Expression and release of IL-18 binding protein in response to IFN-gamma.

IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn's disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-gamma induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-gamma by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-gamma was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-gamma-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-gamma may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.

Macrophage-derived heme-oxygenase-1: expression, regulation, and possible functions in skin repair.

BACKGROUND: Expression and enzymatic activity of heme oxygenase (HO) has been implicated in the development, as well as in the resolution, of inflammatory conditions. Because inflammation is central to tissue repair, we investigated the presence and potential functions of HO in an excisional model of normal and diabetes-impaired wound repair in mice. MATERIALS AND METHODS: Expression of HO-1 during cutaneous healing was analyzed by RNase protection assay, Western blot, and immunohistochemical techniques in a murine model of excisional repair. Furthermore, we determined HO-1-dependent release of proinflammatory cytokines from RAW 264.7 macrophages by enzyme-linked immunosorbent assay (ELISA). RESULTS: Upon injury, we observed a rapid and strong increase in HO-1 mRNA and protein levels at the wound site. By contrast to normal repair, late stages of diabetes-impaired repair were associated with elevated HO-1 expression. Besides a few keratinocytes of the hyperproliferative epithelium, immunohistochemistry revealed infiltrating macrophages as the predominant and major source of HO-1 at the wound site. In vitro studies demonstrated the potency of exogenous and also endogenous nitric oxide (NO) to strongly induce HO-1 expression in RAW 264.7 macrophages. However, L-NIL-mediated enzymatic inhibition of inducible NO-synthase (iNOS) at the wound site in vivo was not paralleled by decreased HO-1 levels. In vitro inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPPIX) in RAW 264.7 macrophages markedly attenuated tumor necrosis factor-alpha (TNF-alpha), but strongly increased interleukin-1beta (IL-1beta) release in RAW 264.7 macrophages in vitro. CONCLUSIONS: The observed injury-mediated increase in HO-1 mRNA and protein at the wound site was due to infiltrating HO-1 expressing monocytic cells. Macrophage-derived HO-1 expression was not under regulatory control by NO in skin repair. We provide evidence that HO-1 might exert a regulatory role in macrophage-derived cytokine release.

A novel keratinocyte mitogen: regulation of leptin and its functional receptor in skin repair.

Wound re-epithelialization represents a tissue movement that crucially participates in wound closure. Recently, we demonstrated that supplemented leptin improved re-epithelialization processes in leptin-deficient ob/ob mice. In this study we investigated regulation of the leptin system during normal repair in healthy animals. We found leptin to be present at the wound site during healing, although leptin levels were clearly reduced upon injury compared with uninvolved control skin. The functional leptin receptor subtype obRb was observed to be constitutively expressed in nonwounded skin. During early healing, the leptin receptor obRb was downregulated, but re-increased again from day 5 postwounding. Immunohistochemistry revealed that highly proliferative keratinocytes of the wound margin epithelia strongly expressed the functional leptin receptor subtype obRb. In vitro studies demonstrated that murine and human primary epidermal keratinocytes responded to exogenously added leptin with a proliferative response. Moreover, specificity of leptin-mediated mitogenic effects on primary keratinocytes could be shown by completely blocking leptin actions by a soluble, nonfunctional chimeric leptin receptor. Finally, we report that leptin, besides the recently described activation of the janus tyrosine kinase signal transducers, also activated extracellular signal-regulated kinase-controlled signaling pathways in primary keratinocytes.

Expressional regulation of angiopoietin-1 and -2 and the tie-1 and -2 receptor tyrosine kinases during cutaneous wound healing: a comparative study of normal and impaired repair.

It has become evident that a closely regulated presence of vascular endothelial growth factor (VEGF) and angiopoietin (Ang) factors determines the fate of blood vessel formation during angiogenesis. As angiogenesis is central to a normal wound-healing process, we investigated the regulation of Ang-1 and -2 and the related tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie)-1 and -2 receptors during normal repair in Balb/c mice and diabetes-impaired wound healing conditions in genetically diabetic (db/db) mice. For both normal and impaired healing conditions, we observed a constitutive expression of Ang-1, which was paralleled by an increase of Ang-2 upon injury. Whereas the observed Ang-2 expression declines from Day 7 after injury in control mice, diabetic-impaired healing was characterized by still increasing amounts of Ang-2 at these time points. Furthermore, Tie-1 was strongly induced during repair with a prolonged expression in diabetic mice, whereas Tie-2 expression was constitutive during normal repair but completely absent in diabetes-impaired healing. The overexpression of Ang-2 in the presence of markedly reduced VEGF in wounds of diabetic mice was associated with a dramatic decrease in endothelial cell numbers compared with normal healing as assessed by analysis of the endothelium-specific markers CD31 and von Willebrand factor, whereas the lymphatic endothelium remained stable as determined by expression of VEGF receptor-3 (VEGFR-3/Flt-4).

Regulation of interleukin-18 (IL-18) expression in keratinocytes (HaCaT): implications for early wound healing.

Keratinocytes display a high basal level expression of IL-18. Tumor necrosis factor-alpha (TNF-alpha) mediated a large decrease in IL-18 mRNA levels in the human keratinocyte cell line HaCaT, which was accompanied by a subsequent accumulation of IL-18 protein in the cell culture supernatants, which was shown to be biologically active. By contrast, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), respectively, strongly decreased IL-18 mRNA expression in HaCaT keratinocytes in the absence of IL-18 protein release from the cells. Notably, a pre-treatment of the cells with EGF, or TGF-alpha clearly attenuated TNF-alpha-induced IL-18 protein, release and bioactivity. For the in vivo situation of cutaneous wound repair, we observed an increase in IL-18 protein, 10 hours post-wounding, that closely correlated to infiltration of neutrophils which are known as producers of TNF-alpha. Our data suggest that bioactive IL-18 might be tightly counter-regulated by platelet- and neutrophil-derived factors at the onset of repair.

Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.

Wound-healing disorders are a therapeutic problem of extensive clinical importance. Leptin-deficient ob/ob mice are characterized by a severely delayed wound healing that has been explained by the mild diabetic phenotype of these animals. Here we demonstrate that systemically and topically supplemented leptin improved re-epithelialization of wounds in ob/ob mice. Leptin completely reversed the atrophied morphology of the migrating epithelial tongue observed at the wound margins of leptin-deficient animals into a well-organized hyperproliferative epithelium. Moreover, topically supplemented leptin accelerated normal wound-healing conditions in wild-type mice. As assessed by immunohistochemistry, proliferating keratinocytes located at the wound margins specifically expressed the leptin-receptor subtype ObRb during repair. Additionally, leptin mediated a mitogenic stimulus to the human keratinocyte cell line HaCaT and human primary keratinocytes in vitro. Therefore, leptin might represent an effective novel therapeutic factor to improve impaired wound-healing conditions.

Keratinocyte-derived chemotactic cytokines: expressional modulation by nitric oxide in vitro and during cutaneous wound repair in vivo.

Inhibition of inducible nitric oxide-synthase (iNOS) enzymatic activity during cutaneous wound repair leads to severely impaired tissue regeneration. To assess whether disturbed leukocyte infiltration might participate in impaired repair, we determined expressional kinetics of neutrophil-attracting macrophage inflammatory protein-2 (MIP-2), and monocyte-attracting macrophage chemoattractant protein-1 (MCP-1) using an excisional wound healing model in mice. MCP-1 was induced in epithelial keratinocytes upon wounding, and our data indicate that NO serves a negative regulatory role for MCP-1 expression in vivo, as clearly reduced numbers of wound margin keratinocytes associated with NO-deficient repair compensate for high MCP-1 expression levels observed during normal healing. MIP-2 expression was restricted to hair follicles which were not reduced in number during NO-deficient repair. In vitro studies confirmed a regulatory role of NO for keratinocyte-derived chemokine expression, as NO attenuated IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression, whereas NO augmented IL-1beta-induced IL-8 (functional human homolog to murine MIP-2) mRNA expression in the human keratinocyte cell line HaCaT.

Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair.

Chemokines are seen as the stimuli that largely control leukocyte migration. To assess whether the severely impaired process of cutaneous repair observed in genetically diabetic db/db mice is associated with a dysregulated infiltration of immune cells, we determined the expressional kinetics for the murine growth-regulated oncogene/melanoma growth stimulatory activity homolog macrophage inflammatory protein-2, and the macrophage chemoattractant protein-1, respectively. Wound repair in db/db mice was characterized by a sustained inflammatory response and a prolonged expression of macrophage inflammatory protein-2 and macrophage chemoattractant protein-1. Immuno-histochemistry revealed that keratinocytes at the wound margins expressed macrophage chemoattractant protein-1, whereas macrophage inflammatory protein-2 immunopositive signals were observed only in keratinocytes of hair follicles located adjacent to the wound site. Inactivation studies using neutralizing antibodies against macrophage chemoattractant protein-1 or macrophage inflammatory protein-2 indicated that sustained expression of these chemokines participated in a prolonged presence of neutrophils and macrophages at the wound site during diabetic repair. Furthermore, our data provide evidence that late infiltration (day 13 after injury) of neutrophils and macrophages into wounds in db/db mice was associated with a simultaneous downregulation of mRNA for receptors specific for macrophage inflammatory protein-2 and macrophage chemoattractant protein-1 in these animals.

Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression.

We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1beta- and tumour-necrosis-factor-alpha-induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-N(G)-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair.

Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation.

Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2 -diolate (DETA-NO)¿, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation.

Interferon-gamma mediates gene expression of IL-18 binding protein in nonleukocytic cells.

Interleukin-18 (IL-18) binding protein is a soluble decoy receptor for IL-18 which efficiently antagonizes biological functions of IL-18 in vitro and in vivo. Since regulation of IL-18 activity likely contributes to the pathogenesis of inflammatory diseases as well as malignancies, we investigated gene expression of IL-18 binding protein (IL-18BP) in different human cell systems, namely in the keratinocyte cell line HaCaT, in the colon carcinoma cell line DLD-1, and in primary renal mesangial cells. In unstimulated cells only minute amounts of mRNA coding for IL-18 binding protein were detectable. However, in all three cell types gene expression was markedly upregulated by interferon-gamma (IFN-gamma). IL-18 is recognized as a pivotal mediator of IFN-gamma production. Therefore, the present data imply that activity of IL-18 is modulated by a negative feedback mechanism which is mediated by IFN-gamma-induced IL-18 binding protein.

Lack of interferon-gamma production despite the presence of interleukin-18 during cutaneous wound healing.

BACKGROUND: Recently, we have reported a rapid and strong induction of interleukin-18 (IL-18) upon cutaneous injury in mice. In this paper, we investigated a possible role of IL-18 in triggering interferon-gamma (IFN-gamma) production at the wound site. MATERIALS AND METHODS: Expression of IFN-gamma during cutaneous wound healing was analyzed by RNase protection assay, Western blot, ELISA, and immunohistochemical techniques in a murine model of excisional skin repair. RESULTS: We could not detect any IFN-gamma mRNA and protein expression during normal skin repair. Additionally, impaired healing in the genetically diabetic db/db mouse, which was used as a model for a prolonged inflammatory phase of repair, was characterized by largely elevated levels of IL-18 during the late phase of repair and an absence of IFN-gamma. Western blot analysis for T-cell- and monocyte/macrophage-specific marker proteins (CD4, F4/80) clearly revealed the presence of these subsets of leukocytic cells at the wound site, that are known to produce IFN-gamma in response to IL-18. Furthermore, we provide evidence that the presence of transforming growth factor-beta1 (TGF-beta1) at the wound site might reflect a counterregulatory mechanism in IL-18-induced IFN-gamma production, as TGF-beta1 strongly suppressed IL-18/phytohaemagglutinin (PHA)-induced IFN-gamma production by peripheral blood mononuclear cells (PBMC) in vitro. CONCLUSIONS: Normal tissue regeneration processes after cutaneous injury were not dependent on the presence of IFN-gamma in vivo, and IL-18 must serve additional roles rather than inducing IFN-gamma during the healing process.

The function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.

Recently, we demonstrated a large induction of inducible nitric oxide synthase (iNOS) during cutaneous wound repair. In this study, we established an in vivo model in mice to investigate the role of NO during the wound healing process. During excisional repair, mice were treated with L-N6-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS enzymatic activity. Compared with control mice, L-NIL-treated animals were characterized by a severely impaired reepithelialization process, as the hyperproliferative epithelia at the wound edges appeared to be delayed and characterized by an atrophied morphology. Immunohistochemical labeling for detection of proliferating cells (BrdU-, Ki67-staining) revealed a strong reduction in proliferating keratinocyte cell numbers during the process of re-epithelialization after inhibition of iNOS activity during repair. Western blot analysis of total wound lysates from PBS- and L-NIL-treated mice clearly demonstrated a reduction in proliferating cell nuclear antigen, representing a marker for cell proliferation, in lysates isolated from L-NIL-treated mice. The dependency between keratinocyte proliferation and NO availability observed during wound repair in vivo is further supported by the observation that proliferation of the keratinocyte cell line (HaCaT) is stimulated by low concentrations of NO-donors also in vitro. In summary, our data demonstrate that the presence of a functionally active iNOS is a crucial prerequisite for normal wound reepithelialization.

Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.

Recently, we demonstrated a large induction of inducible nitric oxide synthase (iNOS) during cutaneous wound repair. In this study, we investigated the role of nitric oxide (NO) for the expression of vascular endothelial growth factor (VEGF), which represents the most important angiogenic factor during the proliferative phase of skin repair. Since keratinocytes are the major source of VEGF production during this process, we used cultured keratinocytes (HaCaT cell line) as an in vitro model to investigate NO action on growth factor- and cytokine-stimulated VEGF expression. Exogenously added NO enhanced transforming growth factor-beta1-, keratinocyte growth factor-, interleukin-1beta-, tumor necrosis factor-alpha-, and interferon-gamma-induced VEGF mRNA and protein synthesis in keratinocytes. We could demonstrate that high-level expression of cytokine-induced VEGF mRNA in keratinocytes is dependent on endogenously produced NO, as inhibition of the coinduced iNOS by N(G)-monomethyl-L-arginine (L-NMMA) markedly decreased cytokine-triggered VEGF mRNA levels in the cells. We also established an in vivo model in mice to investigate the role of NO during wound healing. During excisional wound repair, mice were treated with L-N(6)-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS enzymatic activity. Compared to control mice, L-NIL-treated animals were characterized by markedly reduced VEGF mRNA levels during the inflammatory phase of repair. Immunohistochemistry demonstrated reduced VEGF protein expression and a completely disorganized pattern of VEGF-expressing keratinocytes within the hyperproliferative epithelium at the wound edge in L-NIL-treated mice. We demonstrate that triggering of VEGF expression is a crucial molecular mechanism underlying NO function during wound healing.

Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.

Recent work has suggested interleukin-18 to represent a proinflammatory cytokine that contributes to systemic and local inflammation. As the process of cutaneous wound healing crucially involves an inflammatory phase of repair, we investigated the regulation of interleukin-18 during the repair process. In non-wounded skin we observed high levels of interleukin-18 mRNA, whereas corresponding interleukin-18 protein was expressed only at low basal levels. Upon injury, we found a rapid and large induction of interleukin-18 protein expression, which is directly correlated with decreasing mRNA levels within the wound. Immunohistochemical analysis revealed different sites of expression in the wounded area, with keratinocytes as one major source of interleukin-18 production. The counterregulation of interleukin-18 mRNA and protein expression during wound repair in vivo might represent a general mechanism for interleukin-18 expressional regulation, as cytokine-stimulated keratinocytes exhibit a similar downregulation of interleukin-18 mRNA that is directly associated with increasing interleukin-18 protein levels in vitro. The rapid induction of interleukin-18 during wound healing suggests a role for interleukin-18 within the early phase of repair rather than a role in costimulation of interferon-gamma release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair.

Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells.

Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1-3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1beta mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor-BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells.