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First published online 31 January 2006
doi: 10.1242/jcs.02786

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Androgens modulate the inflammatory response during acute wound healing

Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (68K): [in a new window]   Fig. 1. In rats, incisional wound healing is accelerated and inflammatory cell influx attenuated by 5-reductase inhibition. (A,C) Day-2 and -6 wound sections were subjected to histological analysis and quantification. Wound areas at day 2 post wounding were reduced in wounds of castrated (CSX) animals and those treated with MK-434 (n=5-7 per group). Arrows indicate the wound margins. (B,D) Numbers of ED1-positive macrophages (studied immunohistochemically, quantified and indicated by arrows) in day-2 and -6 wounds of castrated animals were reduced as they were in day-2-wounds of animals treated with MK-434. (B,E) Numbers of His48-immunoreactive granulocytes (arrows) were significantly reduced both in day-2-wounds of castrated animals and those treated with MK-434. Bars, 500 µm (A) or 50 µm (B). Data represent mean ± s.d. *P<0.05; **P<0.01.

View larger version (38K): [in a new window]   Fig. 2. Wound inflammatory cytokine mRNA and protein levels are modulated by MK-434 treatment and castration. (A-D) Incisional day-2-wound sections were analysed immunohistochemically and tissue mRNA and protein extracts were analysed by qPCR and immunoblotting, respectively (n=6-7 per treatment group). Day 6 wound sections were analysed immunohistochemically. Arrows indicate immunoreactive cells. Numbers of IL-6-immunopositive cells, overall protein levels and gene expression were reduced in day-2-wounds of castrated animals and those treated with MK-434 (gene expression in castrated animals only). Numbers of TNF--immunoreactive cells, levels of TNF- protein and mRNA were also reduced in day-2-wounds of castrated animals. Numbers of TGF-ß1-immunopositive cells and TGF-ß1 protein levels were increased in day-2-wounds of castrated rats; also increased were numbers of TGF-ß1-immunopositive cells in day-6-wounds of castrated animals and of those treated with MK-434. Numbers in D represent relative levels of protein based on optical density readings, normalised to the ß-actin signal. Bars, 20 µm. Data represent mean ± s.d. *P<0.05; **P<0.01.

View larger version (26K): [in a new window]   Fig. 3. mRNA levels of inflammatory cytokines in macrophages are attenuated by MAP kinase and PI 3-kinase inhibitors, which additionally block the inhibition of fibroblast cytokine mRNA expression by DHT. Macrophage- and fibroblast-derived mRNA samples were analysed by qPCR and supernatants by ELISA. (A) The increase in macrophage IL-6 mRNA levels in response to DHT was blocked by actinomycin D, PD 98059 and wortmannin. By contrast, TNF- mRNA expression was unaffected by treatment with DHT or testosterone but was attenuated by actinomycin D, PD 98059 and wortmannin. Treatment with wortmannin tended to reduce TGF-ß1 mRNA levels that were otherwise unaffected by androgen treatment. (B) In fibroblasts, the decrease in IL-6 mRNA levels in response to DHT was blocked by wortmannin but not PD 98059. Expression of TNF- mRNA was similarly attenuated by DHT, a response that was blocked by treatment with PD 98059 or wortmannin. Co-treatment with PD 98059 or wortmannin reversed the inhibitory effect of DHT on fibroblast TGF-ß1 mRNA expression. (C) Fibroblast secretion of IL-6 and total TGF-ß1 protein levels (active and inactive) was reduced after treatment with DHT. (D-F) Fibroblast mRNA levels of IL-6 and TNF- were dramatically increased following treatment with cycloheximide. Fibroblast TGF-ß1 mRNA levels were unaffected. Data represent mean ± s.d. *P<0.05; **P<0.01. Con, control; T, testosterone; D, DHT; A, actinomycin D; P, PD 98059; W, wortmannin; CHX, cycloheximide.

View larger version (37K): [in a new window]   Fig. 4. Numbers of macrophages, neutrophils and TNF--positive cells in wounds of Smad3-/- mice are not affected by castration. Day 3 wound sections were analysed immunohistochemically to quantify inflammatory cells, and IL-6- and TNF--immunoreactive cell numbers. (A) Numbers of macrophages were significantly reduced in day-3-wounds of castrated wild-type mice but were not affected in day-3-wounds of Smad3-/- animals (n=4-5 per treatment group). (B) The population of neutrophils was similarly depleted in wounds of castrated wild-type mice but not of Smad3-/- animals. (C) Numbers of TNF--positive cells were decreased in day-3-wounds of castrated wild-type animals but were not affected in day-3-wounds of castrated Smad3-/- mice. (D) Numbers of TGF-ß1-immunoreactive inflammatory cells were increased solely in wounds of castrated wild-type mice. Arrows indicate immunoreactive cells; dashed lines in D indicate the dermal-epidermal boundary. Bars, 20 µm. Data represent mean ± s.d. *P<0.05; **P<0.01. EP, epidermis.

View larger version (38K): [in a new window]   Fig. 5. Smads are regulated by androgens and castration. Day-2-wound sections were analysed immunohistochemically, wound protein samples by western blotting, and tissue and macrophage-derived mRNA samples by qPCR. (A-B) Numbers of Smad3- and Smad4-immunopositive cells and protein levels of phosphorylated (activated) Smad3 (pS3) were unaffected in wounds of castrated animals and those treated with MK-434. Smad7-positive cell numbers were significantly increased in day-2-wounds of castrated animals and those treated with MK-434. (C) Castration of animals reduced Smad4 and Smad7 mRNA levels in wounds. (D) Treatment of peritoneal macrophages with testosterone or DHT for 18 hours significantly increased the levels of Smad3 mRNA, whereas exposure to DHT but not testosterone increased mean Smad4 mRNA levels and significantly increased Smad7 mRNA levels. Arrows indicate immunostained cells; dashed lines surround blood vessels. Bars, 50 µm. Data represent mean ± s.d. *P<0.05; **P<0.01. Con, control; T7 and T8, 10-7 M and 10-8 M testosterone, respectively; D7 and D8, 10-7 M and 10-8 M DHT, respectively.

View larger version (23K): [in a new window]   Fig. 6. Proposed mechanism for the androgenic regulation of wound healing. DHT binds to the AR and the resulting ligand-activated AR represents an effector complex whose activities may be modulated by Smad3. Activated AR stimulates macrophage production of pro-inflammatory cytokines, while at the same time depressing fibroblast cytokine expression and secretion. The increase in macrophage cytokine release, which depends on MAPK and PI 3-kinase signalling, contributes to enhanced inflammation and hence delayed healing.

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