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Endocrinology Vol. 149, No. 11 5747-5757
Copyright © 2008 by The Endocrine Society

Sex Dimorphism in Wound Healing: The Roles of Sex Steroids and Macrophage Migration Inhibitory Factor

Stephen C. Gilliver, Jayalath P. D. Ruckshanthi, Matthew J. Hardman, Toshinori Nakayama and Gillian S. Ashcroft

Faculty of Life Sciences (S.C.G., J.P.D.R., M.J.H., G.S.A.), University of Manchester, Manchester M13 9PT, United Kingdom; and Graduate School of Medicine (T.N.), Chiba University, Chiba 260-8670, Japan

Address all correspondence and requests for reprints to: Gillian S. Ashcroft, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom. E-mail: gillian.s.ashcroft{at}manchester.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
That endogenous sex steroid hormones profoundly influence the response to cutaneous injury is well established. How they and other factors combine to direct repair in male and female animals is much less well understood. Using a murine incisional wound-healing model, we investigated the roles of circulating sex steroids, macrophage migration inhibitory factor (MIF) (the mediator of delayed healing in ovariectomized animals), and hormone- and MIF-independent factors in controlling repair. We report that d 3 wounds, of comparable size in intact male and female mice, are significantly larger in ovariectomized female animals than in castrated males, suggesting that native sex hormones mask inherent underlying differences in the ways in which males and females respond to wounding. Wound MIF levels were comparable in intact male and female mice but greater in ovariectomized females than castrated males. Furthermore, wound levels of Jun activation domain-binding protein 1 (JAB1), a key factor by which MIF activates intracellular responses, were increased through ovariectomy and greater in ovariectomized females than castrated males. This difference in wound JAB1 levels may underscore the marked sex difference we observed in the responses of MIF knockout mice to the local application of MIF: healing was impaired in ovariectomized females but not castrated males. Separately, systemic treatment with androgens and estrogens yielded contrasting effects on repair in male and female animals. Collectively, the presented data indicate sex divergence in wound healing to be multifaceted, being strongly influenced by MIF and seemingly limited by the combined actions of gonadal steroids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLOWING A DECADE of intensive research, a substantial body of work describes the contrasting influences of androgenic and estrogenic sex steroids on the healing of acute skin wounds. Whereas the so called male hormones testosterone and its metabolite 5{alpha}-dihydrotestosterone (DHT) inhibit repair (1, 2), 17β-estradiol accelerates recovery (3, 4). In young male rodents, wound healing may be improved through either bilateral castration or systemic androgen receptor blockade (1), which accelerate repair by increasing collagen deposition and reducing local production of the proinflammatory cytokine TNF-{alpha}. Moreover, in a group of aged men, excisional wounds areas were positively correlated with circulating testosterone levels (1). In elderly women, hormone replacement therapy (HRT) by contrast reversed an age-linked decline in skin regenerative capacity, accelerating reepithelialization and increasing the TGF-β1-stimulated deposition of collagen (3). In both males and females, topically applied estrogen enhanced wound repair, repressing neutrophil recruitment and resultant secretion of the degradative enzyme elastase (4).

With males and females differing markedly in terms of their circulating sex hormone profiles, gender differences in wound repair parameters would not intuitively be unexpected. Those to have been described relate to aged individuals (1). Elderly males display delayed and less regenerative basement membrane type VII collagen deposition than do age-matched females (1), who also boast improved patterns of elastin regeneration (5) as well as increased elastase activity (4). Overall wound proteolytic activity is also greater in elderly females than in males (6).

These studies all relate to the healing of acute skin wounds. In the context of chronic nonhealing ulcers, being male was, through neural network studies, identified as a primary risk factor (7). The healing of mucosal wounds, by contrast, reportedly progresses more slowly in females than males (8).

The mechanisms controlling repair in males and females bear fundamental differences. The TGF-β-activated transcription factor mothers against decapentaplegic homolog 3 (Smad3) is centrally involved in the inhibition by androgens of healing but dispensable for the enhancement effected by estrogens (2, 9). The proinflammatory and pleiotropic cytokine macrophage migration inhibitory factor (MIF), by contrast, is responsible for obstructing repair in ovariectomized (OVX) female mice (10, 11). Its role in males remains unexplored.

Other investigations have sought to evaluate the influence of gender upon repair in artificial models. In one such study, a group of premenopausal females were shown to deposit significantly greater quantities of collagen into sc-implanted Teflon-coated tubes than age-matched male counterparts (12). However, none has attempted directly to probe the comparative contributions of hormonal and nonhormonal factors to such sex divergence.

Collectively, the published data encouraged us to hypothesize that young males and females might heal skin wounds differently, especially under conditions of low circulating sex steroid levels. Moreover, we speculated that MIF may have gender-specific functions during healing. The aim of the present study was to compare wound repair in male and female mice and to evaluate the individual contributions of MIF, estrogens, and androgens. To that end, we studied the healing of skin wounds in intact and gonadectomized mice and the influence of MIF gene disruption, local MIF treatment, and systemic hormone replacement. We report the existence of fundamental sex differences in repair: whereas healing was markedly impaired in both gonadectomized female mice and estrogen-treated males, it was enhanced in gonadectomized male mice and estrogen-treated females. And although exogenous MIF retarded healing in OVX MIF knockout female mice, it elicited no such response in castrated (CSX) males. Furthermore, it appears that increased wound expression of the key MIF signaling effector Jun activation domain-binding protein 1 (JAB1) may contribute to impaired healing in OVX mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Wound-healing experiments
All animal studies were approved by both the University of Manchester institutional Animal Use Committee and the U.K. Home Office. Eight-week-old male and female BALB/c and MIF knockout (13) mice were anesthetized with isoflurane, and their dorsa were shaved and cleansed using ethanol. Dorsal incisions (1 cm long) were made through the skin and underlying panniculus carnosus muscle 1 cm below the base of the skull and the same distance either side of the midline. Subgroups of male and female BALB/C mice and all MIF knockout animals had been gonadectomized 2 wk previously. Subsets of gonadectomized male and female MIF knockout mice received (per wound) 500 ng of recombinant human MIF (R&D Systems, Abingdon, UK), injected intradermally at the site to be incised immediately before wounding. Further subgroups of gonadectomized males and females were treated with 21-d slow-release testosterone (10 mg) or 17β-estradiol (0.05 mg) pellets (Innovative Research of America, Sarasota, FL), implanted sc immediately after gonadectomy using a trocar. Postoperatively, all animals (n = 4–6 per treatment group) received immediate analgesia in the form of buprenorphine (sc; 0.1 mg/kg) and were housed individually. Wounds were excised on d 3 after wounding and immediately bisected, half being snap frozen in liquid nitrogen and stored at –80 C, the other being processed in formaldehyde-based fixative for embedding in paraffin. All procedures were performed in accordance with the U.K. Government Home Office regulations relating to animal care.

Immunohistochemistry and image analysis
Five-micrometer-thick histological sections were prepared from wound tissue fixed in 8% formaldehyde-based fixative and embedded in paraffin. Sections prepared from the central portion of each wound were subjected to hematoxylin and eosin staining or immunohistochemistry with monoclonal IgG antibodies raised against Ly-6G, Mac3, CD44, CD74, and phospho-activated ERK (pERK) (BD Biosciences, Oxford, UK); JAB1 (Sigma-Aldrich, Gillingham, UK); and polyclonal IgG antibodies raised against MIF (R&D Systems) and ERK1/2 (Insight Biotechnology, Wembley, UK). Treatment of tissue sections with PBS in place of primary antibody (negative controls) yielded no signal. Bound primary antibody was detected using Vectastain ABC peroxidase kits (Vector Laboratories, Peterborough, UK) in conjunction with the substrate Novared (Vector). Photographic images were prepared and the quantification of wound areas and widths, the extent of reepithelialization, and cell numbers per unit area undertaken using Image-Pro Plus software (Media Cybernetics, Marlow, UK) as described previously (3). Briefly, the wound area was defined as being the area bounded by the panniculus carnosus muscle at the base of the wound, the scab and migrating epithelium at the top of the wound and the margins of normal skin on either side. Wound widths were measured as the distance between the unwounded dermis margins at the top of the wound. Extent of reepithelialization was calculated as the percentage of this wound width covered by epithelium. Cell populations were determined by calculating the average number of positively stained cells in each of four systematically selected regions of the wound.

Quantification of epidermal protein expression
Wound CD44 staining intensities were compared by four-grade semiquantitative scoring: 0, no neoepidermal staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Data are presented as box-and-whisker plots.

Immunoblotting
Total protein was extracted from mouse wound tissue using a sodium dodecyl sulfate (SDS)-based detergent buffer [0.06 M Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, 1% (vol/vol) protease inhibitor cocktail (P8340; Sigma-Aldrich), 5% (vol/vol) 2-mercaptoethanol (Bio-Rad, Hemel Hempstead, UK)]. Extracted protein samples (n = 4–5 per treatment group; 1 mg/sample) were pooled and then analyzed as has been described previously (6). Briefly, wound samples were separated by denaturing, reducing SDS-PAGE and blotted to 0.2 µm nitrocellulose membrane (Bio-Rad). Membranes were then blocked for 16 h in Tris-buffered saline supplemented with 0.1% Tween 20 and 5% nonfat dry milk. After sequential 1-h incubations with primary and horseradish peroxidase-conjugated secondary IgG antibodies, antigen binding was probed using SuperSignal West Pico chemiluminescent substrate (Perbio, Cramlington, UK). Signal intensities were determined densitometrically and the signal from each protein of interest normalized to that from β-actin, used as a loading control. All experiments were repeated three times, yielding qualitatively identical data. Primary IgG antibodies raised against MIF (raised in goat; R&D Systems); JAB1 (rabbit) and β-actin (mouse) (both Sigma-Aldrich); ERK1/2 (rabbit; Insight Biotechnology); and pERK (mouse; BD Biosciences) were used in conjunction with rabbit antigoat (Dako, Ely, UK), donkey antirabbit, and sheep antimouse (both GE Healthcare, Little Chalfont, UK) secondary IgGs.

Macrophage treatment assays
Peritoneal macrophages were isolated by ip lavage with ice-cold sterile PBS, followed by purification using Ficoll-Paque Plus (GE Healthcare) in accordance with the manufacturer’s instructions. Cells from the animals of each treatment group were pooled and then diluted to a concentration of 2 x 106 cells/ml in phenol red-free DMEM, supplemented with 5% charcoal-stripped fetal bovine serum. Cells were treated with bacterial lipopolysaccharide (1 µg/ml) for 6 h at 37 C, 5% CO2.

Quantitative real-time PCR
Total RNA was extracted from frozen d 3 wound tissue and peritoneal macrophages using Trizol (Invitrogen, Paisley, UK) and was then purified using RNeasy minikits (QIAGEN, Crawley, UK) in accordance with the manufacturer’s instructions. cDNA was synthesized from 1 µg of RNA using a reverse transcriptase kit (Promega, Southampton, UK) and a separate avian myeloblastosis virus reverse transcriptase enzyme (Roche, Lewes, UK). Quantitative real-time PCR (qPCR) analysis was performed using a SYBR Green I core kit (Eurogentec, Romsey, UK), in conjunction with an Opticon qPCR thermal cycler (Genetic Research Instrumentation, Braintree, UK), following the manufacturer’s guidelines. Optimal sample dilutions were determined and the specificity of amplification assessed through the analysis of product melting curves. Each test sample was serially diluted over 3 orders of magnitude, and all samples were analyzed on a single 96-well plate. PCR was carried out using primer pairs designed to amplify specific portions of the genes encoding CD44, CD74, JAB1, and CXCR2 and, for normalization purposes, the housekeeping genes that encode, the 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase , and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein-{zeta}. Primer sequences are listed in Table 1Go.


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  		TABLE 1. Primer sequences and product sizes for qPCR-amplified genes

 
Enzyme immunoassays
Sera were prepared from mouse blood isolated through cardiac puncture. Serum testosterone and 17β-estradiol levels were determined by competitive enzyme immunoassay (EIA; MP Biomedicals, Cambridge, UK) according to the manufacturer’s instructions. Serum DHT levels were measured through competitive EIA (Autogen Bioclear, Calne, UK). All analyses were performed in triplicate.

Statistical analysis
Simfit (version 6.0.12; William Bardsley, University of Manchester, Manchester, UK) was used to determine statistically significant intergroup differences by unpaired Student’s t test and one-way ANOVA. Post hoc testing was performed using Tukey’s Q test. Data determined (by the Shapiro-Wilks test) not to be parametric were analyzed through pairwise Mann-Whitney U testing with Bonferroni correction. The results of these statistical tests are presented as follows: tdf = x, P < y; Fa,b = x, P < y (where a represents between-group degrees of freedom and b residual degrees of freedom); Qn1,n2 = x, P < y; and Un1,n2 = x, P < y. P < 0.05 was considered significant (parametric data).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sex differences exist in fundamental wound-healing parameters
There is growing evidence that endogenous androgens (1, 2) and estrogens (3, 4), respectively, inhibit and enhance skin repair. To assess whether well-characterized sex differences in systemic steroid profiles are associated with divergence in the response to skin wounding, the healing of dorsal incisions in 8-wk-old male and female BALB/c mice was studied. In intact (nongonadectomized) animals, mean d 3 wound areas were comparable in males and females (Fig. 1AGo). However, the average wound width was significantly greater in female mice than male animals. The extent of reepithelialization on d 3 after wounding was similar in male and female mice. In gonadectomized animals, gender differences in each one of these three parameters were identified (Fig. 1BGo). Wound areas and widths were highly significantly greater in OVX females relative to CSX males. Furthermore, reepithelialization was less advanced in OVX mice than CSX animals, in which it was complete. In MIF knockout animals, wound areas, and widths in OVX females exceeded those in CSX males (Fig. 1CGo), although reepithelialization was comparable in these two groups. Based on these observations, it appears that d 3 wound widths in female mice are greater than those in males, irrespective of gonadal and MIF status. By contrast, significant sex differences in wound areas and the progress of reepithelialization emerge only when gonadal sex steroid biosynthesis as a source of divergence is removed.


Figure 1
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  		FIG. 1. The effects of gonadectomy and MIF deletion on healing in male and female mice. Wound areas and widths and extent of reepithelialization were measured on d 3 after wounding in male (M) and female (F) mice: intact (In) BALB/c (WT) (A) (t6 = 3.09; *, P < 0.03); CSX and OVX WT (B) (U5,5 = 0.00; **, P < 0.008) (t8 = 15.8; #, P < 0.001) (U5,5 = 25.0; {dagger}, P < 0.008); and OVX and CSX MIF knockout (KO) (C) (U6,6 = 1.00; **, P < 0.005) U6,6 = 3.00; *, P < 0.02). Values shown are means ± 1 SD (n = 4–6 per treatment group). Arrows (A–C) identify the wound margins.

 
Male and female MIF knockout mice respond differently to MIF
Although the contribution MIF makes to the estrogenic regulation of skin repair in female mice is well defined (10, 11), its roles in males have not been explored. Having found that wounds in male and female gonadectomized MIF knockout mice exhibit distinct properties, we next sought to characterize the responses to MIF treatment and hormonal replacement in these animals. Day 3 wounds were of similar mean area in intact male and female mice (Fig. 2Go, A and B). In males, castration reduced d 3 wound areas. The local administration of MIF to OVX mice elicited a significant increase in d 3 wound areas. In CSX males, by contrast, MIF elicited no such response. Replacement of testosterone in MIF-treated CSX animals did, however, increase d 3 wound areas. In OVX females, systemically delivered 17β-estradiol yielded no measurable response. In summary, whereas OVX MIF knockout mice apparently respond negatively to MIF treatment and are resistant to estrogens’ beneficial properties when cotreated with MIF, healing in CSX animals is impaired by the combination of MIF and testosterone but not by MIF alone. Intriguingly, reepithelialization of wounds in MIF knockout mice was unaffected by MIF or either of the hormone treatments (Fig. 2BGo).


Figure 2
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  		FIG. 2. Repair in MIF knockout mice treated locally with MIF and systemically with sex steroids. A, Day 3 wounds from intact (In), CSX, and OVX MIF knockout (KO) mice, subsets treated locally with MIF (500 ng per wound site) and/not slow-release testosterone (T; 10 mg) or 17β-estradiol (E2; 0.05 mg) pellets. B, Wound areas and extent of reepithelialization on d 3 after wounding in MIF-, E2-, and T-treated CSX and OVX mice [F3,16 = 47.9, P < 0.001; Q5,6 = 13.9, **, P < 0.001 (CSX vs. In); Q4,5 = 9.54, ##, P < 0.001 (CSX + MIF + T vs. CSX + MIF); F3,17 = 4.14, P < 0.03; Q5,6 = 4.29, *, P < 0.04 (OVX + MIF vs. OVX)]. Values shown are means ± 1 SD (n = 4–6 per treatment group). Bars, Wound areas; lines, reepithelialization. Arrows (A) identify the wound margins.

 
Distinct wound inflammatory profiles in male and female mice
Because hormone-linked differences/changes in healing rates have previously been attributed, at least in part, to alterations in the local inflammatory response (1, 2, 4), key wound inflammatory parameters were compared in d 3 wound tissue sections and total protein samples prepared from male and female mice. Staining for the marker Ly-G6 (Fig. 3AGo) revealed neutrophil numbers to be comparable between male and female animals, intact and gonadectomized. In MIF null animals, by contrast, the neutrophil population was significantly greater in OVX females than CSX males as well as being reduced (in both groups) relative to wild-type animals. Mac3-positive macrophage numbers were significantly greater in wild-type female mice than males (both intact and gonadectomized) (Fig. 3BGo). In MIF knockout animals, in contrast, no such sex difference was identified. Overall wound levels of MIF [proinflammatory and implicated in sex steroid regulated repair (3)] were comparable in intact male and female mice but significantly greater in OVX females than castrated males (Fig. 3CGo).


Figure 3
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  		FIG. 3. Gender differences in wound inflammation. Paraffin-embedded wound sections and total wound protein were respectively subjected to immunohistochemical and immunoblotting analyses. Numbers of Ly-6G-immunoreactive granulocytes (t10 = 3.90; **, P < 0.004) (A) and Mac3-positive macrophages (t6 = 6.90; **, P < 0.001; t8 = 6.29; {dagger}, P < 0.001) (B) in d 3 wounds in male (M) and female (F) intact (In), CSX, and OVX wild-type (WT) mice and CSX and OVX MIF knockout (KO) animals. C, Numbers of MIF-expressing cells and overall wound MIF levels in d 3 wounds in In, CSX, and OVX WT M and F mice (t8 = 2.68; *, P < 0.03). Values shown are means ± 1 SD (n = 4–6 per treatment group). Numerical data (immunoblots, C) represent signal ratios (protein of interest/β-actin). U5,6 = 30.0; #, P < 0.005 (CSX, WT vs. KO); U5,6 = 30.0; ##, P < 0.005 (OVX, WT vs. KO). Arrows (A–C) identify immunoreactive cells.

 
Inherent and hormonally controlled sex differences in MIF receptor profiles
Although wound MIF levels were found to be similar in intact male and female BALB/c mice, MIF is tightly regulated by estrogens during skin repair (10), mediating inhibition of healing in OVX mice (11). We hence probed the influence of gender and sex steroids upon expression and activation of MIF signaling intermediates. MIF activates intracellular cascades by binding to its cell surface-expressed receptor CD74 (14). Greater numbers of d 3 wound CD74-expressing cells were present in intact, wild-type, female mice than in males (Fig. 4AGo), suggesting that females may respond more strongly to injury-induced MIF than males. No such gender differences were observed in gonadectomized mice, wild type or MIF knockout, suggesting that the divergence in intact animals has a hormonal basis. CD74 signals intracellularly by forming functional complexes with its binding partners CD44 (15) and CXCR2 (16). In d 3 wounds, CD44 protein was primarily localized to the proliferating epidermis (Fig. 4BGo). Although similarly expressed in intact male and female wild-type mice, it was present in greater quantities in castrated males than OVX females (Fig. 4BGo), as was CD44 mRNA (Fig. 4CGo). The castration-invoked increase in wound CD74 protein levels was accompanied by a parallel increase in CD74 mRNA expression (Fig. 4CGo). CXCR2 mRNA expression was significantly higher in ovariectomized female MIF knockout mice than in castrated males (Fig. 4CGo).


Figure 4
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  		FIG. 4. Wound MIF receptor profiles in male and female mice. Paraffin-embedded wound sections and total wound mRNA were, respectively, analyzed immunohistochemically and by qPCR. A, Numbers of d 3 wound CD74-expressing cells in male (M) and female (F) intact (In), CSX, and OVX wild-type (WT) mice and CSX and OVX MIF knockout (KO) animals (t6 = 3.48; *, P < 0.02). B, Epidermal CD44 expression in d 3 wounds in M and F WT and KO mice (U5,5 = 22.0; *, P < 0.05). C, Day 3 wound expression of CD74, CD44 and CXCR2 mRNA in M and F mice [M vs. F: (U5,5 = 24.0; *, P < 0.02) (U6,5 = 29.0; **, P < 0.009) (t8 = 2.37; {dagger}, P < 0.05) (U6,5 = 5.00; {ddagger}, P < 0.05]. Values shown are means ± 1 SD (n = 4–6 per treatment group). U4,5 = 19.0; #, P < 0.04 (WT, In vs. CSX); t9 = 5.73; ##, P < 0.001 (CSX, WT vs. KO). Arrows (A) identify immunoreactive cells.

 
Dimorphism in the expression of the intracellular MIF signaling intermediate JAB1
JAB1 is an intracellular protein that binds to MIF and inhibits its ability to signal (17). In d 3 wounds, JAB1 was immunolocalized to both immigratory inflammatory cells and the neoepidermis (Fig. 5AGo). Wound numbers of JAB1-positive cells (immunohistochemically assessed) (Fig. 5BGo) were comparable in intact wild-type male and female mice but greater in OVX females than CSX males. In MIF knockout mice, fewer wound JAB1-positive cells were present in CSX males than OVX females. Overall wound JAB1 protein levels (assessed through immunoblotting) (Fig. 5CGo) were reduced through castration such that in OVX mice (wild type and MIF knockout), they far exceeded those in CSX animals. In OVX MIF knockout females, MIF treatment reduced wound JAB1 levels (Fig. 5CGo). No significant differences in total wound JAB1 mRNA levels between different animal groups were detected (Fig. 5DGo). Moreover, JAB1 mRNA expression was similar in lipopolysaccharide-primed macrophages from CSX and OVX MIF knockout mice (Fig. 5EGo). JAB1 mRNA expression was approximately 1000-fold greater in macrophages than total wound tissue, reinforcing the conclusion that this particular cell type is an important wound source of JAB1.


Figure 5
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  		FIG. 5. The effects of sex steroids and MIF on wound JAB1 expression. Paraffin-embedded wound sections and total wound protein were respectively subjected to immunohistochemical and immunoblot analyses. Macrophage and total wound mRNA samples were analyzed by qPCR. A, Day 3 wound JAB1 localization. Numbers of JAB1-expressing cells (B) (U5,5 = 25.0; **, P < 0.008) (t8 = 5.43; ##, P < 0.001) and overall JAB1 expression (C) in male (M) and female (F) intact (In), CSX, and OVX wild-type (WT) and MIF knockout (KO) mice. D, Day 3 wound JAB1 mRNA levels in M and F WT and KO mice. E, Comparative JAB1 mRNA expression in d 3 wounds and lipopolysaccharide-treated macrophages from KO mice. Values shown are means ± 1 SD (n = 4–6 per treatment group). Numerical data (C) represent signal ratios (protein of interest/β-actin). Arrows (A) identify immunoreactive cells. E2, 17β-Estradiol; M, MIF; T, testosterone.

 
ERK activation profiles in male and female mice
Having identified putative hormone-dependent and -independent components to the sexually divergent regulation of wound CD74 and JAB1 expression, we next investigated the potential influence of gender, sex hormones and MIF on wound activation of ERK, which may be activated by MIF via both CD74 (14) and JAB1 (18). In d 3 wounds, ERK1/2 protein was localized to both the neoepidermis and wound inflammatory cells (Fig. 6AGo). Phospho-activated ERK (pERK) immunoreactivity was, however, restricted to the neoepidermis (Fig. 6BGo), suggesting this to be the primary site of ERK signaling activity in healing wounds. Whereas overall wound ERK protein levels were similar in wild-type male and female mice (intact and gonadectomized), pERK activity in females (intact and OVX) exceeded that in males (intact and CSX). Seemingly hormone independent, this sex difference was replicated in gonadectomized MIF knockout mice (Fig. 6CGo). The observed reduction in wound pERK levels in OVX, MIF knockout mice injected locally with MIF seemingly resulted from a prior decrease in overall wound ERK levels.


Figure 6
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  		FIG. 6. Wound ERK activity profiles in male and female mice. Paraffin-embedded wound sections and total wound protein were respectively analyzed immunohistochemically and through immunoblotting. Day 3 wound expression of ERK1/2 (A) and pERK (B) in male (M) and female (F) intact (In), CSX, and OVX wild-type (WT) mice. C, Expression, in d 3 wounds, of ERK1/2 and pERK in MIF (M)-, 17β-estradiol (E2)- and testosterone (T)-treated CSX and OVX MIF knockout mice (n = 4–6 per treatment group). Numerical data represent signal ratios (protein of interest/β-actin). Arrows (A) identify immunoreactive cells.

 
Marked differences in the responses of male and female mice to exogenous sex steroids
Having used CSX and OVX mice to identify and characterize underlying, nonhormonal sex differences in the healing of skin wounds, we lastly sought to compare the responses of male and female animals to systemic treatment with estrogens and androgens. In CSX males, but not OVX females, testosterone increased d 3 wound areas (Fig. 7AGo). Intriguingly, 17β-estradiol enhanced repair in OVX females but retarded healing in CSX males (Fig. 7AGo). These sex differences did not apparently result from global differences in steroid release or metabolism: circulating levels of testosterone, DHT, and 17β-estradiol were comparable in similarly treated males and females (Fig. 7BGo).


Figure 7
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  		FIG. 7. The effects of HRT on wound healing in gonadectomized male and female mice. A, Day 3 wound areas (determined from hematoxylin and eosin stained tissue sections) in CSX and OVX mice treated with testosterone (T) or 17β-estradiol (E2) [F2,13 = 26.9, P < 0.001; Q6,5 = 4.19, *, P < 0.03 (CSX + T vs. CSX); Q6,5 = 10.4, **, P < 0.001 (CSX + E2 vs. CSX)] [F2,12 = 17,4, P < 0.001; Q5,5 = 7.42, ##, P < 0.001 (OVX + E2 vs. OVX)]. B, Serum T, E2, and DHT levels in T- and E2-treated CSX and OVX mice, measured by competitive EIA. Values shown are means ± 1 SD (n = 5–6 per treatment group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The recovery from skin injury is a complex, multifaceted process that requires the cooperation of resident and immigratory cells, cytokines, growth factors, enzymes, and receptors. Although estrogens have been shown to accelerate wound healing (3, 4), primarily by constraining expression of the deleterious factor MIF (10, 11) and androgens to inhibit repair (2, 19), the mechanisms by which males and females heal acute skin wounds have not previously been compared directly.

Surgical castration of male mice lowers serum testosterone to undetectably low levels (20); in females, ovariectomy has a similar effect on circulating estrogen levels (21). However, it is very apparent that equalizing circulating sex hormone levels through gonadectomy does not, in the context of wound healing, yield equivalents. The histological differences identified in d 3 wounds in intact male and female mice (i.e. wounds being wider in the latter) were markedly accentuated in gonadectomized animals (Fig. 8Go). Wound areas were greater and reepithelialization less rapid in OVX female mice than castrated males. These findings corroborate previous observations that castration decreases d 3 wound areas (1, 2), whereas ovariectomy has the opposite effect (10); and that systemic estrogen-based HRT (3) and the blockade of DHT (36), respectively, accelerate wound reepithelialization in elderly females and young male rats. Moreover, in in vitro assays on skin-derived keratinocytes, DHT retards scratch wound-induced migration (36), whereas 17β-estradiol stimulates proliferation (22).


Figure 8
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  		FIG. 8. A summary of the contributions of sex steroids, MIF, and associated factors to gender dimorphism in wound healing. In wild-type (WT) mice, gonadectomy (GDX) improves healing in males (by preventing inhibitory androgens from being synthesized) and worsens healing in females (by removing the restraining effects of estrogens on expression of harmful MIF). In MIF knockout (KO) animals, gonadectomy improves healing in males but is without obvious effect in females. The local administration of MIF worsens repair in GDX, KO females, but not GDX, KO males. Relative wound areas, extent of reepithelialization, and endogenous MIF levels are indicated schematically. T, Testosterone; E2, 17β-estradiol.

 
The fundamental differences we uncovered in the responses of gonadectomized male and female mice to systemic treatment with both androgens and estrogens was surprising. That testosterone did not impair healing in OVX females, as it did in CSX males, may reflect: 1) its local aromatization to estrogens, which may counteract any deleterious effects of testosterone itself; 2) the observation that healing is so retarded in OVX females that further impairment cannot be detected; or 3) the relation of impaired healing in females to high local MIF levels, which are unaffected by testosterone. Our parallel observation that 17β-estradiol, well established as a potent enhancer of repair in females (3, 10), inhibits healing in CSX males may be the manifestation of a fundamental difference in the responses of males and females to sex steroids. The basis of such sex divergence may be differences in hormone dose optima and/or downstream signaling effectors engaged (e.g. MIF).

Key genetic differences direct the dimorphic development of male and female mammals such that they are endocrinologically and physiologically distinct. It is our conclusion that, in the context of wound healing, hormonal factors act to mask underlying differences in repair. The influence on repair of intrinsic gender differences in such processes as tissue morphogenesis and perinatal hormonal imprinting may be exposed only when these hormonal factors are removed. Initial exposure of a receptor to a hormone alters its binding capacity and that of the receptors in progeny cells (23). Such changes are not reversed by subsequent hormone withdrawal. Furthermore, Y chromosome genes may influence repair in males, and it should be noted that skin architecture itself displays inherent gender divergence: the dermis is thicker in males than females and the epidermis and hypodermis both thicker in females (in both intact and gonadectomized animals) (24).

The master regulator of impaired healing in ovariectomized female mice (10, 11), MIF did not influence the rate of healing in castrated MIF knockout males. And whereas testosterone inhibited repair in MIF-treated males, 17β-estradiol was without significant effect in their female counterparts. It therefore appears that the primary role of estrogens during healing is to constrain MIF expression and that when MIF is locally abundant, they are relatively inactive. Such a conclusion is supported by the previous finding that, of the 380 microarray-identified gene expression changes effected by estrogens in mouse wounds, fewer than 10% were realized independently of MIF (11).

In males, the situation appears to be somewhat different. MIF apparently does not possess the same potent inhibitory properties as it does in females. Moreover, disruption of the MIF gene did not affect castration’s capacity to enhance healing. Although in humans serum testosterone and MIF levels have been positively correlated (25), overall wound MIF levels in mice were not measurably affected by castration. MIF, in isolation, seems not to modulate significantly the healing rate of acute skin wounds in males.

Overall wound MIF levels were increased by ovariectomy in wild-type BALB/c mice, as has been shown previously (10, 11). This increase was not paralleled by a similar increase in numbers of macrophages, the principal wound source of MIF and a target of estrogenic suppression of MIF production (10). We report wound macrophage numbers to be higher in wild-type female mice than in males. Increased macrophage numbers have been implicated in suboptimal healing of diabetic and chronic venous leg ulcers in human patients (26) and acute wounds in ovariectomized mice (10). In the present study, a larger wound macrophage population in females relative to males was translated into slower healing in gonadectomized, but not intact, animals.

Additional factors including MIF undoubtedly contribute to defective repair in OVX animals. MIF signals by two major mechanisms: via its cell surface receptor CD74 (14) in partnership with binding partners CD44 (15) and CXCR2 (16) and, after endocytosis, by binding to and inhibiting the activity of the intracellular protein JAB1 (17). In d 3 wounds, CD74 was largely localized to inflammatory cells and cells presenting a dendritic morphology in the epidermis (presumably Langerhans cells). Although greater numbers of CD74-positive inflammatory cells were present in the wounds of intact female mice than males, this difference was abolished through mutual gonadectomy and absent in MIF knockout mice. Hence, CD74 may not be an important contributor to gender dimorphism in the responses to wounding and MIF treatment. Moreover, wound expression of CD44 protein and mRNA was reduced through ovariectomy, through a mechanism that may be the consequence of estrogen withdrawal [because 17β-estradiol has been reported to increase CD44 protein expression in multiple human cell lines (27)], and was similar in castrated and OVX MIF knockout mice. Although CXCR2 mRNA was significantly more highly expressed in OVX MIF knockout mice than castrated animals, altered CD74 signaling most likely does not exacerbate impaired healing in OVX mice, wild-type and MIF-treated MIF knockout.

Having previously been shown to stabilize interaction of the progesterone receptor with the coactivator steroid receptor coactivator-1 (SRC-1) (28), JAB1 was recently shown to coactivate the androgen receptor (29). Its potential involvement in sexually dimorphic injury responses is therefore of significant interest. Our finding that wound JAB1 levels were greater in OVX females than castrated males suggests that JAB1 may be an effector of gender-divergent wound healing. Moreover, depletion of JAB1 in castrated mice may limit MIF’s ability to influence repair. In OVX females, the activities of JAB1 may be limited by the induction of MIF. In male MIF knockout mice, the absence of significant JAB1 activity may contribute to their failure to respond to exogenous MIF. In females, in which JAB1 is highly expressed, MIF may obstruct repair by inhibiting JAB1’s activities. Moreover, overall wound JAB1 levels were decreased as a result of MIF treatment.

Whether the overall effect of JAB1 on wound healing is positive or negative remains to be determined. JAB1 has previously been reported both to stimulate cell growth (17) and prevent apoptotic cell death (30). Increases in these putatively advantageous activities in OVX mice may be prevented by the concurrent increase in wound MIF levels. It is, however, by no means certain that JAB1 is beneficial to wound healing. In alternative contexts, notably cancer, its up-regulation is associated with poor clinical prognosis (31, 32). Moreover, JAB1 has been implicated in the pathology of atherosclerosis, JAB1-MIF complexes having been isolated from atherosclerotic plaques (33).

A necessary factor for reepithelialization (34, 35), ERK may be activated by MIF via either CD74 (14) or JAB1 (18). Our demonstration that pERK localized almost exclusively to wound edge keratinocytes suggests that any activation by MIF is affected by JAB1 (because these cells did not express CD74). Intriguingly, overall wound pERK activity was greater in females than males, irrespective of MIF genotype or gonadal status (intact/gonadectomized). However, that wound pERK levels were respectively reduced and unaltered in MIF knockout females and males treated with MIF and unaffected by the major up-regulation of MIF that follows ovariectomy in wild-type animals prompts us to question the significance of MIF’s role in activating ERK during wound healing.

In summary, our data identify some of the key factors that underscore gender dimorphism in wound healing. The overall picture is highly complex: steroid sex hormones, MIF, and unidentified factors all conspire to minimize gender dimorphism in intact animals such that ablation of gonadal androgens and estrogens (which have broadly opposite effects on healing) generates stark sex differences. Normalization of the hormonal milieu reveals underlying differences in the ways that males and females heal wounds. These differences extend to the response to MIF, which worsens repair in females but not males. Our findings have important implications for the treatment of chronic wound pathologies, characterized by unresolved inflammation and dramatic overexpression of MIF (our unpublished observations).

As the field of pharmacogenetics expands, future prohealing therapies may involve optimization according to each individual’s genotype. In the development of such personalized medicines, patient gender should be an important consideration.


   Footnotes
 
This work was supported by Wellcome Trust Senior Fellowship in Clinical Science Grant GR064256MA.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: CSX, Castrated; DHT, 5{alpha}-dihydrotestosterone; EIA, enzyme immunoassay; HRT, hormone replacement therapy; JAB1, Jun activation domain-binding protein 1; MIF, migration inhibitory factor; OVX, ovariectomized; pERK, phospho-activated ERK; qPCR, quantitative real-time PCR; SDS, sodium dodecyl sulfate; Smad3, mothers against decapentaplegic homolog 3.

Received March 14, 2008.

Accepted for publication July 14, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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