Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells.

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Interconverting receptor states at 4 degrees C for the neutrophil N-formyl peptide receptor.

With the aid of high time resolution kinetic data extracted from a flow cytometer, we determined that there are two N-formyl peptide receptor states for human neutrophils at 4 degrees C: a low affinity and a high affinity state. Competitive binding of FMLP, FNLP, and t-BOC with FNLPNTL-FL revealed different kinetic rate constants for two distinct reactions that control the lifetime of the low affinity ligand-receptor complex. For these ligands, the rate constant for dissociation of ligand from the low affinity receptor state (the first reaction) ranges in order of magnitude from 10(-2) to 1 s-1, and the conversion rate constant from the low affinity receptor state to the high affinity receptor state (the second reaction) ranges from 10(-4) to 10(-2) s-1. The antagonist t-BOC differed most significantly from the three agonists by having an association rate constant for the low affinity receptor on the order of 10(5) M-1 s-1; the value for all three agonists was on the order of 10(7) M-1 s-1. Characterization of the receptor conversion at 4 degrees C revealed that it is irreversible (or very slow) and independent of Gi protein and that neither receptor state is a form of receptor precoupled to Gi protein. The affinity conversion and the dissociation characteristics of each receptor state determine the duration of the signaling complex and may contribute to differences in ligand efficacy.