Altered proximal T-cell receptor signalling events in mouse CD4+ T cells in the presence of anti-CD4 monoclonal antibodies: evidence for reduced phosphorylation of Zap-70 and LAT.

Anti-CD4 monoclonal antibodies are potential therapeutic agents for the prevention of autoimmune disease and treatment of rejection after organ transplantation and are capable of both restoring tolerance to self-antigens and inducing tolerance to antigens introduced under the cover of the antibody therapy in vivo. Tolerance to donor alloantigens can be induced in vivo by administering donor alloantigen in combination with either depleting (YTA 3.1) or nondepleting (YTS 177) anti-CD4, 28 days before heart transplantation in the mouse. The effect of anti-CD4 on proximal T-cell receptor (TCR) signalling pathways and proliferation was investigated in vitro and in vivo in the presence and absence of YTA 3.1 or YTS 177. Anti-CD4 was found to perturb proximal signalling events upon TCR/CD3 ligation, resulting in reduced tyrosine phosphorylation of Zap-70 and LAT (linker for activation of T cells) and reduced association of tyrosine-phosphorylated LAT with lck. This ultimately resulted in severely reduced proliferation of the responding CD4+ T cells. The signalling profile of the anti-CD4-treated cells resembled that of anergic T cells. This could be a result of a common mechanism involving perturbation in the formation of the central supramolecular activation cluster of the immunological synapse by impaired recruitment of CD4 and CD28, thereby resulting in severely reduced lck activation.

Cyclic AMP-dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field.

Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.

Human IgE mediates stimulus secretion coupling in rat basophilic leukemia cells transfected with the alpha chain of the human high-affinity receptor.

Cell surface expression of the alpha chain of the human (h) Fc epsilon receptor type 1 (Fc epsilon R1) can be observed following stable transfection of RBL-2H3 cells with the h alpha chain of the Fc epsilon R1 complex. Association and dissociation constants for hIgE are similar to those previously reported for the ligand and its receptor. The demonstration of mediator release following the cross-linking of hFc epsilon R1 alpha by hIgE and antigen or anti-hFc epsilon R1 alpha antibody suggests that this system will assist the identification of structural motifs and specific amino acids that are involved in the generation of the signal(s) that will initiate and/or propagate the secretion of mediators from mast cells and basophils.