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Physiological relevance of tumor necrosis factor in mediating macrophage-Leydig cell interactions
1Department of Cell Biology and Anatomy, Texas Tech University Health Sciences Center, Lubbock 79430.
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.
Insights
Testicular macrophages do not constitutively release tumor necrosis factor (TNF). Collagenase used in isolation activates macrophages, but testicular macrophages release TNF upon lipopolysaccharide (LPS) exposure, suggesting a role in testicular pathology.
Area of Science:
- Immunology
- Reproductive Biology
- Cell Biology
Background:
- Testicular macrophages were previously reported to constitutively release tumor necrosis factor (TNF) in vitro and be unresponsive to lipopolysaccharides (LPS).
- These properties differ from other tissue macrophages, prompting further investigation into their in vivo behavior and in vitro isolation effects.
Purpose of the Study:
- To determine if testicular macrophages release TNF in vivo.
- To investigate if the isolation procedure influences TNF secretion in vitro.
Main Methods:
- Assessing testicular interstitial fluid for TNF using the L929 cytotoxicity assay.
- Testing the effect of collagenase on macrophage TNF release and LPS responsiveness.
- Utilizing antibodies to TNF alpha and -beta, TNF-resistant L929 cells, and enzyme-linked immunosorbent assay (ELISA) for TNF immunoreactivity.
Main Results:
- Testicular interstitial fluid contained cytotoxic factors, but not TNF or TNF-like molecules.
- Macrophages isolated without collagenase did not release TNF but responded to LPS.
- Collagenase exposure induced constitutive TNF release in peritoneal macrophages and abolished LPS responsiveness.
Conclusions:
- Testicular macrophages do not constitutively release TNF; collagenase significantly activates macrophages.
- Testicular macrophages release TNF upon LPS exposure, indicating potential paracrine regulation of testicular steroidogenesis in pathological conditions.