Showing papers by "Andrew C. Issekutz published in 1990"

An endotoxin-induced factor distinct from interleukin-1 and tumour necrosis factor alpha produced by the THP-1 human macrophage line stimulates polymorphonuclear leukocyte infiltration in vivo.

Abstract: Endotoxin and gram-negative bacteria induce vigorous inflammatory reactions. Our previous work showed that rabbit macrophages (M phi) incubated with endotoxin produce a 45,000 dalton protein that recruited polymorphonuclear leukocytes (PMNL) into the skin of rabbits. This factor was separated from interleukin-1 (IL-1) but could not be unequivocally distinguished from rabbit tumour necrosis factor (TNF alpha). Here we have examined the human M phi cell line, THP-1, for the production of an analogous protein. After exposure to phorbol diester the THP-1 cells assumed the characteristic M phi phenotype and function. During 6 hours of culture with LPS these M phi released a factor(s) that caused PMNL recruitment into the skin of rabbits when injected intradermally, measured using 51Cr-labelled blood leukocytes. This activity, referred to as PMNL recruiting activity (PRA), was heat labile, and its production was blocked by cycloheximide, suggesting that this is most likely a de novo synthesized protein. Sephadex-G 100 and Superose-12 FPLC chromatography indicated a molecular weight in the 45,000-65,000 dalton range. The active fractions were free of IL-1 activity (less than 0.2 U/ml), and Superose-12 chromatography separated the peak of PRA, which eluted around 45,000 daltons, from TNF alpha eluting at 20,000 daltons. The peak PRA was not neutralized by antiserum to IL-1 alpha, IL-1 beta TNF alpha, IL-6, and granulocyte-macrophage colony-stimulating factor (GMCSF), indicating that it was distinct immunologically from these cytokines. The major PRA did not induce migration of rabbit or human PMNLs in vitro in a Boyden chamber chemotaxis assay, although peaks of chemotactic activity and weak PMNL recruitment in vivo were detected in fractions eluting around 15,000 daltons and 800 daltons. The generation of PRA by a human M phi cell line is analogous to that reported previously with rabbit M phi. Here we extend these observations to a human M phi system and confirm that this molecule is distinct from several other M phi cytokines and M phi chemotactic factors with inflammatory properties.

Endotoxin-stimulated human macrophages produce a factor that induces polymorphonuclear leucocyte infiltration and is distinct from interleukin-1, tumour necrosis factor alpha and chemotactic factors.

Abstract: Previously we reported that rabbit macrophages (M phi) in the presence of nanogram quantities of endotoxin (LPS) release factors that induce polymorphonuclear leucocyte (PMNL) infiltration into the skin of rabbits following i.d. injection. The predominant factor was a de novo synthesized protein of 45,000 MW on gel filtration that was distinguishable from IL-1 but not from TNF alpha. Here we examined human monocytes, in vitro monocyte-derived M phi and peritoneal M phi for the production of an analogous protein. Upon stimulation with LPS, they all rapidly (6 hr) produced a factor(s) that caused PMNL accumulation in the skin of rabbits when injected i.d. This activity, referred to as PMNL-recruiting activity (PRA), was heat labile and its production was blocked by cycloheximide. By Sephadex-G100 chromatography the major PRA of cultured M phi or peritoneal M phi had a molecular weight (MW) of 45,000-60,000. The active fractions were free of IL-1 (less than 0.2 U/ml) and Superose-12 FPLC chromatography separated the peak of PRA, which eluted at 45,000 MW, from TNF alpha, eluting at 20,000 MW. The peak PRA was not neutralized by antisera to IL-1 alpha, IL-1 beta, TNF alpha, IL-6 or GM-CSF, indicating that it was distinct from these cytokines. The major PRA did not induce the migration of PMNL in vitro in a filter chemotaxis assay. In contrast to the M phi, the major PRA produced by LPS-stimulated monocytes eluted at 15,000-20,000 MW, contained IL-1 activity and was neutralized by antisera to IL-1 alpha and IL-1 beta. Monocytes from a few donors also produced the 45,000-60,000 MW PRA simultaneously. We conclude that human peritoneal M phi and in vitro monocyte-derived M phi exposed to LPS secrete a protein of 45,000-60,000 MW, which is a potent inducer of PMNL infiltration but is distinct from IL-1, TNF alpha, IL-6, GM-CSF and PMNL chemotactic factors.