1 and markedly augments VCAM-1 expression induced by TNF or IL-1 (1-6). These changes favor the binding of eosinophils and T cells to endothelial cells (ECs). In addition, IL-4-treated ECs secrete the chemokine MCP-1 (7) and selectively stimulate transendothelial migration of eosinophils in vitro (8). The effects of IL-4 on EC differ from those on hematopoietic cell types in that IL-4 is not an endothelial mitogen or survival factor.IL-13 is another pleiotropic immunoregulatory cytokine (9, 10) that shares a number of biological properties with IL-4 (11-16). IL-13, like IL-4, selectively induces VCAM-1 in cultured human ECs (17, 18). IL-13 has been shown recently to stimulate tyrosine phosphorylation of the IL-4-binding subunit of the 140-kD IL-4 receptor (IL-4R) in a number of hematopoietic cell types (19), suggesting that IL-4 and IL-13 may activate a common tyrosine kinase. Two recent findings further suggest that the IL-13-binding subunit of the IL-13 receptor (IL-13R) may share at least one subunit with the IL-4R: ( a ) the IL-4 mutant protein Y124D, which inhibits IL-4-dependent reactions (20) also inhibits effects of IL-13 (21, 22); and ( b ) IL-13 competes with radiolabeled IL-4 for binding to some cells (21-24).The IL-2 receptor (IL-2R) ␥ chain is a functional component of the IL-4R in lymphocytes, required for tyrosine phosphorylation of the insulin receptor substrate-1 in response to IL-4 (25) and for IL-4-induced cell proliferation (26). The (IL-2R) ␥ chain, recently renamed the common ␥ chain ( ␥ c chain), is also a common receptor component of many other members of the cytokine receptor superfamily including , [30][31][32]34). It is not known if IL-13 can signal through ␥ c , but some cell lines of hematopoietic origin are known to respond to IL-4 and IL-13 in the absence of ␥ c (24,35). This evidence suggests two distinct signaling pathways for IL-4, one involving ␥ c , and an alternate pathway, shared with IL-13, that does not use ␥ c .Previous work by our laboratory has shown that IL-4 activates a protein tyrosine kinase that phosphorylates the IL-4R binding subunit in cultured human ECs and that activation of this protein tyrosine kinase may play a part in the signaling pathway leading to increased VCAM-1 expression in response to . We now show that IL-13, like IL-4, causes the activation of a protein tyrosine kinase in ECs that phosphorylates IL-4R, that both cytokines activate JAK2 and the Stat6 transcription factor, and that these responses cannot involve ␥ c , since this protein is not expressed in ECs. MethodsCell isolation and culture. ECs were isolated and cultured as described previously (37,38). ECs used in these experiments were of passage levels 2 to 5 and were free of detectable CD45 ϩ leukocytes. PHA- 1. Abbreviations used in this paper: EC, endothelial cell; JAK, Janus kinase; RT, reverse transcription; Stat, signal transducer and activator of transcription; VCAM-1, vascular cell adhesion molecule-1.
This assumption seems to be based on the growth pattern of a single infant, aged 4 to 7 months, who grew in 1 1 saltuses during 1 18 days of observation, or 8% of the time [figure 1 of (1)]. The actual skewness coefficient calculated from our experimental data for that infant is 1.265 (Fig. 2).
A DNA cloning approach was taken to identify islet cell protein antigens that are recognized specifically by insulin-dependent diabetes mellitus (IDDM) sera. A human islet cDNA library was generated and screened with diabetic sera. In this article, identification of two clones is described. Proteins expressed by these lambda phages appeared to react specifically with newly diagnosed diabetic sera. Islet cell antibody 12 (ICA12) was tested by Western blotting. ICA512 was not reactive with sera in the Western format but was specifically immunoprecipitated by diabetic sera from an Escherichia coli extract.
We have investigated whether recombinant erythropoietin (r-Epo) elicits a change in intracellular free calcium (IFC) in purified Epo-responsive cells in spleens of mice treated with phenylhydrazine. Colony-forming units (CFU-E) were prepared by negative selection through immunologic panning. Anti-Forssman, Mac-1, Ia, and HSA antibodies were used to eliminate nonhematopoietic progenitors. After two pannings, 29 +/- 1.5% (mean +/- 1 SD) of the recovered cells were CFU-E. IFC was measured by labeling cells with the fluorescent dye Indo-1 and analyzing them on a flow cytometer from 15 seconds to 30 minutes after the addition of agonist. At each step of the panning procedure, there was no effect of r-Epo (0 to 10 U/mL) on IFC even in the larger cells that are predominantly CFU-E. As a positive control, calcium ionophore (A23187) significantly increased IFC in greater than 90% of the spleen cells enriched in CFU-E. During growth of CFU-E in methylcellulose, the calcium ionophore did not affect the r-Epo-dependent formation of erythroid colonies. EGTA inhibited the formation of erythroid colonies. This inhibition appeared to be the result of a toxic effect of the chelator because the colony growth could not be restored when Ca2+ was added to the cultures in the presence of the EGTA. We conclude that the biologic action of Epo on responsive erythroid cells does not depend on acute changes in IFC.
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