Macrophages rapidly internalize their tumor necrosis factor receptors in response to bacterial lipopolysaccharide.
The effect of bacterial lipopolysaccharide (LPS) on macrophage receptors for tumor necrosis factor/cachectin (TNF-R) was studied. At equilibrium, iodinated recombinant human TNF alpha (rTNF alpha) bound to 1100 +/- 200 sites/cell on macrophage-like RAW 264.7 cells with a Kd of 1.3 +/- 0.1 x 10(-9) M. Preexposure of RAW 264.7 cells to 10 ng/ml LPS for 1 h at 37 degrees C resulted in complete loss of cell surface TNF alpha binding sites. 50% loss ensued after 1 h with 0.6 ng/ml LPS, or after 15 min with 10 ng/ml LPS. Complete loss of TNF alpha binding sites occurred without change in numbers of complement receptor type 3. No decrease in TNF-R followed preexposure to LPS at 4 degrees C, nor could LPS displace 125I-rTNF alpha from its binding sites. Although TNF-R disappeared from the surface of intact macrophages following exposure to LPS, specific TNF alpha binding sites were unchanged in permeabilized macrophages, indicating that TNF-R were rapidly internalized. Conditioned media from LPS-treated RAW 264.7 cells induced 30% down-regulation of TNF-R on macrophages from LPS-hyporesponsive mice (C3H/HeJ), suggesting that a soluble macrophage product may be responsible for a minor portion of the LPS effect. Additional evidence against endogenous TNF alpha being the major cause of TNF-R internalization was the rapid onset of the effect of LPS on TNF-R compared to the reported onset of TNF alpha production, the relatively high concentrations of exogenous rTNF alpha required to mimic the effect of LPS, and the inability of TNF alpha-neutralizing antibody to block the effect of LPS. LPS-induced down-regulation of TNF-R was complete or nearly complete not only in RAW 264.7 cells, but also in primary macrophages of both human and murine origin, was less marked in human endothelial cells, and was absent in human granulocytes and melanoma cells and mouse L929 cells. Thus, in situ, macrophages and some other host cells may be resistant to the actions of TNF alpha produced during endotoxinemia, because such cells may internalize their TNF-R in response to LPS before TNF alpha is produced.
Ding
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Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York 10021.
Sanchez
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Srimal
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Nathan
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D000818:Animals; D000937:Antigen-Antibody Reactions; D001685:Biological Factors / Q000494:pharmacology; D002460:Cell Line; D016207:Cytokines; D004305:Dose-Response Relationship, Drug; D004537:Eicosanoic Acids / Q000494:pharmacology; D004705:Endocytosis; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D001685:Biological Factors; D016207:Cytokines; D004537:Eicosanoic Acids; D008070:Lipopolysaccharides; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; D010447:Peptide Hydrolases