We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37°C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time-and concentration-dependent manner) when incubated separately with LPS. Lactoferrinreleasing activity was heat (80°C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor a. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor a derived from contaminating monocytes.
The thiol proteinase cathepsin H, isolated and purified from rat liver lysosomes, provokes acute inflammation characterized by the accumulation of polymorphonuclear leukocytes (PMN) when injected intracutaneously into newborn rats. We have examined the possibility that the accumulation of PMN at skin sites injected with cathepsin H is due, in part, to generation locally of C-derived chemotactic factors. We have found that cathepsin H acts in a concentration- and time-dependent fashion in whole human (and rat) EDTA-plasma to generate C5-derived peptides with chemotactic activity for PMN. Chemotactic activity was not generated in EDTA-plasma by either heat-inactivated cathepsin H or by a combination of active enzyme and a thiol proteinase inhibitor isolated from rat epidermis. Cathepsin H also acted in a concentration- and time-dependent fashion on isolated (functionally pure) human C5 to yield chemotactic activity for PMN as well as PMN lysosomal enzyme-releasing activity. Whereas 10 ng/ml cathepsin H generated significant chemotactic activity from isolated C5 (1000 CH50 U/ml), 7 to 10 micrograms/ml were required to generate chemotactic activity in whole EDTA-plasma. Cathepsin H not only was capable of generating biologically active, C5-derived peptides, but also was capable of degrading these peptides. Incubation of either whole EDTA-plasma or isolated C5 with high concentrations of cathepsin H (e.g., 25 micrograms/ml and 100 ng/ml, respectively) caused the rapid appearance of chemotactic activity followed by an equally rapid disappearance. PMN accumulated more rapidly in the skin of newborn rats injected with cathepsin H-treated C5 than in the skin of animals injected with cathepsin H alone. These data suggest that generation by cathepsin H of C-derived chemotactic activity contributes to the ability of this enzyme to induce dermal inflammation.
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