidonic acid metabolism inhibitors 4-bromophenacyl bromide, indomethacin and caffeic acid; microtubule disrupters vinblastine, colchicine and colcemide. On the contrary, microfilament disrupters, cytochalasin B and phalloidin, enhance PAO-activated Ca2+ entry. Our data suggest that the dynamic balance between tyrosine kinase and phosphatase activity may play a central role in the maintenance of homeostatic levels of [Ca2+]i both in unstimulated cells and after agonist application.     

Development and function of T cells in T cell antigen receptor/CD3 zeta knockout mice reconstituted with Fc epsilon RI gamma

Engagement of alpha-beta T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and zeta-zeta or zeta-eta dimers. Previous experiments using knockout (KO) mice that lacked zeta (zeta KO) showed that zeta is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in zeta KO mice, a subset of T cells in the gut of both zeta KO and normal mice bore nearly normal levels of TCR on its surface. This was because zeta was replaced by the Fc epsilon RI gamma (FcR gamma). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that zeta replacement by the FcR gamma chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of zeta substitution by FcR gamma for T cell development and function in vivo, we produced zeta KO mice expressing FcR gamma in all of their T cells (FcR gamma TG zeta KO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcR gamma + lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcR gamma TG zeta KO mice were lower than those of controls. This was particularly true for the CD4+ T cells. We conclude that FcR gamma can replace the functions of zeta in T cell development in vivo but that TCR/CD3 complexes associated with FcR gamma rather than zeta are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4+ and CD8+ T cells.