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An Endotoxin‐lnduced Factor Distinct From lnterleukin‐1 and Tumour Necrosis Factor α Produced by the THP‐1 Human Macrophage Line Stimulates Polymorphonuclear Leukocyte Infiltration In Vivo
Author(s) -
Megyeri Pal,
Issekutz Thomas B.,
Issekutz Andrew C.
Publication year - 1990
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.47.1.70
Subject(s) - biology , infiltration (hvac) , in vivo , tumor necrosis factor alpha , thp1 cell line , macrophage , immunology , microbiology and biotechnology , cell culture , in vitro , biochemistry , genetics , physics , thermodynamics
Endotoxin and gram‐negative bacteria induce vigorous inflammatory reactions. Our previous work showed that rabbit macrophages (M⊘) incubated with endotoxin produce a 45,000 darton protein that recruited polymorphonuclear leukocytes (PMNL) into the skin of rabbits. This factor was separated from lnterleukin‐1 (IL‐1) but could not be unequivocally distinguished from rabbit tumour necrosis factor (TNFα). Here we have examined the human Mo cell line, THP‐1, for the production of an analogous protein. After exposure to phorbol diester the THP‐1 cells assumed the characteristic M⊘ phenotype and function. During 6 hours of culture with LPS these Mo released a factor(s) that caused PMNL recruitment into the skin of rabbits when injected intradermally, measured using 51 Cr‐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 (< 0.2 U/ml), and Superose‐12 chromatography separated the peak of PRA, which eluted around 45,000 daltons, from TNFα eluting at 20,000 daltons. The peak PRA was not neutralized by antiserum to IL‐1α, IL‐1β TNFα, 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 Me cell line is analogous to that reported previously with rabbit Me. Here we extend these observations to a human Me system and confirm that this molecule is distinct from several other Me cytokines and Me chemotactic factors with inflammatory properties.