Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-proteincoupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca 2؉. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca 2؉ . These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Fever is frequently observed in cancer patients treated with high-dose recombinant human interleukin-2 (rIL-2). The preincubation of rIL-2 with polymyxin B, an antibiotic that inhibits the biologic effects of endotoxins, did not diminish the pyrogenicity of IL-2 in New Zealand rabbits, indicating that IL-2-induced fever is not due to contaminating endotoxins. In contrast to interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon alpha, which cause fever through their effects on arachidonic acid metabolism in the hypothalamus, IL-2 was unable to induce prostaglandin E2 synthesis in hypothalamic cells or fibroblasts in vitro, suggesting that IL-2 is not intrinsically pyrogenic. To determine if IL-2-induced fever is mediated indirectly through the generation of pyrogenic cytokines, culture supernatants from IL-2-stimulated human peripheral blood mononuclear cells were screened for the presence of pyrogens by direct injection into rabbits and by measuring the amounts of IL-1 alpha, IL-1 beta, and TNF alpha by specific radioimmunoassays (RIA). All three cytokines were readily detected by RIA in these supernatants, which in turn caused fever when injected into rabbits. Furthermore, in six of six cancer patients treated with rIL-2, elevated levels of TNF alpha were detected in the plasma by RIA 2 hr after IL-2 administration. Plasma TNF levels increased from pretreatment values of 14 +/- 7 to 765 +/- 150 pg/ml 2 hr after an IL-2 injection. These results strongly implicate IL-2-induced pyrogenic cytokines, in particular TNF alpha, as a major cause of the fever and possibly other aspects of the acute-phase response associated with IL-2 therapy.

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