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Suppression of macrophage NO Synthesis by wound fluid
Author(s) -
Witte M.B.,
Barbul A.
Publication year - 2008
Publication title -
wound repair and regeneration
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.847
H-Index - 109
eISSN - 1524-475X
pISSN - 1067-1927
DOI - 10.1111/j.1067-1927.2005.130215aw.x
Subject(s) - arginase , nitric oxide synthase , nitric oxide , nitrite , wound healing , microbiology and biotechnology , blot , chemistry , cell culture , fibroblast , macrophage , arginine , biochemistry , biology , immunology , in vitro , amino acid , genetics , organic chemistry , gene , nitrate
Background: Essential parameters of wound healing are regulated by the wound environment., as reflected by the bathing wound fluid (WF). WF increases fibroblast proliferation, collagen synthesis, and also inhibits Nitric oxide (NO) synthesis in wound‐derived fibroblasts. The aim of this study was to investigate whether WF influences NO metabolism in other wound cells, namely macrophages. Methods: The mouse hybridoma macrophage cell line RAW 267.4 was employed. Cells were seeded at 2 × 10 5 /well into 24 well plates or at 2 × 10 6 in Petri dishes. Cells were stimulated with 1 μg/ml LPS and 10 U/ml IFN‐gamma to induce inducible nitric oxide synthase (iNOS). iNOS expression was investigated by Northern blotting after 6 h, enzyme secretion by western blotting after 20 h and NO production by nitrite synthesis in cell culture supernatants after 24 h. WF was harvested at different days postwounding from PVA sponges implanted into male Lewis rats. WF was added at 10% v/v to the cell media. Cells were incubated in the presence of 60 mM L‐valine, an arginase inhibitor, to rule out substrate (arginine) depletion by arginase as a mechanism. Results are expressed as mean ± SD, n = 5. Results: Following inflammatory cytokine stimulation, RAW cells produced 57.36 μmol nitrite/ml/24 h. Nitrite synthesis was significantly inhibited to 23.89 ± 13.3 by day 7 and to 6.79 ± 1.28 by day 10 WF, respectively. iNOS expression was almost completely blocked by addition of 10% WF from day 10 as assessed by Northern and western blotting. WF from the later days (day 10 and 14) had larger inhibitory effect than WF from early days (day 2, 5, and 7). Although omitting L‐arginine from the media abolished iNOS expression, incubation of WF in the presence of 60 mM L‐valine did not restore the inhibitory effect suggesting that arginase is not involved. Conclusion: There is a factor in WF which strongly inhibits NO synthesis in RAW cells; the effect becomes more marked over time after wounding. The effect appears to be transcriptionally regulated since mRNA expression is impaired. Further characterization of this inhibitory factor is under way.