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Published as doi: 10.1096/fj.06-5837fje.
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(The FASEB Journal. 2006;20:2366-2368.)
© 2006 FASEB

Mast cells are required for normal healing of skin wounds in mice

Karsten Weller*,{dagger}, Kerstin Foitzik{ddagger}, Ralf Paus§, Wolfgang Syska*,|| and Marcus Maurer*,||,1

* Department of Dermatology and Allergology, Allergie-Centrum-Charité, Charité – Universitätsmedizin Berlin, Berlin, Germany;

{dagger} Department of Dermatology, University Hospital Würzburg, Würzburg, Germany;

{ddagger} Department of Dermatology, University Hospital Hamburg-Eppendorf, Hamburg, Germany;

§ Department of Dermatology, University Hospital Lübeck, Lübeck, Germany; and

|| Department of Dermatology, University Hospital Mainz, Mainz, Germany

1Correspondence: Department of Dermatology and Allergy, Charité – Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. E-mail: marcus.maurer{at}charite.de

ABSTRACT

Mast cells (MCs) have recently been reported to play a pivotal role in the elicitation of inflammatory reactions that are beneficial to the host, e.g., during innate immune responses to bacteria. To explore whether MCs also contribute to wound repair, we studied experimentally induced skin wounds in MC-deficient KitW/KitW-v mice, normal Kit+/+ mice, and MC-reconstituted KitW/KitW-v mice. Wound closure was significantly impaired in the absence of MCs during the first 6 days of wound healing and histomorphometric analyses of MC degranulation at the wound edges revealed distance-dependent MC activation, i.e., MC degranulation was most prominent directly adjacent to the wound. In addition, KitW/KitW-v mice showed impaired extravasation and recruitment of neutrophils to the wounded areas. Notably, wound closure, extravasation, and neutrophil recruitment were found to be normal in MC-reconstituted KitW/KitW-v mice. Therefore, we examined whether MCs promote wound healing by releasing histamine or TNF-{alpha}. Interestingly, wound closure was reduced in mice treated with an H1-receptor antagonist but not after treatment with an H2-receptor antagonist or in the absence of TNF-{alpha}. Taken together, our findings indicate that MC activation and histamine release are required for normal cutaneous wound healing.—Weller, K., Foitzik, K., Paus, R., Syska, W., Maurer, M. Mast cells are required for normal healing of skin wounds in mice.


Key Words: inflammation • neutrophil

MAST CELLS (MCS) ARE WIDELY recognized to contribute to the pathology of inflammatory conditions including allergies and autoimmune diseases (1) . In addition, MCs have recently been shown to promote inflammatory responses that are beneficial to the host. For example, peritoneal MCs control bacterial infections by initiating protective innate immune responses to bacteria, at least in part, by releasing TNF-{alpha} and recruiting neutrophils (PMNs; refs 2 , 3 , 4 , 5 ).

Cutaneous inflammatory responses, e.g., to allergens or autoantigens, are also associated with the activation of MCs and their release of proinflammatory mediators. This is not surprising as large numbers of MCs are expressed in the skin, where MCs represent {approx}2–8% of dermal cells (6) . Also, cutaneous MCs are preferentially localized in the vicinity of the epidermis, hair follicles, blood vessels, and nerves (7 , 8 , 1) , i.e., skin compartments, where MCs can be readily activated by environmental and endogenous triggers. Even though skin MCs are thought to promote resistance to ticks, control hair growth, and induce foreign body granuloma formation (9 , 10 , 11 , 12) , their role and functional relevance in cutaneous physiology remain largely unclear (1) .

Because of their large repertoire of proinflammatory and growth-promoting mediators such as histamine, leukotrienes, prostaglandins, proteases, and cytokines (13 , 14 , 15) , MCs have been repeatedly hypothesized to contribute to the healing of skin wounds (16 , 17 , 18 , 19 , 20) . Cutaneous wound healing (WH) is characterized by three sequential phases (21 , 22) : 1) Inflammation as a direct consequence of wounding, 2) proliferation, and 3) remodeling and MCs have been suggested to be involved in all three WH phases (23 , 24 , 19) .

During early WH, the MC mediators histamine, serotonin, and TNF-{alpha} are thought to contribute to the induction of localized coagulation, extravasation, and leukocyte recruitment, important features of the initial inflammatory phase of WH (25 , 26 , 27 , 21 , 22) . Subsequently, MCs reportedly promote the proliferation of fibroblasts, endothelial cells, and keratinocytes during the proliferative phase of WH. For example, we and others (28 , 29) have shown that the MC products histamine and serotonin exert mitogenic effects on murine epidermal keratinocytes in situ. Also, MCs can promote the conversion of fibroblasts to a myofibroblast phenotype (20) , which facilitates wound contraction and closure. Studies on in vitro wounds, furthermore, have shown that MCs can stimulate fibroblast proliferation and migration (30) and that MCs, which accumulate at sites of neovascularization (31 , 32) , can induce neoangiogenesis (33) .

Finally, MCs may be involved in tissue remodeling, the key feature of late WH responses. Skin MCs produce and release potent proteolytic enzymes, such as matrix metalloproteinases (MMP, e.g., MMP9), which facilitate tissue remodeling by initiating the degradation of extracellular matrix (ECM; ref 34 ). Also, we and others have previously shown that cutaneous remodeling, e.g., during hair follicle growth and regression, is importantly modulated by skin MCs and MC-derived histamine (10 , 11) . Furthermore, the inhibition of skin MC histamine synthesis in wounded rats has been shown to decrease the hydroxyproline content of granulation tissue, to delay epithelization, and to reduce wound breaking strength (35) .

Despite this extensive body of suggestive evidence in support of MC functions during WH, a role of MCs in this context, at least to our knowledge, has yet to be proven, and the relevance and mechanisms of MC effects on WH remain to be characterized in detail. In light of this lack of direct evidence for a critical role of MCs in WH, the view that MCs are key WH initiators has recently been challenged by several authors who suggest that MCs are dispensable in uncomplicated wound repair responses (36 , 37) . As a consequence of these uncertainties, a recent landmark review on WH by Werner and Grose (21) does not even list MCs as potential players in the execution and control of WH.

Therefore, we have used in the current study the well-established in vivo model of genetically MC-deficient KitW/KitW-v mice, and their reconstitution with functional MCs (12) , to probe whether direct and conclusive evidence for MC functions during skin WH can be obtained. Our results provide the first definitive proof that MCs are indeed functionally important during early WH and that wound closure and inflammatory wounding responses (including extravasation and neutrophil recruitment to sites of injury) are MC dependent.

MATERIALS AND METHODS

Animals
Genetically MC-deficient WBB6F1-KitW/KitW-v (KitW/KitW-v) mice and congenic normal WBB6F1-Kit+/+ (Kit+/+) mice were purchased from Jackson Laboratories (Bar Harbor, ME). TNF-{alpha} –/– mice and TNF-{alpha} +/+ mice on a mixed 129/Sv x C57BL/6 genetic background (38) as well as C57BL/6 mice were bred and housed in community cages at the Animal Care Facilities of the Department of Dermatology, Mainz. Mice were used at 6–11 wk of age, when all hair follicles of the back skin were in the telogen (resting) phase of the hair cycle. All animal care and experimentation were conducted in accordance with current federal, state, and institutional guidelines.

MC reconstitution of MC-deficient KitW/KitW-v mice
The MC deficiency of KitW/KitW-v mice (female, 4–6 wk old) was repaired locally and selectively by the injection of growth factor-dependent bone marrow derived cultured mast cells (BMCMCs; 39 , 5 ) into the lower back skin. Briefly, femoral bone marrow cells from Kit+/+ mice were grown in vitro for 4 wk in interleukin (IL)-3 containing medium until MCs represented >95% of total cells as determined by Giemsa staining. MCs (106 in 200 µl Dulbecco’s modified Eagle’s medium per cm2, 20 injections of 10 µl each) were injected into an area of 4 cm2 of the lower back skin of KitW/KitW-v mice. Four weeks after adoptive transfer of BMCMCs, mice were used for experiments, together with gender- and age-matched Kit+/+ mice and KitW/KitW-v mice that had not been MC reconstituted. In all cases, local reconstitution of dermal MCs was confirmed by histomorphometric analysis of paraffin-embedded, Giemsa-stained sections of the reconstituted area (5) . For these analyses, MC numbers were assessed at the periphery of the wounded skin obtained (see below) to avoid distortions of MC numbers due to the response to wounding, which are to be expected to occur in the central areas. The results show an average of 6.38 MCs per microscopic field (MF) in wild-type (WT) mice (n=12) and 3.51 MCs/MF (n=7) in MC-reconstituted KitW/KitW-v mice (at x400 magnification), indicating that the adoptive transfer of BMMCs to the back skin results in the repopulation of these sites with skin MCs. The distribution of MCs in the repopulated sites was largely comparable with those in WT controls.

Wounding and measurements of wound area
Full-thickness wounds (one per mouse) were induced on the lower back skin of mice as described previously by Werner and coworkers (40) . Briefly, the dorsal hair of mice was shaved 24 h before wounding. Mice were anesthetized intraperitoneally (ip; ketamine/xylazine), and the skin was wiped with 70% ethanol and wounded using a 6 mm diameter biopsy punch (Sklar Instruments, West Chester, PA). Wound area was assessed by measuring vertical and perpendicular diameter at distinct time points after wounding and calculating the area by using the formula for an ellipse: (vertical diameter/2) x (perpendicular diameter/2) x {pi}.

Detection of MC degranulation
Skin of the wound edge and back skin distant to the wound area serving as control was harvested 1 h after wounding. Tissue samples were processed for 1 µm Epon-embedded, alkaline Giemsa stained sections as described previously (41) . Dermal MCs in the first five microscopic fields adjacent to the wound edge and in control skin were classified (at x1000 magnification) as extensively degranulated (>50% of the cytoplasmic granules exhibiting fusion, staining alterations, and/or extrusion from cell), moderately degranulated (10 to 50% of granules exhibiting fusion or discharge), or not degranulated (41) .

Detection of keratinocyte proliferation
Skin wounds were harvested 24 and 72 h after wounding, and tissue samples were processed for immunostaining following standard protocols. Briefly, the tissue was fixed in 5% buffered formalin overnight and the paraffin-embedded sections from the central portion of the wound and the surrounding tissue were heated for 1 h in citrate buffer pH 6.0. Sections were then washed with TBS (DAKO S3001, DAKO Deutschland GmbH, Germany) before incubation for 1 h at room temperature with the primary antibody (Ab) against Ki67 (DAKO M7249) diluted 1:25 in Ab diluent (DAKO S3022). After a second washing, sections were incubated with a biotinylated immunoglobulin (Ig; DAKO E0468) and streptavidine (DAKO P0397), each for 1 h at room temperature. Subsequently, 3-amino-9-ethyl carbazole+substrate-chromogen (DAKO K3469) was applied to all sections according to the instructions of the supplier and sections were counterstained with hemalaun. No staining was observed in parallel sections treated with control IgG instead of the primary Ab. The first 100 cells of the basal keratinocyte layer from the wound edge were classified (at x400 magnification) as Ki67 positive or negative cells, and the percentage of positive cells was calculated. The histopathologist performing theses analyses was blinded to the identity of the sections.

Evans blue extravasation assay
For measurement of vascular permeability, the back skin of mice was shaved. 24 h later, mice were anesthetized intraperitoneally and injected intravenously (iv) with 200 µl 1% Evans blue (Sigma-Aldrich Chemie) in PBS 10 min before wounding. Animals were killed 30 min after wounding, and the wound edge and control back skin were harvested, i.e., skin rings of 2 mm width were obtained by measuring 2 mm of skin using a ruler at a right angle to the wound edge. Subsequently, the skin was cut at the 2 mm distance around the wound down to the fascia and weighed [lsq]mean wt of skin samples for Evans blue and myeloperoxidase (MPO) assays: 0.025 g]. Evans blue was extracted by incubating the skin samples in 99% N,N-dimethyl-formamide (Merck-Schuchardt, Germany) at 37°C overnight (42 , 43) and measured at 650 nm photometrically (Zeiss DMR 10, Germany). Extravasated Evans blue per gram of tissue was calculated using a standard curve and the weight of the individual skin samples.

MPO assay
For the quantification of neutrophil accumulation, tissue MPO activity was assayed in back skin from the wound edge and control back skin, both obtained 0, 6, 12, 24, and 48 h after wounding as described above. MPO is the most abundant enzyme in primary neutrophils and has been shown to be a useful and reliable marker for neutrophil infiltration in inflammatory diseases such as asthma and rhinitis (44 , 45) . MPO was extracted from the homogenized tissue by suspending the material in extraction buffer (0.5% hexadecyltrimethylammonium bromide (Sigma, Germany) in 50 mM potassium phosphate buffer, pH 6) before sonication in an ice bath. After three freeze-thaw-cycles, sonication was repeated and suspensions were centrifuged at 4000 g for 30 min at 4°C. Triplicate aliquots of 100 µl supernatant were added to 2.9 ml of 50 mM potassium phosphate buffer (pH 6) containing 0.167 mg/ml o-dianisidine dihydrochloride (Sigma, Germany) and 0.0005% hydrogen peroxide (Sigma, Germany). MPO activity was measured by the change in optical density (OD) at 460 nm resulting from the decomposition of H2O2 in the presence of o-dianisidine dihydrochloride. MPO content was calculated as units per gram tissue by using a standard curve, which was established using purified MPO (Sigma, Germany).

Pharmacological inhibition of histamine receptors
The histamine receptor subclass-specific antagonists dimethindene (H1-receptor, 0.25 mg in 250 µl) or ranitidine (H2-receptor, 0.5 mg in 250 µl) or vehicle (saline) were injected intraperitoneally twice daily for 10 days, starting 12 h before wounding. Treatment protocols (concentrations, frequency and duration) are similar to those of previous reports (46) and based on the results of pilot experiments on mice subjected to passive systemic anaphylaxis (data not shown).

Statistical analysis
Data are presented as means ± SE. Statistical differences were determined using the unpaired two-tailed Student‘s t test, except for analysis of differences in MC degranulation, which were assessed using the {chi}2 test.

RESULTS

Early skin WH is impaired in the absence of mast cells
To compare the differences in skin wound closure between KitW/KitW-v mice and normal Kit+/+ mice, we measured the wound area of full-thickness back skin wounds at defined time points after wounding with a 6 mm biopsy punch (Fig. 1 ). Wound areas in Kit+/+ mice were found to decrease in size immediately after wounding (24.9±2.1 at +6 h vs. 27.9±1.6 mm2 at 0 h). Skin wounds continued to heal, as reflected by a persistent reduction in wound size, until day 10 after wounding, when virtually all wounds in Kit+/+ mice were closed (Fig. 1) .


Figure 1
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  		Figure 1. Healing of excisional full-thickness skin wounds is impaired in mast cell-deficient KitW/KitW-v mice. KitW/KitW-v mice (n=11) and WT (Kit+/+) littermates (n=18) were anesthetized, and their dorsal hair was shaved. A single full-thickness skin wound was prepared on the lower back of each mouse using a 6 mm biopsy punch. Wound area was assessed by measuring vertical and perpendicular diameter at distinct time points after wounding. Data pooled from 2 independent experiments. Results are mean ± SE; *P < 0.05, ***P < 0.005 comparing KitW/KitW-v and Kit+/+ mice.

In contrast, wounds in MC-deficient KitW/KitW-v mice did not decrease, but slightly increased, in size during the first hours after wounding (32.6±3.1 at +6 h vs. 30.2±1.5 mm2 immediately after wounding) and wound sizes only started to decrease after 12 h postwounding. A significant reduction of wound area sizes (as compared with sizes of initial wounds) was first achieved after 2 days. Significant differences in wound sizes of KitW/KitW-v mice and Kit+/+ mice were detectable at all time points assessed until day 6 after wounding. Differences in skin wound areas were most prominent 48 h after wounding, when KitW/KitW-v mice exhibited wounds that were almost twice as big as those in Kit+/+ mice (21.7±2.7 vs. 11.8±1.2 mm2, P<0.005). Interestingly, KitW/KitW-v mice showed discretely faster WH than Kit+/+ mice after day 2 postwounding (albeit not significantly) and completed wound closure at the same time as Kit+/+ mice, i.e., day 10 after wounding.

In a series of pilot experiments, we next examined the rates of proliferation of basal keratinocytes at the wound edge 24 and 72 h after wounding in KitW/KitW-v and Kit+/+ mice. At 24 h after wounding, KitW/KitW-v mice showed significantly reduced numbers of proliferating Ki67 positive basal keratinocytes as compared with WT mice [27 vs. 41% (P<0.02) Ki67 positive in KitW/KitW-v mice (n=4) vs. WT mice (n=9), respectively]. Interestingly, both types of mice exhibited similar numbers of Ki67 positive basal keratinocytes 72 h after wounding [75% vs. 70% (P=0.65) Ki67 positive cells in KitW/KitW-v mice (n=3) vs. WT mice (n=5), respectively].

MCs degranulate in response to skin wounding
To test whether MCs are activated by skin wounding in vivo, we assessed the extent of MC degranulation in skin wounds of C57BL/6 mice 1 h after wounding by quantitative histomorphometry. As shown in Fig. 2 , the majority of skin MCs (73%) directly adjacent to the wound exhibited signs of extensive degranulation. The extent of MC activation was found to correlate inversely with the distance of MCs from the wound edge, with MCs showing signs of significantly increased degranulation at skin sites as far as 300 µm distant from wound edges (Fig. 2) .


Figure 2
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  		Figure 2. Degranulation of mast cells after wounding is most prominent in areas directly adjacent to the wound. C57BL/6 mice (n=16) were subjected to full-thickness wounds (6 mm diameter biopsy punch) on the lower back skin. Plastic embedded 1 µm sections of the wound area obtained 1 h after wounding were stained with alkaline Giemsa and MC degranulation was assessed by quantitative histomorphometry in the first 5 microscopic fields adjacent to the wound margin at x1000 magnification. MCs were classified as "extensively degranulated" (>50% of cytoplasmic granules exhibiting staining alterations, fusion and/or extrusion from cell), "moderately degranulated" (10–50% of granules affected), "not degranulated" (<10% granules affected). Data pooled from 2 independent experiments. Results are mean ± SE; n.s. = not significant; ***P < 0.005 as compared with control skin.

Vascular permeability and neutrophil recruitment after wounding are decreased in the absence of mast cells
As skin MC degranulation can result in increased vascular permeability, a hallmark feature of early WH responses, we measured extravasation of Evans blue after wounding in KitW/KitW-v and Kit+/+ mice. Kit+/+ mice showed markedly increased levels of Evans blue extravasation at sites of wounding (30 min after wounding) as compared with control skin (>400%, P<0.005, Fig. 3 A). Notably, increases in vascular permeability in Kit+/+ skin wounds were found to be significantly larger than those in KitW/KitW-v mice (P<0.005), where wounding increased Evans blue skin concentrations <1-fold (Fig. 3A ).


Figure 3
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  		Figure 3. Extravasation and neutrophil accumulation at skin wound sites are reduced in the absence of mast cells. A) MC-deficient KitW/KitW-v mice (n=10) and normal Kit +/+ littermates (n=8) were injected iv with 200 µl 1% Evans blue. 30 min after wounding, skin at the wound edge was harvested, and Evans blue was extracted and measured photometrically. Data pooled from 2 independent experiments. Results are mean ± SE; *P < 0.05, ***P < 0.005 comparing control and wounded skin of same genotype; {dagger}{dagger}{dagger}P < 0.005 comparing wounded skin of KitW/KitW-v and Kit+/+ mice. B) MPO was extracted from skin at wound edge of KitW/KitW-v and Kit+/+ mice, and MPO-activity was assayed spectrophotometrically. Data pooled from at least 2 independent experiments. Results are mean ± SE; *P < 0.05, ***P < 0.001 comparing KitW/KitW-v and Kit+/+ mice.

To test whether the impaired up-regulation of vascular permeability at skin wound sites in KitW/KitW-v mice is associated with a reduction in subsequent PMN recruitment (compared with Kit+/+ mice), we measured levels of the PMN enzyme MPO in skin wounds of both genotypes (Fig. 3B ). Increase in MPO activity in wounded skin was detectable in both types of mice as early as 6 h after wounding, when values in Kit+/+ mice were already higher than in KitW/KitW-v mice, but no statistical significance could be detected; 12 h after wounding, MPO-levels in Kit+/+ mice exhibited a maximum increase (1.1±0.1 U MPO/g tissue) as compared with control skin (0.2±0.3 U MPO/g tissue, P<0.005). In contrast, MPO levels of KitW/KitW-v mice increased to a much lesser extent (0.5±0.1 U MPO/g tissue at 12 h after wounding vs. 0.2±0.4 U MPO/g tissue in control skin, P<0.005). Levels of PMN recruitment in KitW/KitW-v mice never reached Kit+/+ levels, at any of the time points studied, and differences of MPO-levels between both types of mice were statistically significant at 12, 24, and 48 h after wounding (P<0.005, P<0.05, and P<0.05, respectively). These findings demonstrate that PMN recruitment to sites of WH is markedly impaired in the absence of skin MCs.

Closure of skin wounds, up-regulation of vascular permeability, and PMN recruitment after wounding are MC dependent
In addition to their profound MC deficiency, KitW/KitW-v mice, as a consequence of their loss of function c-kit mutations, are also sterile, anemic, and deficient for melanocytes and interstitial cells of Cajal, and the Kit ligand, stem cell factor (SCF), is not able to adequately stimulate its cognate receptor on selected keratinocyte populations (47, 48, 49). Thus, in theory, impaired WH in KitW/KitW-v mice, i.e., failure to reduce skin wound areas during the initial phase of healing as well as defects in up-regulating extravasation and PMN recruitment in response to wounding, could be due to any or a combination of these Kit-dependent defects and independent of MCs.

To test this possibility, we reconstituted KitW/KitW-v mice locally and selectively with bone marrow-derived cultured MCs (BMCMCs) and assessed these mice for wound closure, extravasation, and PMN accumulation. Notably, the adoptive transfer of functional MCs to KitW/KitW-v mice led to a complete normalization of wound closure (Fig. 4 A). Moreover, extravasation and PMN accumulation (Fig. 4B,C ) were found to be fully restored in MC-reconstituted KitW/KitW-v mice. This provides definitive evidence that early cutaneous WH as well as up-regulation of vascular permeability and PMN recruitment is MC dependent.


Figure 4
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  		Figure 4. MCs are required for normal wound closure. A) Impaired wound closure of KitW/KitW-v mice (n=9) is repaired by prior reconstitution of the dermis with BMCMCs (n=11). Data pooled from 3 independent experiments. Results are mean ± SE; **P < 0.01, ***P < 0.005 comparing KitW/KitW-v and Kit+/+ mice (n=19); {dagger}P < 0.05, {dagger}{dagger}{dagger}P < 0.005 comparing KitW/KitW-v mice and mast cell-reconstituted KitW/KitW-v mice (KitW/KitW-v mice + BMCMCs). B) KitW/KitW-v mice + BMCMCs (n=11) exhibit normal extravasation responses to wounding. Data pooled from 3 independent experiments. Results are mean ± SE; ***P < 0.005 comparing control and wounded skin of the same group of mice; {dagger}P < 0.05, {dagger}{dagger}{dagger}P < 0.005 comparing wounded or control skin of KitW/KitW-v mice (n=11) and KitW/KitW-v mice + BMCMCs or Kit+/+ mice (n=18). C) Influx of neutrophils to sites of skin wounds as assessed by measuring skin MPO-levels is MC dependent. Data pooled from 2 independent experiments. Results are mean ± SE; ***P < 0.001 comparing control and wounded skin of same group of mice; {dagger}P < 0.05, {dagger}{dagger}{dagger}P < 0.005 comparing wounded or control skin of KitW/KitW-v mice (n=6) and KitW/KitW-v mice + BMCMCs (n=6) or Kit+/+ mice (n=6).

Inhibition of histamine, but not the absence of TNF-{alpha}, results in delayed skin wound closure
To assess mechanistically whether the MC products histamine and/or TNF-{alpha}, both of which promote extravasation and PMN recruitment, are involved in MC dependent skin WH responses, we analyzed skin wound closure in mice genetically deficient for TNF-{alpha} (TNF-{alpha}–/– mice) and in C57BL/6 mice treated with selective histamine receptor antagonists. Surprisingly, we found that TNF-{alpha}–/– mice exhibit significantly accelerated wound closure as compared with TNF-{alpha}+/+ (20.3 mm2 vs. 26.1 mm2 at 24 h, P < 0.05 and 15.5 mm2 vs. 20.3 mm2 at 48 h, P<0.05, Fig. 5 A) indicating that the absence of TNF-{alpha} does not result in impaired skin wound closure.


Figure 5
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  		Figure 5. Skin WH is impaired in mice treated with an H1-receptor antagonist but not after treatment with an H2-receptor antagonist or in the absence of TNF-{alpha}. TNF-{alpha} –/– mice (A; n=23) and TNF-{alpha} +/+ littermates (A; n=20) as well as C57BL/6 mice treated ip with 0.25 mg of the histamine-H1-receptor-antagonist dimethindene (B; anti-H1R, n=10), 0.5 mg of the histamine-H2-receptor-antagonist ranitidine (C; anti-H2R, n=8), or sterile saline solution (B and C; vehicle, n=11) were subjected to single full-thickness wounds using a 6 mm biopsy punch. Wound area was assessed 48 h after wounding by measuring vertical and perpendicular diameter. Data pooled from 2 independent experiments. Results are mean ± SE; *P < 0.05 (Student’s t test).

In contrast, C57BL/6 mice treated with the selective histamine-H1-receptor antagonist dimethindene showed significantly delayed skin wound closure as compared with vehicle treated mice (21.4±1.7 mm2 vs. 15.8±1.6 mm2 at 48 h, P<0.05), whereas treatment with the H2-receptor antagonist ranitidine did not affect WH (Fig. 5B and C). Interestingly, wound closure impairment in both dimethindene-treated mice and KitW/KitW-v mice was limited to the initial phase of WH and more pronounced in the latter group, suggesting that histamine is one important, but not the only, WH-promoting mediator provided for by skin MCs.

DISCUSSION

Here, we show that the early closure of murine skin wounds is impaired in the absence of MCs. Moreover, we demonstrate that normal WH is MC dependent, i.e., that the local and selective repair of skin MC deficiency by the adoptive transfer of functional MCs leads to the complete normalization of wound closure.

Our finding that early skin WH is impaired in genetically MC-deficient KitW/KitW-v mice is in line with earlier observations by Egozi and coworkers, who found impaired re-epithelization of excisional skin wounds in the absence of MCs as compared with wounds in normal mice (37) . However, these differences did not reach statistical significance, most likely due to the small number of animals used in this study, which also does not provide the decisive evidence that MC reconstitution repairs this WH defect. The differences between KitW/KitW-v mice and Kit+/+ mice in skin wound sizes reported by these authors would probably have been even more pronounced and statistically significant, if skin wounds had been assessed during the initial WH response (i.e., during the first 3 days after wounding). In contrast, Iba and coworkers (36) , who characterized the late phase of cutaneous WH using KitW/KitW-v mice and Kit+/+ mice, failed to detect size differences in healing skin wounds, which is most likely explained by the substantial differences of the model used in this study (1 cm diameter, occlusive dressing, 20 days) and the wounding procedures used in our study (6 mm diameter, no dressing, 10 days) and by Egozi et al. (3 mm diameter, no dressing, 7 days).

Because we had observed that WH impairment in MC-deficient mice was most prominent during the initial response to wounding, we speculated that MCs in early skin wounds undergo activation and degranulation. Quantitative histomorphometric analyses of skin wound MCs indeed revealed a striking "activation gradient": the closer MCs are localized to the edge of a skin wound, the more activation and degranulation they exhibited. This observation suggests that the MC-activating signals induced by wounding are rapidly generated within the wound edge, which is subsequently exposed to high extracellular concentrations of proinflammatory MC mediators. In follow-up studies, it will be important to identify the signals that induce MC degranulation after wounding. Because of the large number of potential candidates that must be considered and systematically tested in this context, including neuropeptides (e.g., substance P and endothelin-1), neurohormones (e.g., ACTH, CRH, and ß-endorphin), growth factors (NGF), and complement components (e.g., C3a and C5a), the dissection of these MC activating signals was far beyond the scope of the current study (1 , 50) .

Therefore, we focused on the mechanisms of MC-regulated WH and asked whether MCs contribute to extravasation and/or the recruitment of PMNs, two key features of early skin WH responses. Plasma extravasation after wounding was markedly reduced in the absence of MCs yet was restored when the skin MC-deficiency of KitW/KitW-v mice had been repaired by the local transfer of MCs before wounding. These findings show that MCs are responsible for increasing vascular permeability in early skin wounds. Interestingly, the absence of MCs did not completely abrogate extravasation in our experiments, suggesting that MCs are largely, but not entirely, responsible for this event. Next, we studied PMN influx into wounded skin areas and found this element of initial WH also to be MC dependent, i.e., the accumulation of neutrophils in skin wounds of MC-deficient KitW/KitW-v mice was fully restored by the adoptive transfer of MCs. Our demonstration of a role for MCs in PMN recruitment to sites of cutaneous injury is in line with earlier observations made in other cutaneous inflammatory processes, such as granuloma formation (12) and IgE-dependent cutaneous late phase reactions (27) . Also, Egozi and coworkers (37) have recently reported that 1 and 3 days after wounding PMN numbers are lower in the absence of mast cells as assessed by immunohistochemistry.

Likely mediators of MC-dependent extravasation and PMN recruitment include histamine and TNF-{alpha} (51) . MCs are the only resident skin cell population known to contain relevant amounts of preformed TNF-{alpha}, which can be rapidly released on stimulation (52) . Surprisingly, no defects in wound closure occurred in TNF-{alpha} deficient mice subjected to skin wounding as compared with their WT controls. On the contrary, wound closure was found to be slightly improved in the absence of TNF-{alpha}, which clearly demonstrates that TNF-{alpha} does not promote MC-dependent WH. One explanation for this finding could be our previous demonstration that TNF-{alpha} inhibits murine epidermal keratinocyte proliferation in skin organ culture (28) .

Histamine, a potent vasoactive mediator in several inflammatory settings, is also stored in large amounts by cutaneous MCs (53) , and earlier reports had suggested this MC mediator to support cutaneous WH (54) . Our findings from experiments using histamine antagonists in mice subjected to skin wounding support this notion: WH is impaired by dimethindene, an H1-receptor antagonist, but not ranitidine, an H2-receptor antagonist. Interestingly, wound closure in dimethindene-treated mice was impaired to a much lesser extent as compared with MC-deficient mice, suggesting that histamine is one of several MC mediators that accounts for delayed wound closure in the absence of MCs. Our results complement earlier reports demonstrating that H1-receptor antagonists suppress skin WH. For example, Bairy and coworkers (35) have shown that the H1 blockers mepyramine and promethazine decrease breaking strength and collagen content in rat skin wounds. In our studies, WH impairment in dimethinden-treated mice peaked at 48 h after wounding, i.e., when KitW/KitW-v mice and Kit+/+ or MC-reconstituted KitW/KitW-v mice exhibited the most prominent difference in wound area size, indicating that WH-promoting histamine is largely derived from skin MCs.

Taken together, our data demonstrate that cutaneous WH responses in mice are critically controlled by activated MCs, which after skin wounding promote increased extravasation, PMN influx, and normal wound closure. In addition to our ongoing attempts to identify the key MC activating signals in wounded murine skin, the evident next step now is to explore the relevance of our findings in the context of WH in human skin, and to probe whether skin WH in patients can be promoted, e.g., by topical application of selected MC secretagogues and/or MC products. That this may actually be the case is already suggested by reports in the older literature that topical administration of histamine can accelerate WH (55) . Likewise, it will be important to test whether long-term systemic therapy with antihistamines, which rank among the most widely prescribed and consumed drugs on the market, have any negative effects on human skin WH, comparable with the WH-inhibitory effects we show here for a clinically widely used -H1-antagonist in murine skin.

ACKNOWLEDGMENTS

We thank D. Benner, S. Dinges, and A. Bolch for excellent technical support, Jodie Urcioli for proofreading the manuscript, and Sven Guhl and Martin Metz for helpful discussion. This study was supported in part by grants from ECARF and the Deutsche Forschungsgemeinschaft to M. Maurer (MA 1909/4–1 and SFB548B10) and from the Federal Ministry of Education and Research to R.P. (BMBF01GN0517). This work benefited from the experience gained in the European Community Programme GA2LEN, Global Allergy and Asthma European Network.

Received for publication February 9, 2006. Accepted for publication June 2, 2006.

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