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B Activation and a Reciprocal Suppression of TGF-
Signal Pathway1
* Department of Forensic Medicine, Wakayama Medical University, Wakayama, Japan;
Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Tokyo, Japan; and
Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
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Abstract |
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, IL-1
, and IL-1 receptor antagonist (IL-1ra) during wound healing has been observed, the pathophysiological roles of these factors, particularly IL-1ra, still remain elusive. We explored skin wound-healing processes in IL-1ra-deficient mice. Compared to wild-type (WT) mice, IL-1ra-deficient mice exhibited impaired wound healing, as evidenced by attenuated collagen deposition and delayed neovascularization. In contrast, neutrophil recruitment was significantly exaggerated, with the augmented expression of IL-1s, TNF-, and CXC chemokines, MIP-2 and KC, in IL-1ra-deficient mice compared with WT mice. Because the transcription of these proinflammatory cytokines and CXC chemokines requires the activation of NF-
B, a major target of IL-1- and TNF--mediated signal pathway, we examined the activation states of NF-B. Nuclear translocation of NF-B p65 was significantly enhanced and prolonged in IL-1ra-deficient mice, compared to that in WT mice. The cross-talk between NF-B and TGF--mediated signals has been proposed based on in vitro observations. Indeed, compared to WT mice, the amounts of total and phosphorylated Smad2 and Smad3 were decreased with a reciprocal increase in the amount of Smad7 in skin wound sites of IL-1ra-deficient mice. Moreover, the gene expression of vascular endothelial growth factor, a target gene of TGF-1, was decreased in IL-1ra-deficient mice. Thus, the absence of IL-1ra may suppress TGF--mediated signaling pathway, which is crucial for collagen deposition and vascular endothelial growth factor-mediated neovascularization in wound healing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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IL-1, a pleiotropic inflammatory cytokine, is produced by various kinds of cells such as neutrophils, macrophages, and fibroblasts. Two distinct gene products, IL-1 and IL-1, have similar biological activities after binding to a common IL-1 receptor and are implicated as essential mediators of tissue destruction in various inflammatory diseases such as septic shock and rheumatoid arthritis ( 3, 4). IL-1 receptor antagonist (IL-1ra)3 is a member of the IL-1 family and exhibits an identical -pleated sheet structure as IL-1s ( 5, 6). Due to its similar structure, IL-1ra can also bind to IL-1R at the same sites with a similar affinity as IL-1s ( 7), but fails to associate with IL-1R accessory protein, which is indispensable for the biological activities of IL-1s ( 8, 9). Thus, IL-1ra antagonizes IL-1s at the receptor levels. Moreover, IL-1ra is aberrantly produced in various diseases, such as rheumatoid arthritis and infectious diseases, to negatively regulate the bioactivities of IL-1s ( 7, 10). This notion was supported by phenotypic changes in mice lacking IL-1ra (IL-1ra KO mice). IL-1ra KO mice on a BALB/c background exhibited spontaneously polyarthritis resembling rheumatoid arthritis ( 11) and developed exaggerated bacteria-induced intrahepatic granuloma formation ( 12).
Several lines of evidence indicate aberrant expression of IL-1s and IL-1ra at wound sites ( 13, 14). Given the ability of IL-1s to induce the expression of various growth factors and chemokines ( 15), it is reasonable to assume that IL-1s have roles in the wound-healing process. Nevertheless, it still remains controversial on the roles of IL-1s in wound healing. Topical application of IL-1 accelerated epidermal wound healing ( 16). In contrast, IL-1R KO mice exhibited retarded oral but not dermal wound healing ( 17), and short-term but not long-term blockade of IL-1 can promote periodontal wound healing ( 18). Moreover, to date, the biological role of IL-1ra in skin wound healing has not yet been investigated. Thus, we examined the biological roles of the IL-1 system, particularly IL-1ra, by the use of IL-1ra KO mice.
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Materials and Methods |
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The following mAbs or polyclonal Abs (pAbs) were used in this study; rabbit anti-myeloperoxidase (MPO) pAb (Neomarkers), rabbit anti-mouse IL-1ra pAb ( 10), rat anti-mouse CD31 mAb (BD Pharmingen), rabbit anti-mouse phosphorylated Smad2 (P-Smad2) pAb (Upstate Biotechnology), rabbit anti-mouse TGF-1 pAbs, goat anti-mouse Smad2 pAb, rabbit anti-mouse Smad3 pAb, goat anti-mouse P-Smad3 pAb, goat anti-mouse Smad7 pAb, mouse anti--tubulin mAb, and rabbit anti-mouse NF-B p65 pAb (Santa Cruz Biotechnology), mouse anti--smooth muscle actin (SMA) mAb (clone 1A4; DakoCytomation), cyanine dye 3 (Cy3)-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG pAb (Jackson ImmunoResearch Laboratories).
Mice
IL-1ra KO mice were generated and backcrossed to BALB/c mice for more than eight generations ( 12). Pathogen-free BALB/c mice were obtained from Clea Japan and were designated as wild-type (WT) mice. Age- and sex-matched IL-1ra KO and WT mice were housed individually in cages under specific pathogen-free conditions during the whole course of the study. All animal experiments complied with the standards set out in the Guidelines for the Care and Laboratory Animals at Wakayama Medical University.
Excisional wound preparation and macroscopic examination
Full-thickness skin wounds were made in the dorsal skin under sterile conditions as described previously ( 19, 20). Briefly, after being anesthetized with i.p. administration of pentobarbital (50 µg/g weight), the dorsal skin was picked up at the midline and was punched through two layers of skin with a sterile disposable biopsy punch (diameter 4 mm; Kai Industries). This procedure generated two excisional full-thickness wounds with one on each side of the midline. The same procedure was repeated three times, generating six wounds on each animal. Each wound site was digitally photographed at the indicated time intervals, and wound areas were determined on photographs using PhotoShop (version 7.0; Adobe Systems). Changes in wound areas were expressed as the proportion of the initial wound areas. In some experiments, wounds and their surrounding areas including the scab and epithelial margins were cut for further analyses with a sterile disposable biopsy punch with a diameter of 8 mm (Kai Industries) at the indicated time intervals.
Histopathological analyses of wound sites
Wound specimens were fixed in 4% formaldehyde buffered with PBS (pH 7.2) and then embedded with paraffin. Sections of 6-µm in thickness were stained with H&E. Other sections were further processed for immunohistochemical analyses to evaluate leukocyte infiltration or neovascularization ( 19, 21) or for a double-color immunofluorescence analysis to identify IL-1ra-expressing cells or to determine the localization of NF-B p65 proteins ( 22).
MPO assay
Wound samples were homogenized for determination of MPO activities as described previously. MPO activities in the resultant homogenates were assayed using the SUMILON peroxidase assay kit (Sumitomo Bekuraito), according to the manufacturer’s instructions, to evaluate neutrophil recruitment at the wound sites ( 21). The data were expressed as units per wound.
Measurement of hydroxyproline (Hyp) contents at wound sites
After being dried for 16 h at 120°C, the contents of Hyp, a major constituent of collagen in skin wound samples, were measured as the index of collagen accumulation at the wound sites, as described previously ( 19, 20). Hyp content was calculated by comparison to standards and expressed as the amount (µg) per wound.
ELISA
Wound samples were homogenized with PBS containing Complete Protease Inhibitor Cocktail (Roche Diagnostics) and centrifuged at 5000 x g for 10 min. Supernatants were used to determine IL-1ra, IL-1, IL-1, and TNF- levels with commercial ELISA kits (IL-1ra, IL-1, and IL-1; R&D Systems; TNF-, BioSource International) according to the manufacturer’s recommendation. Pellets were also collected to extract nuclear proteins as described below. The detection limits in each method were as follows: IL-1ra >7 pg/ml, IL-1 >2.5 pg/ml, IL-1 >3 pg/ml, and TNF- >5 pg/ml. The data are expressed as the target molecule (nanogram or picogram) per wound for each sample.
Extraction of nuclear proteins and measurement of NF-B p65 protein
Nuclear proteins were extracted from the obtained cell pellets using the NE-PER method (Pierce). The multiwell chemiluminescent assay was performed to detect NF-B p65 protein, using an EZ-Detect Transcription Factor kit (Pierce), according to the manufacturer’s instructions. The p65 contents were expressed as relative light units divided by total protein contents for each sample.
Western blotting analysis
Wound samples were homogenized, and the resultant lysates (30 µg) were electrophoresed in a 7.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane as previously described ( 19). The membrane was then incubated with Abs to TGF-1, Smad2, Smad3, phosphorylated Smad2, phosphorylated Smad3, or Smad7 diluted 1000-fold. After the incubation of HRP-conjugated secondary Abs, the immune complexes were visualized using an ECL Plus System (Amersham Biosciences) according to the manufacturer’s instructions.
Extraction of total RNAs and RT-PCR
Total RNAs were extracted from skin samples using ISOGENE (Nippon Gene) and 5 µg of the obtained total RNA was reverse-transcribed as previously described ( 19, 20). The resultant cDNAs were amplified together with Taq polymerase (Takara Bio) using the specific sets of primers (Table I). PCR amplification of each gene was conducted with the optimal cycles consisting of 94°C for 30 s, optimal annealing temperature shown in Table I for 30 s, and 72°C for 1 min, followed by incubation at 72°°C for 5 min. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities were measured using NIH Image Analysis software version 1.63 and the ratios to -actin were calculated.
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The means and SEMs were calculated for all parameters determined in this study. Statistical significance was evaluated by using ANOVA or the Mann-Whitney U test. A value of p < 0.05 was accepted as statistically significant.
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Results |
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IL-1ra mRNA expression was markedly enhanced at 1 day after the injury and remained elevated until 6 days after the injury (Fig. 1, a and b). Consistently, IL-1ra protein contents were significantly increased at 1 day after the injury and remained at high levels at 10 days after the injury (Fig. 1c). Immunohistochemical analysis demonstrated that IL-1ra proteins were detected in keratinocytes in uninjured and injured skin (Fig. 1d). In contrast, at 1 and 3 days after the injury, IL-1ra proteins were observed in both MPO-positive neutrophils and F4/80-positive macrophages (Fig. 1, e and f), in addition to keratinocytes. At 6 days after the injury, immunopo-sitive IL-1ra was found also in SMA-positive myofibroblasts (Fig. 1g). These observations implied that IL-1ra protein expression was enhanced at the wound sites.
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We next made excisional skin wounds in IL-1ra KO and WT mice, to compare the healing processes between these two strains. In WT mice, the wound areas were reduced to <40% at 6 days after the injury and wound closure was almost complete at 14 days after the wound. In contrast, the wound areas in IL-1ra KO mice remained over 50% of the original wound area even at 6 days after the injury, and the wounds were not closed at 14 days (Fig. 2). These observations indicate that the absence of IL-1ra delayed wound healing.
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We next examined the effects of IL-1ra deficiency on leukocyte infiltration into the wound sites. Consistent with our previous observations, a large number of neutrophils infiltrated to the wound sites immediately after the injury, and MPO activity reached a peak 1 day after the injury in WT mice (Fig. 3, a and c). In IL-1ra KO, neutrophils were recruited to a similar extent until 1 day after the injury. In contrast, later than 2 days after the injury, neutrophil recruitment and increases in MPO activities were markedly exaggerated in IL-1ra KO mice, compared to those in WT mice (Fig. 3, b and c). Similarly, F4/80-positive macrophage infiltration was more prominent in IL-1ra KO than WT mice (data not shown). Thus, the lack of IL-1ra exaggerated a characteristic feature of inflammatory reaction, leukocyte infiltration into wound sites.
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, IL-1, and TNF- expression in IL-1ra KO mice
Because IL-1ra is a natural antagonist for IL-1-mediated signals, its absence may affect IL-1 and IL-1 expression. Moreover, various stimuli can simultaneously induce the production of TNF- as well as IL-1 ( 23). Thus, we examined the expression of IL-1, IL-1, and TNF- at mRNA and protein levels. In the uninjured skin, mRNA of IL-1 and TNF- was detected faintly but to a similar extent in both IL-1ra KO and WT mice, while IL-1 mRNA was barely found in both strains (Fig. 4a). In WT mice, skin injury caused a transient increase in IL-1 mRNA expression at 3 days after the injury, whereas the gene expression of IL-1 and TNF- was enhanced, starting at 1 day after the injury and remaining at similar levels until 6 days after the injury (Fig. 4). Consistent with our previous reports ( 24, 25), an immunohistochemical analysis demonstrated that neutrophils and macrophages expressed IL-1s and TNF-, while keratinocytes and fibroblasts expressed IL-1 and TNF- (data not shown). The absence of IL-1ra enhanced the recruitment of neutrophils and macrophages, the main cellular sources of IL-1 and TNF-. This may result in exaggerated gene expression of these inflammatory cytokines in IL-1ra KO mice, compared to those in WT mice (Fig. 4). Concomitantly, the protein contents of IL-1, IL-1, and TNF- were higher in IL-1ra KO than in WT mice after the injury (Fig. 5). Thus, the absence of IL-1ra may enhance IL-1s and TNF- production at the skin wound sites.
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Enhanced leukocyte recruitment in IL-1ra KO mice prompted us to evaluate the expression of two neutrophilic CXC chemokines, MIP-2 and KC, at the wound sites. The gene expression of MIP-2 and KC was barely detected in uninjured skin of both IL-1ra KO and WT mice (Fig. 6a). At 1 day after the injury, the gene expression of both MIP-2 and KC was augmented to a similar extent to that in IL-1ra and WT mice. However, at 3 and 6 days after the injury, IL-1ra KO mice exhibited exaggerated gene expression of both CXC chemokines, compared to that in WT mice (Fig. 6, b and c). These observations implied that the absence of IL-1ra augmented CXC chemokine gene expression, thereby leading to enhanced neutrophil recruitment.
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B p65 contents in wound sites of IL-1ra KO and WT mice
NF-B is a major target of the IL-1- and TNF--mediated signal pathway ( 26, 27, 28, 29, 30, 31, 32), whereas NF-B activation is indispensable for inducible gene expression of MIP-2 and KC as well as TNF- ( 33, 34, 35). Thus, aberrant expression of IL-1, TNF-, and these CXC chemokines prompted us to examine NF-B in the wound sites of IL-1ra KO mice. There was no significant difference in the nuclear amounts of NF-B p65 in uninjured skin between IL-1ra KO and WT mice. The nuclear amounts of p65 were increased in WT mice transiently at 6 days after the injury (Fig. 7a). On the contrary, in IL-1ra KO mice, the nuclear amounts of p65 were increased progressively later than 3 days after the injury (Fig. 7a). Consistently, the nuclear p65 in anti-SMA-positive myofibroblasts was more obvious in IL-1ra KO mice than WT mice 10 days after the injury (Fig. 7b). Thus, these observations would imply that NF-B activation was prolonged in the absence of IL-1ra.
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Delayed wound healing suggested an impairment in collagen accumulation in IL-1ra KO mice. Thus, we next examined collagen accumulation process. Collagen type I (Col I) mRNA was detected faintly but to a similar extent in uninjured skin samples of WT and IL-1ra KO mice (Fig. 8, a and b). Skin wound enhanced Col I gene expression in WT mice, starting at 1 day after the injury, and progressively thereafter, whereas increases in Col I gene expression was marginal in IL-1ra KO mice (Fig. 7). Moreover, Hyp contents, an indicator for collagen accumulation, were consistently less in IL-1ra KO than in WT mice (Fig. 8c). Histopathological analysis further demonstrated that granulation tissue was less prominent in IL-1ra KO than WT mice at 6 days after the injury (Fig. 8, d and e). Collectively, the absence of IL-1ra reduced collagen deposition and eventually delayed granulation tissue formation.
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1/Smad-mediated signaling
Several lines of evidence indicated that NF-B activation could down-regulate Smad system, a main signal transduction system of TGF-1, which is a major regulator of collagen biosynthesis ( 36, 37, 38). Moreover, we observed that TGF-1 was detected in macrophages and fibroblasts (data not shown), consistent with our previous report. Thus, we examined TGF-1 and Smads contents at the wound sites by Western blot analysis. TGF-1, Smad2, phosphorylated Smad2, Smad3, phosphorylated Smad3, and Smad7 were detected faintly but to similar extents in IL-1ra KO and WT mice before the injury (Fig. 9). Moreover, skin injury increased the amount of TGF-1, Smad2, Smad3, and Smad7 in IL-1ra KO and WT mice but in a differential way. There were no apparent differences in TGF-1 protein amounts between WT and IL-1ra KO mice. The increases in the amount of total Smad2 and Smad3 were prominent in WT mice than IL-1ra KO mice. Moreover, the amounts of phosphorylated Smad2 and Smad3 were increased in WT but not IL-1ra KO mice. Reciprocally, the increases in the amount of Smad7, which prevent phosphorylation of Smad2/3, were more conspicuous in IL-1ra KO mice than in WT mice. These observations indicate that the absence of IL-1ra may negatively regulate TGF-1/Smad signaling partly by inducing the expression of Smad7 and eventually reduce collagen production.
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Several lines of evidence indicated that TGF-/Smad signals regulated VEGF gene expression ( 39, 40). Thus, we examined the gene expression of angiogenic factors such as VEGF and basic fibroblast growth factor (bFGF). The gene expression of VEGF and bFGF was detected faintly but, to similar extents, in uninjured skin of both WT and IL-1ra KO mice (Fig. 10a). In WT mice, the gene expression of both angiogenic factors was enhanced at the wound sites at 1 or 3 days after the injury (Fig. 10, a–c). However, the enhanced gene expression of VEGF but not bFGF was remarkably attenuated in IL-1ra KO mice, compared to that in WT mice (Fig. 10, a–c). Moreover, neovascularization in skin wound sites was significantly depressed in IL-1ra KO mice, compared to that in WT mice, when vessel density was evaluated by the immunohistochemical analysis with anti-CD31 mAb (Fig. 10, d and e). These observations imply that the lack of IL-1ra may impair angiogenesis in skin wound sites, at least partly by reducing VEGF expression.
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Discussion |
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and IL-1, proinflammatory cytokines with pleiotropic activities ( 3, 4), are presumed to have crucial roles in various types of inflammatory reactions ( 41). IL-1ra, a member of the IL-1 family, is an endogenous antagonist against IL-1 by competition of receptor binding ( 7). Along with IL-1, IL-1ra was massively produced in endotoxemia ( 42) and bacteria-induced fulminant hepatitis models ( 10), presumably to counteract the actions of IL-1. However, the clarification on the roles of IL-1ra in various pathologies was obstructed by the requirement of a large excess amount of IL-1ra to inhibit the activities of IL-1 even in vitro. Our previous observation (unpublished data), that IL-1ra was expressed in human skin wound sites, prompted us to investigate the roles of IL-1ra in the skin wound-healing process by using IL-1ra KO mice. Our observation suggests that endogenously produced IL-1ra may be beneficial for the wound-healing process probably by counteracting IL-1- and TNF--mediated signals. Neutrophil infiltration is a hallmark of the inflammatory phase of skin wound healing and is presumed to have beneficial roles in the process by eradicating microbes and clearing cellular debris ( 1, 2). However, IL-1ra KO mice showed a retarded wound-healing process, despite exaggerated neutrophil infiltration. These observations raise questions on the protective roles of neutrophils. Indeed, neutropenic mice exhibited accelerated closure upon an aseptic excisional skin wound ( 43). Moreover, secretory leukocyte protease inhibitor-deficient mice showed impaired wound healing with enhanced neutrophil infiltration and neutrophil-derived elastase activities ( 44). Furthermore, we did not observe any correlation between neutrophil infiltration and healing rates of the aseptic excisional skin wound in various gene-deficient mice ( 19, 20, 45). Thus, neutrophil infiltration per se may not be necessary for healing of skin wounds under the aseptic conditions. This notion was further supported by the observations on PU.1-deficient mice, which showed effective skin wound healing, similarly as WT mice, without leukocyte infiltration ( 46). In this study, we demonstrated that IL-1ra deficiency delayed the proliferative phase, as evidenced by delayed granulation tissue formation and reduced neovascularization. Because these processes are governed by fibroblasts and endothelial cells, IL-ra deficiency has profound effects on these resident cells in skin besides leukocytes.
Exaggerated neutrophil infiltration in IL-1ra KO mice can arise from augmented expression of CXC chemokines with potent neutrophil chemotactic activities, MIP-2 and KC, and adhesion molecules such as ICAM-1 and VCAM-1 (data not shown). Accumulating evidence indicates that the activation of a transcription factor, NF-B, is indispensable for the inducible expression of these genes ( 33, 34, 35, 47). The lack of IL-1ra resulted in the enhancement in expression and probably biological functions of IL-1, which can activate NF-B efficiently. These notions were substantiated by the observations that nuclear translocation of NF-B p65 was prolonged and exaggerated at wound sites of IL-1ra KO mice compared to that in WT mice.
Collagen deposition is an indispensable step for skin wound healing. We observed that Col I gene expression and Hyp contents were significantly reduced in IL-1ra KO mice, along with impaired wound healing. Several lines of evidence implied that the TGF--mediated signal pathway has crucial roles in collagen expression ( 48, 49, 50, 51). TGF- binding activates the kinase activities of its receptor, which phosphorylates transcription factors, Smad2 and Smad3. Phosphorylated Smad2 and Smad3 associate with Smad4 and translocate into nucleus, thereby inducing the expression of the target genes ( 52, 53, 54, 55). Simultaneously, Smad2 and Smad3 induced the expression of Smad7, which can interfere with the phosphorylation of Smad2 and Smad3 by ligand-bound TGF- receptors ( 52, 53, 54, 55). The important roles of this pathway in collagen expression is substantiated by the observation that adenovirus-mediated gene transfer of Smad7 attenuated the phosphorylation of Smad2, resulting in the attenuation of pulmonary fibrosis induced by bleomycin treatment ( 56).
Of interest is that the TGF-/Smad pathway can be regulated negatively by other signal pathways such as IFN-
/Stat1 signals ( 57, 58). We have provided definitive evidence on the presence of cross-talk between the TGF-/Smad and IFN-/Stat1 signal pathway even in the skin wound-healing process ( 19). The NF-B pathway can negatively regulate TGF- signaling in vitro ( 36, 37, 38). Nevertheless, it remains to be investigated on the presence of the cross-talk between TGF-/Smad and NF-B in vivo. The aberrant NF-B activation in wound sites of IL-1ra KO mice prompted us to investigate TGF-/Smad pathways in skin wound sites. We demonstrated that, at the wound sites of IL-1ra KO mice, the phosphorylation of Smad2 and Smad3 was depressed with a reciprocal increase of Smad7 proteins, compared to that of WT mice. We previously observed that collagen deposition and the wound-healing process was accelerated in mice deficient in TNF receptor p55 ( 20), which can mediate NF-B activation as efficiently as the IL-1 receptor. Considering that NF-B activation may be reduced at wound sites of TNF receptor p55 KO mice, the activation status of NF-B may modulate the Smad pathway also in these mice, thereby modulating the skin wound-healing process.
Nagrajan et al. ( 37) demonstrated that NF-B inhibited Smad signaling independently of Smad7 expression in HEK 293 cells. On the contrary, Bitzer et al. ( 38) demonstrated that NF-B inhibited TGF- signaling by up-regulating Smad7 expression in fibroblasts. Consistent with the latter observation, NF-B activation was observed mainly in fibroblasts in wound sites and the amount of Smad7 was increased with depressed phosphorylation of Smad2 and Smad3, compared with WT mice. Thus, it is reasonable to speculate that the NF-B pathway can down-regulate the TGF-/Smad pathway in fibroblasts, in a Smad7-dependent manner.
Angiogeneis, an indispensable step for wound healing, is regulated by several angiogenic growth factors, particularly VEGF and bFGF ( 1, 2). Because the TGF-/Smads signal pathway can up-regulate the gene expression of VEGF ( 39, 40), we examined the gene expression of these angiogenic factors. Indeed, enhanced gene expression of VEGF but not bFGF was retarded in IL-1ra KO mice, compared with that in WT mice. Thus, VEGF gene expression may be depressed by NF-B-mediated attenuation of the TGF-/Smads signal pathway. If this is the case, the cross-talk between NF-B and TGF-/Smads has crucial roles in wound healing by modulating angiogenesis as well as collagen production.
Alternative splicing generates an intracellular form of IL-1ra mRNA without a leader sequence in addition to the secreted form ( 59). Moreover, the intracellular form of IL-1ra protein was detected in epithelial cells, including keratinocytes but not leukocytes and fibroblasts ( 59, 60). Because most of the coding region of the IL-1ra gene was replaced with a neomycin-resistant gene, our IL-1ra KO mice lack both secretory and intracellular forms of IL-1ra proteins ( 12). Thus, it is difficult to evaluate precisely the relative contribution of these two isoforms of IL-1ra proteins in skin wound healing. However, immunohistochemical analysis detected IL-1ra proteins predominantly in infiltrating leukocytes and fibroblasts after the injury. These observations suggest that, in this process, secretory IL-1ra has more important roles than intracellular IL-1ra.
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Disclosures |
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Footnotes |
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1 This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Science, and Technology of the Japanese government. 
2 Address correspondence and reprint requests to Dr. Toshikazu Kondo, Department of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan. E-mail address: kondot{at}wakayama-med.ac.jp
3 Abbreviations used in this paper: IL-1ra, IL-1 receptor antagonist; Hyp, hydoxyproline; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; pAb, polyclonal Ab; MPO, myeloperoxidase; P-Smad, phosphorylated Smad; SMA, -smooth muscle actin; WT, wild type; Col I, collagen type I.
Received for publication October 4, 2005. Accepted for publication February 14, 2006.
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, WT; 
, IL-1ra KO (n = 12 animals). **, p < 0.01; *, p < 0.05, WT vs IL-1ra KO mice.
