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Effect of tumor necrosis factor on granule release and LTB4 production in adherent human polymorphonuclear leukocytes
1Department of Biological Sciences, University of California, Santa Barbara 93106.
Abstract:
Tumor necrosis factor (rTNF) has previously been shown to induce PMN chemotaxis, stimulate PMN adhesion to vascular endothelium and stimulate hydrogen peroxide secretion from PMNs adhered to biological surfaces. We investigated the activity of both rTNF alpha and rTNF beta on adherent and suspension cultures of human PMNs. rTNF alpha selectively stimulated the release of the specific granule in a dose dependent manner. Exocytosis of the specific granule was measured with an enzyme-immunoassay for lactoferrin and a radioassay for vitamin B12-binding protein. Adherent PMNs released up to 60% of the total lactoferrin content of the cells with no increase in myeloperoxidase (MPO) secretion when stimulated with 0.1-10 nM rTNF alpha. The PMNs in suspension cultures also selectively released the specific granule, although total release was reduced suggesting that adherence of PMNs increased their ability to respond to physiological stimuli. When PMNs in suspension cultures or adherent cells were stimulated with rTNF alpha, no LTB4 production was detectable, yet the cells retained the ability to synthesize LTB4 when stimulated with calcium ionophore A23187. Neither rTNF alpha or rTNF beta stimulated the release of the azurophilic granule, measured by the secretion of MPO and neutrophil elastase activity. These results suggest that a function of rTNF alpha and rTNF beta on PMNs is the release of the contents within the specific granule.
Insights
Tumor necrosis factor-alpha (rTNF alpha) selectively triggers the release of specific granules from human neutrophils (PMNs). This action, observed in both adherent and suspension cells, highlights a key function of TNF in neutrophil activation.
Area of Science:
- Immunology
- Cell Biology
Background:
- Tumor necrosis factor (TNF) is known to influence neutrophil functions like chemotaxis and adhesion.
- The specific roles of TNF-alpha and TNF-beta in human neutrophil activation require further elucidation.
Purpose of the Study:
- To investigate the effects of recombinant TNF-alpha (rTNF alpha) and TNF-beta (rTNF beta) on human neutrophil (PMN) degranulation.
- To determine if rTNF alpha and rTNF beta induce the release of specific granules or azurophilic granules from PMNs.
Main Methods:
- Human PMNs were cultured in suspension and as adherent monolayers.
- Cells were stimulated with varying concentrations of rTNF alpha and rTNF beta.
- Degranulation was assessed by measuring the release of specific granule contents (lactoferrin, vitamin B12-binding protein) and azurophilic granule contents (myeloperoxidase, neutrophil elastase).
- Leukotriene B4 (LTB4) production was also measured.
Main Results:
- rTNF alpha selectively stimulated the dose-dependent release of specific granules from both adherent and suspension PMNs.
- Adherent PMNs showed a greater release of lactoferrin compared to suspension cells.
- Neither rTNF alpha nor rTNF beta stimulated the release of azurophilic granules.
- No LTB4 production was detected upon rTNF alpha stimulation, though cells retained the capacity to produce LTB4 when stimulated with calcium ionophore A23187.
Conclusions:
- rTNF alpha and rTNF beta primarily induce the release of specific granule contents from human neutrophils.
- Neutrophil adherence enhances the response to rTNF alpha-induced specific granule exocytosis.
- TNF's role in neutrophil activation involves targeted degranulation, specifically releasing contents from specific granules.