Homogeneous Escherichia coli chaperonin 60 induces IL-1 beta and IL-6 gene expression in human monocytes by a mechanism independent of protein conformation.
Escherichia coli chaperonin (cpn) 60 (groEL) is a protein-folding oligomer lacking tryptophan residues that copurifies with tryptophan-containing proteins and peptides. Cpn 60 is a major immunogen in infectious diseases, and evidence suggests that groEL and mycobacterial cpn 60s can induce cytokine synthesis, stimulate cytokine-dependent bone resorption, and up-regulate expression of vascular endothelial cell adhesion molecules. Whether such activities are due to the cpn 60 or to the copurifying/contaminating proteins/peptides has not been determined. Here we report a method for removing the protein contaminants of groEL and demonstrate that this, essentially homogeneous, groEL remains a potent inducer of human monocyte IL-1beta and IL-6 production. Contaminating peptides had no cytokine-inducing activity and did not synergize with purified groEL. The LPS inhibitor polymyxin B and the CD14-neutralizing Ab MY4 had no inhibitory action on groEL demonstrating that activity is not due to LPS contamination. Heating groEL had no effect on its capacity to stimulate human monocytes to secrete IL-6. Proteolysis of groEL with trypsin, sufficient to produce low molecular mass peptides, also had no inhibitory effect. Thus, we conclude that groEL is a potent inducer of monocyte proinflammatory cytokine production, which acts through the binding of nonconformational peptide domains that are conserved after proteolysis. These data suggest that if groEL was released from bacteria it could induce prolonged tissue pathology by virtue of its cytokine-inducing activity and its resistance to proteolytic inhibition of bioactivity.
Cellular Microbiology Research Group, Eastman Dental Institute, University College London, United Kingdom.
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P
Tabona
K
K
Reddi
S
S
Khan
S P
SP
Nair
S J
SJ
Crean
S
S
Meghji
M
M
Wilson
M
M
Preuss
A D
AD
Miller
S
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Poole
S
S
Carne
B
B
Henderson
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D010450:Endopeptidases; D004926:Escherichia coli; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics*; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics*; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D011112:Polymyxin B / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018834:Chaperonin 60; D007375:Interleukin-1; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010450:Endopeptidases; D011112:Polymyxin B