Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-decient mice

Zi-Qing Lin,*, Toshikazu Kondo,* Yuko Ishida,* Tatsunori Takayasu,* and Naofumi Mukaida Faculty of Forensic Medicine, China Criminal Police College, Shenyang, China; and *Department of Forensic & Social Environmental Medicine, Graduate School of Medical Science, and Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Japan

Abstract: To clarify interleukin (IL)-6 roles in wound healing, we prepared skin excisions in wildtype (WT) and IL-6-decient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte inltration was evident at wound sites. Furthermore, the re-epithelialization rate was 80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL-1, chemokines, adhesion molecules, transforming growth factor1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was signicantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte inltration, re-epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti-IL-6 monoclonal antibody signicantly delayed wound closure in WT mice. These observations suggest that IL-6 has crucial roles in wound healing, probably by regulating leukocyte inltration, angiogenesis, and collagen accumulation. J. Leukoc. Biol. 73: 713721; 2003.
Key Words: leukocyte recruitment angiogenesis collagen production chemokines TGF-1 VEGF

INTRODUCTION
Wound healing immediately starts after an injury and proceeds with a complicated but well-organized interaction among various types of tissues and cells. The injury causes a gap, which is immediately lled by clots in the presence of platelet aggregates. Then, the inammatory phase follows. In this phase, leukocytes such as neutrophils and monocytes inltrate to the site, remove the breakdown products from the injured cells and clots, and release various growth factors and cytokines [1, 2]. In response to growth factors and cytokines, the proliferative phase starts. In this phase, epidermal cells migrate and proliferate to ll the wound gap, displace the remnants of the original clots, and secrete basement membrane components

such as collagen. Thus, it is generally accepted that leukocyte inltration is mandatory to induce wound healing. Various types of cells, including macrophages, T cells, broblasts, keratinocytes, and endothelial cells, produce interleukin (IL)-6, which exhibits various activities on a wide variety of cells including lymphocytes, hepatocytes, and neuronal cells [3]. Several lines of evidence suggest that IL-6 has crucial roles in inammation, particularly at the early phase [4]. This notion is supported by the observations on IL-6decient mice. These mice did not show any abnormalities under nonchallenging conditions. However, IL-6-decient mice showed an impaired immune response against Listeria monocytogenesis infection and a diminished, inammatory, acute-phase response after tissue damage or infection [5]. Although IL-6 lacks in vitro chemotactic activities for leukocytes, IL-6-decient mice showed a reduction in leukocyte inltration to the site injected with IL-1 or carageenan [6]. We also observed that IL-6-decient mice exhibited a reduction in leukocyte inltration and brotic changes in liver brosis induced by chronic, intermittent injection of carbon tetrachloride [7]. Thus, IL-6 may regulate leukocyte recruitment to the inammatory sites and eventually, brotic changes. Evidence is accumulating that IL-6 may be involved in the pathogenesis of several types of skin diseases, including psoriasis, scleroderma, and systemic lupus erythematosus [8 11]. We previously observed that IL-6 mRNA and protein were detected in neutrophils, macrophages, and broblasts in the wound sites after skin incisions [12, 13]. In streptozotocininduced, diabetic mice, IL-6 levels in wound uids correlated with wound-healing rates [14], and the exogenous IL-6 administration reversed impaired wound healing in immunosuppressed mice by glucocorticoid [15], suggesting that IL-6 is presumed to be involved in skin wound healing. Moreover, Gallucci and his colleagues [16] observed that mice lacking IL-6 exhibited impairment in skin wound healing concomitantly with a reduced activation of a transcription factor, acti-

Correspondence: Naofumi Mukaida, M.D., Ph.D., Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, 920-0934 Kanazawa, Japan. E-mail: naofumim@kenroku.kanazawau.ac.jp Received August 16, 2002; revised February 6, 2003; accepted February 7, 2003; doi: 10.1189/jlb.0802397.

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vated protein-1 (AP-1), at the wound site. However, they did not examine in detail the pathological and cell biological consequences of the wound-healing process in the absence of IL-6. As there are additional signal pathways mediated by IL-6 in addition to AP-1 activation, reduced AP-1 activity in wound sites cannot completely explain the molecular mechanism of a delayed wound healing in IL-6-decient mice. The skin wound-healing process requires leukocyte recruitment, angiogenesis, and collagen accumulation. Various effector molecules, such as chemokines, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)1, regulate these processes. However, Gallucci and his colleagues [16] did not examine those points in their paper. Therefore, we made excisional skin wounds in wild-type (WT) and IL-6-decient [knockout (KO)] mice to clarify the effects of IL-6 deciency on the skin-wound process at the pathological and cell biological levels, particularly focusing on the expression of effector molecules. We provided denitive evidence that IL-6 has crucial roles in the wound-healing process by regulating leukocyte inltration, angiogenesis, and collagen deposition.

starting immediately after wound preparation to evaluate the wound-healing process. In some series of experiments, wounds and their surrounding areas, including the scab and epithelial margins, were cut with a sterile, disposable biopsy punch with a diameter of 8 mm (Kai Industries) at the indicated time intervals after being killed with an overdose of pentobarbital.

Histopathological analyses of wound sites

Skin-wound specimens were xed in 4% formaldehyde buffered with phosphate-buffered saline (PBS; pH 7.2) and were then embedded with parafn. Sections (6-m thick) were stained with hematoxylin and eosin for histological analysis. Immunostaining was also performed using anti-MPO, anti-F4/80, anti-CD3, or anti-CD31 Ab as described previously [17]. In addition, a double-color immunouorescent analysis was also performed for the determination of IL-6-expressing cell types as described previously [18].

Analysis of re-epithelialization
We analyzed the degree of re-epithelialization as described previously [17]. Briey, the central portion of the wound was viewed at the magnication of x100 or x400, and the width of the wound and the distance that epithelium had traversed were measured. Then, the percentage of re-epithelialization was calculated.

Measurements of leukocyte inltration and angiogenesis in wound sites

The wound bed, dened as the area surrounding the wound on both sides, including unwounded skin, fascia, regenerated epidermis, and eschar, was demarcated. The numbers of inltrating neutrophils, macrophages, and T cells within the wound beds were enumerated on 10 randomly chosen, visual elds (magnication, x400) of the sections stained with anti-MPO, anti-F4/80, and anti-CD3 Ab, respectively, and the average of the selected 10 elds was calculated. Among the 10 elds, three elds were selected from each edge of the wound bed, and the remaining four elds were from the middle of the wound bed [17]. Using the free hand tool of PhotoShop, vascular areas, dened as CD31-positive ones, were measured in the wound beds at 6 and 14 days after the wound and were expressed as the percentage of the entire wound-bed areas [17]. All measurements were performed without a prior knowledge of the experimental procedures.

MATERIALS AND METHODS Antibodies

The following monoclonal or polyclonal antibodies (mAb or pAb) were used in this study: rat anti-mouse F4/80 mAb and rat anti-CD-3 mAb (Dainippon Pharmaceutical Co., Osaka, Japan); rat anti-mouse CD31 mAb (PharMingen, Burlington, CA); mouse anti- smooth-muscle actin mAb and rabbit antimyeloperoxidase (anti-MPO) pAb (NeoMarkers, Fremont, CA); rabbit 1-antitrypsin (1-AT) pAb (Dako, Kyoto, Japan); and rat anti-IL-6 mAb (clone 6B4, a kind gift from Dr. Jacque Van Snick, Ludwig Institute of Cancer Research, Belgium).

Mice
Pathogen-free, 8-week-old male BALB/c mice were obtained from Sankyo Laboratories (Tokyo, Japan) and were designated as WT mice in the following experiments. Age- and sex-matched IL-6 KO mice, backcrossed to BALB/c mice for more than eight generations, were used in the experiments [7]. The animal experiments were compiled with the Guidelines for the Care and Laboratory Animals at the Takara-machi Campus of Kanazawa University, and animals were housed individually in cages under specic, pathogen-free conditions during the entire course of the experiments.

MPO assay
MPO activity was measured for the quantitation of neutrophil recruitment at the wound sites [17]. Briey, the excised wound samples were washed in PBS and homogenized in 1 ml 50 mM potassium phosphate-buffer solution with 0.5% hexadecyl trimethyl ammonium bromide (Sigma Chemical Co., St. Louis, MO) and 5 mM EDTA. The samples were sonicated for 20 s, freeze-thawed three times, and centrifuged at 12,000 rpm at 4C. MPO activities in the supernatants were assayed using Sumilon peroxidase assay kit (Sumitomo Bekuraito, Tokyo, Japan), according to the manufacturers instructions. The data were expressed as absorbance divided by total dry weight (mg).

Wound preparation and macroscopic examination

Skin wounds were prepared as described previously [17]. Briey, mice were deeply anesthetized with intraperitoneal (i.p.) administration of pentobarbital (50 g/g weight). After shaving and cleaning with 70% alcohol, excisional, full-thickness skin wounds were aseptically made on the dorsal skin by picking up a fold skin at the midline and using a sterile, disposable biopsy punch with a diameter of 4 mm (Kai Industries, Tokyo, Japan) to punch through the two layers of skin. In this manner, two wounds were made on each side of the midline at the same time. The same procedure was repeated for two additional times, resulting in a total of a six-wound formation, three wounds at each side. Each wound site was digitally photographed using the Nikon FX-35A (Nikon, Tokyo, Japan) at the indicated time intervals, and wound areas were determined on photographs using PhotoShop (Version 5.5, Adobe Systems, Tokyo, Japan) without a prior knowledge of the experimental procedures. Changes in wound areas were expressed as the percentage of the initial wound areas. In another series of experiments, WT mice received an i.p. injection of neutralizing anti-IL-6 mAb or control Ab (250 g/mouse) once a day for 7 days,

Measurement of hydroxyproline (HP) contents in wound sites

Skin wound sites were excised using a sterile, disposable biopsy punch with a diameter of 8 mm and were then dried for 16 h at 120C. HP contents were as described previously [19]. HP amount was calculated by comparison with standards and was expressed as the amount (g) per wound.

Extraction of total RNAs and reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNAs were extracted from uninjured and injured skin samples using Isogene (Nippon Gene, Toyama, Japan) according to the manufacturers instructions. Total RNA (5 g) was reverse-transcribed at 42C for 1 h in 20 l reaction mixture containing mouse Moloney leukemia virus RT (Toyobo, Osaka, Japan) with oligo(dT) primers (Amersham Pharmacia Biotech Japan, Tokyo), followed by PCR amplication. Nonradioisotopic, quantitative RTPCR was performed as described previously [17, 20]. In brief, cDNA was

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amplied together with Taq polymerase (Nippon Gene) using the sets of specic primers for intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), IL-1, IL-1, IL-6, macrophage-inammatory protein-1 (MIP-1), MIP-2, KC, TGF-1, VEGF, and -actin as described in Table 1. PCR conditions for the amplication of all these molecules were 20 40 cycles at two-cycle intervals at 94C for 1 min, optimized annealing temperature shown in Table 1 for 1 min, and 72C for 1 min, followed by incubation at 72C for 3 min. The amplied PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensity of ethidium bromide uorescence was measured using a charge-coupled device imaging system (GelDoc 2000, Bio-Rad, Hercules, CA) and NIH Image Analysis software version 1.61 (National Institutes of Health, Bethesda, MD). The intensity of each reaction mixture was plotted against the cycle numbers on semilogarithmic graphs for each molecule. The number of PCR cycle where uorescence intensity of PCR products increased exponentially was determined as the optimal cycle for each molecule. The intensities of the bands were determined with the use of NIH Image Analysis software, and the ratios to -actin were determined. To standardize the condition of gel staining, a constant amount of control DNA marker was electrophoresed every time. The PCR procedure was performed at least three times for each sample.

enhanced chemiluminescence system (Amersham Pharmacia Biotech Japan), according to the manufacturers instructions.

Statistical analysis
The mean and SEM were calculated for all parameters determined in this study. Statistical signicance was evaluated by using ANOVA or Mann-Whitneys U test. P 0.05 was accepted as statistically signicant.

RESULTS Wound closure and histological re-epithelialization

Skin excisions up-regulated IL-6 gene expression at the wound sites, and a double-color immunouorescent or immunohistochemical analysis revealed that keratinocytes, neutrophils, macrophages, and myobroblasts were the cellular source of IL-6 in wound healing (data not shown). To evaluate the pathophysiological role of locally produced IL-6 in woundhealing processes, IL-6 KO and WT control mice were subjected to full-thickness skin excisions on the dorsal shaved skin and were monitored for up to 14 days. In WT mice, the wound areas were reduced to 50% at 6 days after injury. However, IL-6 KO mice showed an impairment in wound closure (Fig. 1A). The wound areas in IL-6 KO mice remained 50% of the original wound area even at 10 days after the injury. Moreover, between 3 and 14 days after the injury, the wound areas in IL-6 KO mice were consistently larger than in WT mice (Fig. 1B). Furthermore, the administration of a neutralizing anti-IL-6 mAb also retarded wound closure rates in WT mice (Fig. 1D). These observations demonstrate that IL-6 depletion retarded wound closure and subsequent wound healing. At 1 day after the injury, re-epithelialization was scarce in IL-6 KO and WT mice. In WT mice, the re-epithelialization rate was 30% and 75% at 3 and 6 days after the injury,

Determination of blood platelet numbers and plasma brinogen

At the indicated time intervals after the injury, whole blood samples were collected from WT and IL-6 KO mice and were diluted (1:9 v/v) with 4% sodium citrate. An automatic counter for animals (Celltac, MEK-5128; Nihon Kohden, Tokyo, Japan) counted platelets within 1 h after sampling, according to the manufacturers instructions. Moreover, citrated plasma samples were obtained by centrifuging whole blood samples. Plasma brinogen levels were determined by using a commercially available kit (Mitsubushi Kagaku BioClinical Laboratories, Tokyo, Japan).

Serum 1-AT levels in WT and IL-6 KO mice

At the indicated time intervals after the injury, whole blood samples were collected from WT and IL-6 KO mice, and serum samples were obtained by centrifugation. Serum 1-AT levels were examined by Western blotting. In brief, 50 g total protein from the plasma serum samples was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferred to a nylon membrane, the membrane was incubated with anti1-AT pAb diluted 1000-fold. After the incubation of horseradish peroxidaseconjugated secondary Ab, the immune complexes were visualized using an

TABLE 1.

Sequences of the Primers Used for RT-PCR Annealing temperature (C) 55 56 64 60 59 62 50 60 62 Product size (bp) 488 504 469 258 204 454 447 435 456

Transcript IL-1 IL-1 IL-6 MIP-1 MIP-2 KC VCAM-1 ICAM-1 -actin

Sequence (F)5-TGGCCAAAGTTCCTGACTTGTTTG-3 (R)5-CAGGTCATTTAACCAAGTGGTGCT-3 (F)5-GAAATGCCACCTTTTGACAG-3 (R)5-CAAGGCCACAGGTATTTTGT-3 (F)5-CGTGGAAATGAGAAAAGAGTTGTGC-3 (R)5-ATGCTTAGGCATAACGCACTAGGT-3 (F)5-GCCCTTGCTGTTCTTCTCTGT-3 (R)5-GGCAATCAGTTCCAGGTCAGT-3 (F)5-GAACAAAGGCAAGGCTAACTGA-3 (R)5-AACATAACAACATCTGGGCAAT-3 (F)5-GGATTCACCTCAAGAACATCCAGAG-3 (R)5-CACCCTTCTACTAGCACAGTGGTTG-3 (F)5-CAGCTAAATAATGGGGAACTG-3 (R)5-GGGCGAAAAATAGTCCTTG-3 (F)5-GGAGCAAGACTGTGAACACG-3 (R)5-GAGAACCACTGCTAGTCCAC-3 (F)5-TTCTACAATGAGCTGCGTGTGGC-3 (R)5-CTCATAGCTCTTCTCCAGGGAGGA-3

Cycle 32 30 36 32 34 28 36 36 26

(F), Forward primer; (R), reverse primer.

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served at 6 days in WT mice (Fig. 2C). In IL-6 KO mice, MPO activity and macrophage inltration were reduced signicantly, compared with WT mice, at all of the time intervals examined (Fig. 2, B and DF). On the contrary, there was no signicant difference in T cell recruitment between WT and IL-6 KO mice (data not shown). Thus, these results demonstrated that neutrophil and macrophage inltration, in the inammatory phase after skin excision, was markedly reduced in the absence of IL-6.

Angiogenesis at wound sites

Angiogenesis is one of the important morphological changes observed in the proliferative phase of the wound-healing process. By immunostaining for CD31, angiogenesis was assessed in normal and injured skin of WT and IL-6 KO mice (Fig. 3, AE). No signicant difference was found in vessel density of the uninjured skin between WT and IL-6 KO mice (2.20.4% vs. 2.80.5%). Six days after the injury, the vessel density within the wound bed signicantly increased in the WT mice (8.50.2%). On the contrary, IL-6 KO mice showed signicantly impaired angiogenesis compared with WT mice (3.60.8%; P0.01). However, by 14 days after the injury, there were no signicant differences between WT and IL-6 KO mice in terms of the vessel density. These observations demonstrated that the absence of IL-6 interfered with angiogenesis at the wound sites.

Fig. 1. Skin wound healing in IL-6 KO and WT mice. (A) Macroscopic changes in skin wound sites in an IL-6 KO mouse and a WT mouse. The wounds were photographed at the time indicated. Representative results from 12 individual animals in each group are shown here. (B) Changes in percentage of wound area at each of the time points to the original wound area. Values represent mean SEM. Open bars, WT; solid bars, IL-6 KO (n12 animals). *, P 0.05; **, P 0.01, IL-6 KO compared with WT. (C) The ratio of re-epithelialization was evaluated in WT and IL-6 KO mice. All values represent the mean SEM (n6 animals). *, P 0.05, IL-6 KO compared with WT. (D) Changes in percentage of wound area at each of the time points to the original wound area in WT mice administered with a control and an anti-IL-6 mAb. Values represent mean SEM. Open bars, WT treated with control immunoglobulin G (IgG); solid bars, WT treated with anti-IL-6 IgG (n6 animals). *, P 0.05; **, P 0.01, Anti-IL-6 compared with control.

HP content in wound site

An increased collagen content in extracellular matrix is another characteristic change observed in the proliferative process of wound healing. As HP is a major constituent of and found almost exclusively in collagen [17], we determined HP content in the wound sites. There were no signicant differences in terms of HP content between uninjured WT and IL-6 KO mice (data not shown). HP content in wound sites increased progressively in WT mice and peaked at 6 days after the injury. In contrast, an increase in HP content in IL-6 KO mice was delayed at 3 and 6 days after the injury compared with WT mice (Fig. 3F). These results suggested that collagen production was also reduced as a result of the absence of IL-6.

respectively. At 10 days after the injury, histological re-epithelialization was completed (Fig. 1C). In contrast, IL-6 KO mice showed a signicant delay in re-epithelialization of wounds compared with WT mice. At 6 and 10 days after the injury, the re-epithelialization rate was still 50 and 90%, respectively. Re-epithelialization was complete only at 14 days after the injury. These observations suggested that the absence of IL-6 is detrimental to re-epithelialization in skin wound healing.

Leukocyte inltration in wound sites

In the inammatory phase of the wound-healing process, different types of leukocytes inltrate into the wound site, depending on the types of wound and the time intervals after the injury. In WT mice, a large number of neutrophils inltrated to the wound site 1 day after the injury (Fig. 2A) and disappeared at 6 days after the injury. As trapping neutrophils within clots hindered the correct determination of neutrophil numbers, we measured MPO activity to evaluate neutrophil recruitment in a more quantitative manner. In uninjured skin samples of WT and IL-6 KO mice, MPO activity was undetectable under the used experimental conditions (data not shown). MPO activity increased at 6 h and remained at similar levels until 1 day after the injury, consistent with histological ndings. F4/80-positive macrophages inltrated the wound site, starting at 1 day after the injury, and massive inltration of macrophages was ob716 Journal of Leukocyte Biology Volume 73, June 2003

Gene expression of adhesion molecules, cytokines, chemokines, and growth factors

Under the used condition, RT-PCR analysis failed to detect the mRNA of ICAM-1, VCAM-1, IL-1, IL-1, MIP-1, MIP-2, KC, TGF-1, and VEGF in uninjured skin specimens derived from WT and IL-6 KO mice. Skin excision induced the gene expression of all of these molecules in wound sites from WT mice at every time point examined (Figs. 4 and 5). In IL-6 KO mice, the enhanced gene expression of cytokines and chemokines was signicantly attenuated compared with WT mice (IL-1, IL-1, and MIP-2 at 3 and 6 days; MIP-1 and KC at 1, 3, and 6 days; Fig. 4, AF). Moreover, IL-6 KO mice showed signicantly reduced gene expression of adhesion molecules only at several time points (VCAM-1: 6 h, 1 and 3 days; ICAM-1: 3 and 6 h, 1 and 3 days; Fig. 5). Thus, reduced neutrophil and macrophage inltration in IL-6 KO mice was observed along with reduced expression of these adhesion
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Fig. 2. Immunohistochemical analyses of skin wound samples of WT (A and C) and IL-6 KO (B and D) at 1 (A and B; anti-MPO Ab) and 6 days (C and D; anti-F4/80 Ab) after the injury (original magnication, x200). (E) MPO activity at the wound site of IL-6 KO (solid bars) and WT (open bars) was determined at 6 hours, 1 and 3 days after the injury for the evaluation of neutrophil recruitment. All values represent the mean SEM (n6 animals). **, P 0.01; *, P 00.5, IL-6 KO compared with WT. (F) Macrophage recruitment in the wounded skin of IL-6 KO (solid bars) and WT (open bars) at the indicated time intervals after the injury. The number of macrophages per high-power microscopic eld (original magnication, 200) was counted. All values represent the mean SEM (n6 animals). **, P 0.01, IL-6 KO compared with WT.

molecules and cytokines. Moreover, the gene expression of TGF-1 and VEGF was also signicantly attenuated in IL-6 KO mice compared with WT mice; VEGF at 1, 3, and 6 days; and TGF-1 at 3 and 6 days (Fig. 4, G and H). These observations suggest that the diminished expression of TGF-1 and VEGF is responsible for reduced collagen accumulation and angiogenesis in IL-6 KO mice, respectively.

DISCUSSION
Wound healing consists of three phases: inammation, proliferation, and maturation and remodeling. These phases proceed, overlapping each other, and are regulated by a complicated array of cytokines and growth factors secreted by inammatory and resident cells [1, 2]. IL-6 is a pleiotropic cytokine produced by inammatory and resident cells and has a crucial role in the pathogenesis of various inammations [3, 4]. We previously observed the up-regulation of IL-6 at wound sites [12, 13]. Gallucci and his colleagues [16] have recently reported that IL-6 KO mice exhibited impairment in wound healing with reduced activation of a transcription factor AP-1 at the wound sites. However, they did not examine the effects of IL-6 deciency on the effector molecules in the woundhealing process, including cytokines, chemokines, and growth factors. Hence, we examined the wound-healing processes in IL-6 KO mice in comparison with WT mice, particularly focusing on chemokines and growth factors. Skin injury immediately causes clot formation and local inammation characterized by an inltration of neutrophils and macrophages into the wound sites. These pathological changes are hallmarks of the inammatory phase of wound healing. The administration of IL-6 at the wound sites induced massive leukocyte inltration in mice [21]. Consistent with this obserLin et al. Impaired wound healing in IL-6-decient mice 717

Platelet numbers and acute-phase protein levels

Platelets and brinogen are presumed to be important for the initiation of wound healing. There was no signicant difference in the platelet number between uninjured WT and IL-6 KO mice (WT vs. IL-6 KO: 29.81042.4104 vs. 27.11043.6104). Moreover, there was no signicant difference in the plasma brinogen levels between untreated WT and IL-6 KO mice (WT vs. IL-6 KO: 11610 vs. 1119 mg/dl). Although, at 1 day after injury, the plasma concentration of brinogen was marginally increased in both mice, no signicant difference was observed between WT and IL-6 KO mice (WT vs. IL-6 KO: 14316 vs. 13813 mg/dl). Furthermore, there was also no signicant difference in serum 1-AT levels before or 1 day after injury between WT and IL-6 KO mice (data not shown). These observations imply that in this wound-healing model, the absence of IL-6 may have no effect on platelet number, brinogen levels, and 1-AT levels.

Fig. 3. Immunohistochemical sections of skin wound samples of WT (A and C) and IL-6 KO mice (B and D) at 6 days after the injury. The sections were stained with a mAb for the endothelium (CD-31; A and B: original magnication, 10; C and D: original magnication, 100). Representative results from six animals in each group are shown here. (E) Vascular densities within the wound bed at 6 and 14 days after the injury and that in uninjured skin samples of IL-6 KO (solid bars) and WT (open bars) mice were determined using PhotoShop. All values represent the mean SEM (n6 animals). **, P 0.01, IL-6 KO compared with WT. (F) HP contents in the excisional wounds in WT (open bar) and IL-6 KO (solid bar) mice. All values represent the mean SEM (n6 animals). **, P 0.01; *, P 0.05, IL-6 KO compared with WT.

vation, we herein demonstrated that IL-6 KO mice exhibited a reduction in neutrophil and macrophage numbers at the wound sites compared with WT mice. As IL-6 KO mice exhibited a reduced leukocyte inltration in several types of inammations, IL-6 may regulate leukocyte recruitment in inammatory conditions [6, 7]. As IL-6 fails to exhibit a direct chemotactic activity against leukocytes, the reduced leukocyte inltration in IL-6 KO mice is ascribable to its indirect actions on leukocytes. Accumulating evidence indicates the crucial involvement of chemokines in leukocyte inltration. Moreover, IL-6 can induce the expression of several chemokines such as MIP-2 and MIP-1, which are chemotactic for neutrophils and monocytes/macrophages, respectively [2226]. Furthermore, the gene expression of MIP-2, KC, and MIP-1 was reduced at the wound sites of IL-6 KO mice compared with WT mice. Several lines of evidence implied that chemokines were secreted by platelets accumulating at wound sites. In this study, there was no signicant difference in platelet number between WT and IL-6 KO mice. Thus, independent of platelet number, the ablation of the IL-6 gene reduced chemokine gene expression and eventually, leukocyte inltration. Besides chemokines, adhesion molecules have essential roles in leukocyte extravasation. Especially, ICAM-1 and VCAM-1 expressed on endothelium have crucial roles in tight adhesion between leukocytes and endothelium and subsequent
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leukocyte extravasation. The expression of these adhesion molecules is up-regulated by various inammatory stimuli such as IL-1 and tumor necrosis factor (TNF) [27]. IL-6 can up-regulate IL-1 expression, and IL-1 gene expression was reduced at the wound site of IL-6 KO mice compared with WT mice. Thus, in IL-6 KO mice, the attenuated IL-1 expression down-regulated the expression of VCAM-1 and ICMA-1 and dampened leukocyte extravasation synergistically with reduced chemokines gene expression. Collagen deposition is indispensable for granulation tissue formation, a prerequisite step for wound healing. Here, we observed a reduction in collagen deposition at wound sites in IL-6 KO mice compared with WT mice. Several lines of evidence demonstrated that IL-6 can induce collagen production and/or procollagen gene expression in several types of cells including dermal broblasts and fat-storing cells in liver [28, 29]. Thus, IL-6 deciency directly reduced collagen deposition in wound sites. In addition, the expression of TGF-1, a cytokine with a potent brogenic activity [30 32], was reduced with carbon tetrachloride-induced liver brosis in IL-6 KO mice compared with WT mice [7]. A reduction in TGF-1 gene expression was also observed at the wound site in IL-6 KO mice. Thus, IL-6 may also induce collagen deposition indirectly by induction of TGF-1 gene expression. Collagen deposition leads to re-epithelization in the skinwound repair process. Overexpression of IL-6 in normal rat
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angiogenesis by inducing VEGF production. As we also observed that VEGF gene expression was reduced at wound sites of IL-6 KO mice compared with WT mice, similar mechanisms must work in angiogenesis in the skin wound-healing process. Accumulating evidence implies the crucial involvement of various cytokines, chemokines, and growth factors in the wound-healing process [1, 2]. We previously reported that wound healing was accelerated with enhanced angiogenesis and collagen deposition in TNF receptor p55 (TNF-Rp55)decient mice [17]. In IL-6 KO mice, wound healing was delayed, accompanied with delayed angiogenesis and collagen deposition, by the reduced expression of angiogenic and brogenic growth factors. Collectively, angiogenesis and collagen deposition are indispensable for wound healing. Of interest is that leukocyte inltration was attenuated in TNF-Rp55-decient mice, in spite of accelerated wound healing. Although wound healing was delayed with reduced leukocyte inltration in IL-6 KO mice, leukocyte inltration may have few, if any, roles in aseptic wound healing in this model. However, we do

Fig. 4. (A) RT-PCR analysis of gene expression for IL-1 and IL-1, MIP1, MIP-2, KC, TGF-1, and VEGF at wound sites in WT and IL-6 KO mice. Under the conditions used, RT-PCR analysis did not detect the mRNA of these molecules in uninjured skin samples of WT and IL-6 KO mice. Representative results from 10 animals in each group are shown here. The ratios of IL-1 (B), IL-1 (C), MIP-1 (D), MIP-2 (E), KC (F), TGF-1 (G), and VEGF (H) to -actin of WT (open bars) and IL-6 KO mice (solid bars) were determined by RT-PCR at 1, 3, and 6 days after the injury. Each value represents mean SEM (n10 animals). *, P 0.05; **, P 0.01, IL-6 KO compared with WT.

skin induced epidermal proliferation and inammation [33]. TGF-1 can induce epithelial migration and proliferation [34]. Moreover, TGF-1 promoted re-epithelization of skin wounds in rats [31]. Thus, the ablation of the IL-6 gene may delay re-epithelization indirectly through TGF-1. Angiogenesis is also an indispensable step for granulation tissue formation. Several lines of evidence suggest the involvement of IL-6 in neovascularization. IL-6 in vitro induced the proliferation of brain microvascular endothelial cells with accelerated formation of tube-like structures [35, 36]. Moreover, IL-6 can induce the gene expression of a potent angiogenic factor, VEGF, at transcriptional and post-transcriptional levels [37]. Furthermore, the administration of IL-6 promoted the healing of brain injury mainly by inducing VEGF production [35, 36]. These observations suggest that IL-6 can induce

Fig. 5. RT-PCR analysis of gene expression for adhesion molecules (VCAM-1 and ICAM-1) at wound sites in WT and IL-6 KO mice. Under the conditions used, RT-PCR analysis did not detect the mRNA of these adhesion molecules in uninjured skin samples of WT and IL-6 KO mice. Representative results from 10 animals in each group are shown here (A and D). The ratios of VCAM-1 (B and E) and ICAM-1 (C and F) to -actin of WT (open bar) and IL-6 KO mice (solid bar) were determined by RT-PCR. Each value represents mean SEM (n10 animals). *, P 0.05; **, P 0.01, IL-6 KO compared with WT.

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not exclude the possibility that the inltration of leukocytes, particularly neutrophils, has essential roles in the healing process of wounds complicated with bacterial infections. This possibility should be claried by additional experiments using septic skin wounds. Three phases of wound healinginammation, proliferation, and remodelingproceed consecutively. IL-6 deciency had profound effects on all three phases. We cannot exclude the possibility that reduced inammatory responses were responsible for the subsequent delay in wound healing in IL-6 KO mice. However, it is more probable that IL-6 had direct, crucial roles in proliferation and remodeling phases of wound healing by promoting collagen deposition and angiogenesis.

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ACKNOWLEDGMENTS
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The work is supported in part from Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese government. Z-Q. L. and T. K. contributed equally to this work. We express our gratitude to Dr. Hidesaku Asakura [Department of Internal Medicine (III), Kanazawa University School of Medicine, Japan] for his kind help with platelet counting.

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