title: Physiological relevance of tumor necrosis factor in mediating macrophage-Leydig cell interactions.
abstract: Previously, we reported that testicular macrophages constitutively release tumor necrosis factor (TNF) in vitro and are unresponsive to bacterial endotoxins [lipopolysaccharides (LPS)]. These properties are not typical of other tissue macrophages. The goals of the present study were, therefore, to establish 1) if testicular macrophages also release TNF in vivo, and 2) if secretion of TNF in vitro is influenced by the isolation procedure. In vivo TNF production was assessed by assaying testicular interstitial fluid for TNF. Using the L929 cytotoxicity assay for TNF, we found that interstitial fluid contained a cytotoxic factor(s), but this bioactivity was not due to either authentic TNF or a TNF-like molecule acting through the TNF receptor. This was established by showing that 1) antibodies to TNF alpha and -beta could not neutralize interstitial fluid cytotoxicity; 2) interstitial fluid was cytotoxic to TNF-resistant L929 cells; and 3) there was no detectable TNF immunoreactivity in interstitial fluid, as measured by enzyme-linked immunosorbent assay. Therefore, we evaluated whether the release of TNF in vitro was induced by the isolation procedure, particularly by collagenase, which is used to free interstitial cells. Testicular macrophages obtained without the use of collagenase (agitation of testes in buffer) did not release TNF, but responded to the TNF-releasing effect of LPS. Exposure of peritoneal macrophages to collagenase resulted in constitutive TNF release in vitro and lack of responsiveness to LPS. There was no evidence that a non-TNF cytotoxic factor was released in the conditioned medium by any macrophage preparation. Taken together, our findings show that testicular macrophages do not constitutively release TNF, and collagenase has a significant activating effect on macrophages. Testicular macrophages will, however, release TNF when exposed to LPS, indicating that TNF could be a paracrine regulator of testicular steroidogenesis under pathological conditions.
authors.affiliation: Department of Cell Biology and Anatomy, Texas Tech University Health Sciences Center, Lubbock 79430.
authors.forename: C
authors.initials: C
authors.lastname: Moore
authors.forename: J C
authors.initials: JC
authors.lastname: Hutson
mesh_terms: D000818:Animals; D001681:Biological Assay; D002450:Cell Communication / Q000502:physiology*; D002469:Cell Separation; D017364:Collagenases / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005110:Extracellular Space / Q000378:metabolism; D007985:Leydig Cells / Q000502:physiology*; D008264:Macrophages / Q000502:physiology*; D017737:Macrophages, Peritoneal / Q000378:metabolism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013737:Testis / Q000166:cytology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000502:physiology*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D017364:Collagenases