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Counterregulation of Interleukin-18 mRNA and Protein Expression During Cutaneous Wound Repair in Mice

1999; Elsevier BV; Volume: 113; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.1999.00704.x

1523-1747

Heiko Kämpfer, Heiko Mühl, Josef Pfeilschifter, Stefan L. Frank, Uwe Kalina,

IL-33, ST2, and ILC Pathways

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-γ release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair. 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-γ release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair. The process of cutaneous wound repair is characterized by four overlapping phases involving hemostasis, inflammation, proliferation, and the remodeling phase of repair. After injury, new tissue formation starts with re-epithelialization and is followed by granulation tissue formation. The latter process encompasses macrophage accumulation, fibroblast ingrowth, matrix deposition, and angiogenesis (Clark, 1996Clark R.A.F. Wound repair: overview and general considerations.The Molecular and Cellular Biology of Wound Repair. In: Clark, RAF. (ed.). New York, Plenum Press1996: 3-50Google Scholar). Inflammation, re-epithelialization, and granulation tissue formation are driven in part by a complex mixture of growth factors and cytokines, which are released into the area of injury. Especially, proinflammatory cytokines are important mediators of cellular actions during tissue regeneration. Very recently, the group of proinflammatory cytokines is extended by a new member, first described as interferon (IFN) -γ-inducing factor (IGIF) (Nakamura et al., 1989Nakamura K. Okamura H. Wada M. Nagata K. Tamura T. Endotoxin-induced serum factor that stimulates gamma interferon production.Infect Immun. 1989; 57: 590-595Crossref PubMed Google Scholar). After purification of IGIF in 1995 (Okamura et al., 1995Okamura H. Nagata K. Komatsu T. et al.A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock.Infect Immun. 1995; 63: 3966-3972Crossref PubMed Google Scholar), cDNA of murine and human IGIF were cloned subsequently (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2316) Google Scholar;Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar). As recombinant IGIF exhibited pleiotropic immunologic activities, the conventional name interleukin (IL)-18 was proposed. The gene for IL-18 encodes a precursor polypeptide (pro-IL-18), which is biologically inactive (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2316) Google Scholar). IL-18 resembles structural similarity to the IL-1 family of proteins (Bazan et al., 1996Bazan J.F. Timans J.C. Kastelein R.A. A newly defined interleukin-1?.Nature. 1996; 379: 591Crossref PubMed Scopus (253) Google Scholar). Similar to IL-1β, pro-IL-18 is cleaved by IL-1β converting enzyme at the authentic processing site to generate the mature and active form of IL-18 (Ghayur et al., 1997Ghayur T. Banerjee S. Hugunin M. et al.Caspase-1 processes IFN-γ-inducing factor and regulates LPS-induced IFN-γ production.Nature. 1997; 386: 619-623Crossref PubMed Scopus (983) Google Scholar;Gu et al., 1997Gu Y. Kuida K. Tsutsui H. et al.Activation of interferon-γ inducing factor mediated by interleukin-1β converting enzyme.Science. 1997; 275: 206-209Crossref PubMed Scopus (967) Google Scholar). Target specificities and receptors of IL-18 and IL-1, however, are different from each other. Immune cell-derived IL-18 is described to possess two main biologic functions. Primarily, IL-18 is a costimulant for T helper cell 1 cytokine production by acting as a costimulant for IFN-γ production with IL-12, IL-2, microbial agents, or mitogens (Kohno et al., 1997Kohno K. Kataoka J. Ohtsuki T. et al.IFN-γ-inducing factor (IGIF) is a co-stimulatory factor on the activation of Th1 but not Th2 cells and exerts its effects independently of IL-12.J Immunol. 1997; 158: 1541-1550PubMed Google Scholar;Robinson et al., 1997Robinson D. Shibuya K. Mui A. et al.IGIF does not drive Th1 development but synergizes with IL-12 for interferon-γ production and activates IRAK and NFκB.Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). Additionally, IL-18 is able to directly induce tumor necrosis factor (TNF)-α, IL-1β, both CXC and CC chemokines (Puren et al., 1998Puren A.J. Fantuzzi G. Gu Y. Su M.S. Dinarello C.A. Interleukin-18 (IFN gamma-inducing factor) induces IL-8 and IL-1beta via TNF alpha production from non-CD14+ human blood mononuclear cells.J Cin Invest. 1998; 101: 711-721Crossref PubMed Scopus (508) Google Scholar), and nuclear translocation of nuclear factor-κB (Matsimoto et al., 1997Matsimoto S. Tsuji-Takayama K. Aizawa Y. Koide K. Takeuchi M. Ohta T. Kurimoto M. Interleukin-18 activates NFκB in murine T helper type I cells.Biochem Biophys Res Commun. 1997; 234: 454-457Crossref PubMed Scopus (180) Google Scholar;Robinson et al., 1997Robinson D. Shibuya K. Mui A. et al.IGIF does not drive Th1 development but synergizes with IL-12 for interferon-γ production and activates IRAK and NFκB.Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar), placing IL-18 among other proinflammatory cytokines as a most likely contributor to systemic and local inflammation. In this study, we have investigated the expression pattern of IL-18 during the highly dynamic process of cutaneous wound repair. We provide evidence for a counterregulatory mechanism of IL-18 mRNA and protein expression during the inflammatory phase of repair. Furthermore, we describe a dramatic increase in IL-18 protein with highest levels early after injury. As T cell numbers peak at day 7 in skin wounds (Fishel et al., 1987Fishel R.S. Barbul A. Beschorner W.E. Wasserkrug H.L. Efron G. Lymphocyte participation in wound healing: Morphologic assessment using monoclonal antibodies.Ann Surg. 1987; 206: 25-29Crossref PubMed Scopus (82) Google Scholar), our data suggest that the initial role of IL-18 during the repair process is most likely to be independent from its ability to trigger the release of T cell-derived IFN-γ. To examine IL-18 expression during the wound healing process, six full-thickness wounds were created on the backs of female BALB/C mice (3 mo old). Mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg per kg body weight)/Xylazin (10 mg per kg body weight). The hair on the back of these mice was cut, and the back was subsequently wiped with 70% ethanol. Six full-thickness wounds (5 mm in diameter, 3–4 mm apart) were made on the backs of these mice by excising the skin and the underlying panniculus carnosus. The wounds were allowed to form a scab. Skin biopsy specimens from four animals were obtained 1, 3, 5, 7, and 13 d after injury. An area of 7–8 mm in diameter which included the scab and the complete epithelial margins was excised at each time point. As a control, a similar amount of skin was taken from the backs of four non-wounded mice. In every experiment, the wounds from four animals (16 wounds) and the non-wounded back skin from four animals, respectively, were combined, frozen immediately in liquid nitrogen, and stored at –80°C until used for RNA or protein isolation. All animal experiments were carried out according to the guidelines and with the permission from the local government of Hessen. Mice were wounded as described above. Animals were killed at day 5 after injury. Complete wounds were isolated from the middle of the back, bisected, and frozen in tissue freezing medium. Six micrometer sections from the middle of the wound were stained with hematoxylin and eosin. Mice were wounded as described above. Animals were killed at days 1, 3, and 13 after injury. Complete wounds were isolated from the middle of the back, bisected, and frozen in tissue freezing medium. Six micrometer frozen sections were fixed with acetone and treated for 10 min at room temperature with 1% H2O2 in phosphate-buffered saline to inactivate endogenous peroxidases. They were subsequently incubated for 60 min at room temperature with a polyclonal anti-serum against human IL-18 (PeproTech, Frankfurt, Germany) (1:100 diluted in phosphate-buffered saline, 0.1% goat serum albumin). The slides were subsequently stained with the avidin–biotin–peroxidase complex system from SantaCruz (Heidelberg, Germany) using 3-amino-9-ethylcarbazole as a chromogenic substrate. After development, they were rinsed with water, counterstained with hematoxylin (Sigma, Deisenhofen, Germany), and mounted. Skin samples and keratinocytes from the cell culture experiments were homogenized in lysis buffer (1% Triton X-100, 20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM ethylenediamine tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 15 μg leupeptin per ml). The tissue extract was cleared by centrifugation. Fifty micrograms of total protein from these lysates was separated using sodium dodecyl sulfate–gel electrophoresis. After transfer to a polyvinylidene fluoride membrane, IL-18 protein was detected using a monoclonal antibody raised against human IL-18 (Kalina et al., 1998Kalina U. Klebba C. Kauschat D. Pape M. Hoelzer D. Ottmann O.G. HIV-1 replication is enhanced by pro- and eukaryotically expressed recombinant interleukin-18.Eur Cytokine Netw. 1998; 9: S428Google Scholar). A secondary antibody coupled to horseradish peroxidase and the enhanced chemiluminescence detection system were used to visualize IL-18 protein. Phenylmethylsulfonyl fluoride, aprotinin, and leupeptin were from Sigma and the enhanced chemiluminescence detection system was obtained from Amersham (Freiburg, Germany). RNA isolation was performed as described (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62250) Google Scholar). Thirty micrograms of total RNA from wounded or non-wounded skin were used for RNase protection assays. RNase protection assays were carried out as described (Werner et al., 1992Werner S. Peters K.G. Longaker M.T. Fuller-Pace F. Banda M.J. Williams L.T. Large induction of keratinocyte growth factor expression in the dermis during wound healing.Proc Natl Acad Sci USA. 1992; 89: 6896-6900Crossref PubMed Scopus (512) Google Scholar). Protected fragments were separated on 5% acrylamide/8 M urea gels and analyzed using a PhosphoImager (Fuji, Straubenhardt, Germany). The human keratinocyte cell line HaCaT (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreuz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3269) Google Scholar) was cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. For the induction experiments, cells were grown to confluency without changing the medium and rendered quiescent by a 24 h incubation in Dulbecco’s modified Eagle’s medium without serum. Cells were then incubated for varying time periods in fresh Dulbecco’s modified Eagle’s medium containing a combination of proinflammatory cytokines (2 nM IL-1β, 2 nM TNF-α, and 100 U IFN-γ per ml). Aliquots of cells were harvested before and at different time points after treatment and used for RNA and protein isolation. The murine and human IL-18 cDNA probes, and the cDNA probe for murine vascular endothelial growth factor, respectively, were cloned by polymerase chain reaction. The cloned cDNA fragments correspond to nucleotides 318–605 (for murine IL-18), nucleotides 335–627 (for human IL-18), and nucleotides 139–455 (for murine vascular endothelial growth factor) of the published sequences (Breier et al., 1992Breier G. Albrecht U. Sterrer S. Risau W. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation.Development. 1992; 114: 521-532Crossref PubMed Google Scholar;Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2316) Google Scholar;Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar). Data are shown as mean ± SD. The data are presented either as x-fold induction or as percentage change compared with the control (100%). For each reagent, in each set of conditions, data were analyzed by Student’s t test using the software SigmaPlot (Jandel Scientific, Erkrath, Germany). To determine a possible role for IL-18 in cutaneous wound repair, we first investigated the time course of IL-18 mRNA and protein expression during this process. We isolated total RNA and total protein from full-thickness excisional wounds at different intervals after injury and performed RNase protection assays, or western blot analysis, respectively. Sixteen wounds (for RNA) and four wounds (for protein) from the backs of four mice were excised for each time point, combined and used for RNA, or protein isolation. Normal skin from the back of non-wounded mice was used as a control. The area of injured tissue which has been removed from the animals for the isolation of RNA and protein is shown in Figure 3a. Exactly one-half of a 5 d wound is shown in Figure 3a, and the site where the tissue was cut is marked by arrows indicating that the wound margins have been included within the area of wound tissue which has been removed for analysis. As shown in Figure 1 (ctrl), IL-18 was highly expressed at the mRNA level in normal back skin. Notably, these observed high basal levels of IL-18 mRNA did not subsequently result in high IL-18 protein levels in unwounded controls (Figure 2a,ctrl), as we could detect only low levels of IL-18 protein in normal skin. Upon injury, a strong and rapid decrease of IL-18 mRNA levels was observed within 24 h after wounding, reaching reduced minimum levels (40–50% of control) between days 1 and 3 after injury. The late inflammatory phase of repair (days 5–7 after injury) is characterized by re-increasing levels of IL-18 mRNA, as, within the wounded area, cellular pools of IL-18 mRNA started to fill up again. At day 13 after injury, all wounds were completely re-epithelialized, but the granulation tissue still revealed a high cellularity (Figure 3f). At this stage of the repair process, expression of IL-18 mRNA again reached the high basal levels detected in unwounded skin.Figure 2Large induction of IL-18 protein expression during wound repair. (A) Total protein (50 μg) from lysates of non wounded and wounded back skin (days 1, 3, 5, 7, and 13 after injury, indicated at the top of each lane) were analyzed by immunoblotting for the presence of IL-18 protein. Four wounds (n = 4) from the backs of four animals were excised for each experimental time point and used for protein isolation. IL-18 protein is indicated by an arrow. Staining of the PVDF membrane after immunostaining using Ponceau S (Serva, Heidelberg, Germany) was used as a loading control (lower panels). Total protein (50 μg) from lysates of human myelomonocytic KG-1 cells and human skin, respectively, were analyzed for IL-18 protein as a positive control (right panels). The degree of IL-18 protein induction assessed by scanning densitometry (Molecular Analyst, BioRad, Munich, Germany) is shown schematically in (B). Data are expressed as mean ± SD and presented as percentage induction compared with the control (100%). *p < 0.05; **p < 0.01.View Large Image Figure ViewerDownload (PPT) As a next step, we examined whether the observed decrease of IL-18 mRNA expression after skin injury correlates with decreased levels of immunoreactive IL-18 protein in wounds. For this purpose, lysates of non-wounded skin and from 1, 3, 5, 7, and 13 d wounds were analyzed by western blotting for the presence of IL-18 protein (Figure 2). Protein lysates of human myelomonocytic KG-1 cells, which are known to produce IL-18 protein (Konishi et al., 1997Konishi K. Tanabe F. Taniguchi M. et al.A simple and sensitive bioassay for the detection of human interleukin-18/interferon-gamma-inducing factor using human myelomonocytic KG-1 cells.J Immunol Methods. 1997; 209: 187-191Crossref PubMed Scopus (51) Google Scholar), and human skin, respectively, are used as a positive control for a functional antibody. As mentioned above, we detected a differential regulation of IL-18 mRNA and protein in non-wounded control skin, as high IL-18 mRNA did not correlate with the low amounts of IL-18 protein expressed in normal skin (Figure 1 and Figure 2). Upon injury, we were surprised to observe a dramatic counterregulation of IL-18 protein expression compared with IL-18 mRNA levels. In contrast to decreased mRNA levels, IL-18 protein levels were high in the lysates obtained from 1, 3, 5, and 7 d wounds (Figure 2). The strong increase (about 4-fold) in IL-18 protein was maximal at day 1, representing the time point of tissue repair when IL-18 mRNA had declined to a minimum (Figure 1). It is remarkable that rising levels of IL-18 mRNA, starting to increase again at day 5 postwounding, are directly correlated with a reduction of IL-18 protein during the remaining process of repair. Thirteen days after injury, when wounds are completely re-epithelialized, the observed counterregulation finally leads to the expressional state of unwounded skin: high IL-18 mRNA levels are associated with low IL-18 protein levels. Remarkably, a second band which is recognized by the monoclonal anti-IL-18 antibody (Figure 2a, right panels) fits the expected size (22 kDa) of unprocessed pro-IL-18. Thus, IL-18 protein which could be detected during the early and late inflammatory phase of wound repair, and during granulation tissue formation and re-epithelialization (Figure 2) is most likely to represent the biologically active, mature protein (18.3 kDa), as the result of an highly effective processing of pro-IL-18. To investigate the localization of IL-18 in unwounded skin and during wound healing, sections from normal back skin and 1, 3, and 13 d full-thickness mouse wounds were stained with a monospecific, polyclonal antibody against IL-18 protein. The observed constitutive, but low level basal expression of IL-18 protein in unwounded skin (Figure 2) was due to expression in the epidermis and hair follicles, whereas the underlying dermal cells did not express IL-18 protein (Figure 3b). At early time points after wounding (1 and 3 d), IL-18 was expressed at high levels (Figure 2). The late 13 d time point was chosen to determine the cell types which are responsible for the remaining low-level expression of IL-18 protein after re-epithelialization. In 1 d wounds, a remarkably strong expression of IL-18 was seen in a population of cells infiltrating the wounded area, thereby forming invading “groups” of strongly labeled cells and a population of labeled cells localized directly beneath the freshly formed blot clot (Figure 3c). These cells are most likely to represent polymorphonuclear neutrophils, which are the first cells invading the injured tissue. During the repair process, however, localization of IL-18 expression changed, as the protein could be detected in the granulation tissue and in the epidermis in 3 d wounds (Figure 3d,e, indicated by arrows). With the antibody, immunopositive signals were obtained in keratinocytes, particularly within the hyperproliferative epithelium at the wound edge (Figure 3d). Furthermore, strong signals were detected in cells invading towards the granulation tissue. Owing to cell shape, these strongly labeled cells seem to represent migrating fibroblasts and infiltrating mononuclear immune cells, and therefore, most likely macrophages (Figure 3e). Finally, we determined the epidermis of the re-epithelialized wounds to be the source of the remaining low-level expression of IL-18 protein, as the underlying granulation tissue revealed nearly no labeled cells (Figure 3f). Owing to the particularly strong labeling of the observed immunopositive cell types within the wound, the anti-IL-18-antibody was most likely to stain IL-18-synthesizing cells rather than receptor-bound IL-18 protein, for which we would expect a much weaker and peripheral staining of immunopositive cells. We used a cell culture approach to strengthen further the novel counterregulatory mechanism for IL-18 mRNA and protein expression observed during the wound healing process in vivo. As keratinocytes turned out to be one of the main sources of IL-18 expression during wound repair, we have chosen the human keratinocyte cell line HaCaT (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreuz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3269) Google Scholar) to induce IL-18 expression. As the infiltration of polymorphonuclear leukocytes followed by macrophages is a crucial event in wound repair, we tested the ability of cytokines produced by these cells to alter IL-18 mRNA and protein levels in keratinocytes. As shown in Figure 4, we could confirm the counterregulatory mechanism observed for IL-18 expression in vivo, as keratinocytes in vitro respond to a cytokine-mix (TNF-α, IL-1β, IFN-γ) in a similar manner. The keratinocytes potently responded to the cytokine stimulus, as the high basal levels of IL-18 mRNA observed in untreated cells were dramatically reduced within 12 h after stimulation. At this time point, IL-18 mRNA was reduced to a minimum (5% of control) and only hardly detectable (Figure 4a). Similar to the in vivo situation, IL-18 mRNA levels re-increased and mRNA expression levels were high again in keratinocytes after 32 h of cytokine treatment. We could observe a large increase in IL-18 protein expression (Figure 4b) during that time period after cytokine stimulation, which is characterized by a strong decrease in IL-18 mRNA (Figure 4a). Therefore, these data clearly support the counterregulation of IL-18 mRNA and protein expression which could be observed in the wound healing experiments. The wound healing process, initiated upon injury to overcome tissue damage, represents a highly dynamic process. This repair process requires the coordination between involved tissues and cell lineages at the molecular and cellular level. Proliferation, migration, matrix synthesis, contraction of the wound, and the presence of signaling molecules within the wound site are crucial events for the ongoing repair process that have to be closely regulated to sustain a healing process that finally leads to an at least partial reconstruction of the tissue. Proinflammatory cytokines and various peptide growth factors are known to be key players in this process (Moulin, 1995Moulin V. Growth factors in skin wound healing.Eur J Cell Biol. 1995; 68: 1-7Abstract Full Text PDF PubMed Google Scholar;Martin, 1997Martin P. Wound healing—Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3423) Google Scholar). First described as IGIF (Nakamura et al., 1989Nakamura K. Okamura H. Wada M. Nagata K. Tamura T. Endotoxin-induced serum factor that stimulates gamma interferon production.Infect Immun. 1989; 57: 590-595Crossref PubMed Google Scholar), a novel cytokine has been identified and cloned: IL-18 (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2316) Google Scholar;Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar). Recent studies revealed two main functions for IL-18. Whereas, on one hand, IL-18 acts as a costimulant for the production of IFN-γ and other cytokines in T helper type 1 cells, on the other hand, IL-18 could be placed among the other known proinflammatory cytokines due to its ability to induce TNF-α, IL-1β, CC chemokine, and CXC chemokine in monocytic cells (Dinarello et al., 1998Dinarello C.A. Novick D. Puren A.J. et al.Overview of interleukin-18: more than an interferon-gamma inducing factor.J Leukoc Biol. 1998; 63: 658-664PubMed Google Scholar;Puren et al., 1998Puren A.J. Fantuzzi G. Gu Y. Su M.S. Dinarello C.A. Interleukin-18 (IFN gamma-inducing factor) induces IL-8 and IL-1beta via TNF alpha production from non-CD14+ human blood mononuclear cells.J Cin Invest. 1998; 101: 711-721Crossref PubMed Scopus (508) Google Scholar;Yoshimoto et al., 1998Yoshimoto T. Takeda K. Tanaka T. et al.IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production.J Immunol. 1998; 161: 3400-3407PubMed Google Scholar). For these reasons we postulated that this recently described cytokine might serve a role during the inflammatory phase of cutaneous wound repair. There is, however, no information available yet dealing with a possible role of IL-18 in processes of tissue regeneration. For unwounded skin, the situation of IL-18 expression is characterized by high intracellular pools of IL-18 mRNA that are not subsequently translated into the corresponding protein, as IL-18 protein was only detectable at low basal levels in lysates of normal skin. Using a model of excisional wound repair in mice, we observed a strong increase in IL-18 protein expression early upon injury. IL-18 protein levels were highest at day 1, remained elevated until day 5, and subsequently declined when re-epithelialization and granulation tissue formation occurs. It is remarkable that increasing amounts of IL-18 within the wound are associated with decreased IL-18 mRNA pools. This finding suggests a very potent regulatory mechanism for IL-18 expression after tissue injury: IL-18 becomes available early after injury, as a transcriptional activation of producer cells is not necessary. High levels of IL-18 mRNA are subsequently translated and IL-18 protein secreted into the wound. Our data suggest that, in part, IL-18 mRNA transcripts seem to be degraded, as IL-18 protein synthesis moves on. This might reflect a regulatory mechanism to avoid an overshooting IL-18 synthesis from the high pools of mRNA. In line with the rapid induction of IL-18 after injury, the precursor molecule of IL-18 was only hardly detectable: pro-IL-18, which is biologically inactive and has to be cleaved by the IL-1β converting enzyme to generate the mature active form of IL-18 (Ghayur et al., 1997Ghayur T. Banerjee S. Hugunin M. et al.Caspase-1 processes IFN-γ-inducing factor and regulates LPS-induced IFN-γ production.Nature. 1997; 386: 619-623Crossref PubMed Scopus (983) Google Scholar;Gu et al., 1997Gu Y. Kuida K. Tsutsui H. et al.Activation of interferon-γ inducing factor mediated by interleukin-1β converting enzyme.Science. 1997; 275: 206-209Crossref PubMed Scopus (967) Google Scholar). Thus, cleavage of pro-IL-18 by IL-1β converting enzyme is likely to be highly efficient during repair. The observation that IL-18 release is regulated via a post-transcriptional mechanism from high intracellular pools of IL-18 mRNA is further strengthened by cell culture experiments using a keratinocyte cell line (HaCaT). This mechanism seems to be a very potent alternative to another mechanism described for IL-1β and keratinocyte growth factor-2 to circumvent a transcriptional lag-phase after injury: IL-1β and keratinocyte growth factor-2 are stored in large amounts in intact skin and released postwounding (Raines et al., 1989Raines E.W. Dower S.K. Ross R. Interleukin-1 mitogenic activity for fibroblast and smooth muscle cells is due to PDGF-AA.Science. 1989; 243: 393-396Crossref PubMed Scopus (497) Google Scholar;Nicosia et al., 1993Nicosia R.F. Bonnano M. Smith M. Fibronectin promotes the elongation of microvessels during angogenesis in vitro.J Cell Physiol. 1993; 154: 654-661Crossref PubMed Scopus (126) Google Scholar;Beer et al., 1997Beer H.D. Florence C. Dammeier J. McGuire L. Werner S. Duan D.R. Mouse fibroblast growth factor 10: cloning, protein characterization, and regulation of mRNA expression.Oncogene. 1997; 15: 2211-2218Crossref PubMed Scopus (105) Google Scholar). The cell types known until now to express IL-18 are heterogeneous, indicating that the potency to produce IL-18 is not restricted to immune cells. Besides macrophages, IL-18 is reported to be expressed in keratinocytes after contact with an allergen (Stoll et al., 1997Stoll S. Muller G. Kurimoto M. et al.Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes.J Immunol. 1997; 159: 298-302PubMed Google Scholar), in cells of the adrenal gland and the neurohypophysis (Conti et al., 1997Conti B. Jahng J.W. Tinti C. Son J.H. Joh T.H. Induction of interferon-γ inducing factor in the adrenal cortex.J Biol Chem. 1997; 272: 2035-2037Crossref PubMed Scopus (148) Google Scholar), in intestinal epithelial cells (Takeuchi et al., 1997Takeuchi M. Nishizaki Y. Sano O. Ohta T. Ikeda M. Kurimoto M. Immunohistochemical and immune-microscopic detection of interferon-γ-inducing factor (“interleukin-18”) in mouse intestinal epithelial cells.Cell Tissue Res. 1997; 289: 499-503Crossref PubMed Scopus (107) Google Scholar), and osteoblasts (Udagawa et al., 1997Udagawa N. Horwood N.J. Elliott J. et al.Interleukin-18 (interferon-γ inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-γ to inhibit osteoclast formation.J Exp Med. 1997; 185: 1005-1012Crossref PubMed Scopus (347) Google Scholar). Therefore, we could confirm previous reports, as keratinocytes and macrophages exhibited a particularly strong IL-18-positive staining within the wound. Immunohistochemistry further revealed most likely infiltrating polymorphonuclear neutrophils (day 1) as producers of IL-18. This is in line with a recent report which clearly identifies this cell type as a potent source of cytokines of the IL-1 family (Hübner et al., 1996Hübner G. Brauchle M. Smola H. Madlener M. Fässler R. Werner S. Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.Cytokine. 1996; 8: 548-556Crossref PubMed Scopus (357) Google Scholar). In addition, our immunohistochemistry data suggest migrating fibroblasts (day 3) to produce large amounts of IL-18 as well. Staining of IL-18-positive fibroblasts is in agreement with detection of IL-18 mRNA and protein in the dermis of normal murine skin (Xu et al., 1998Xu B. Aoyama K. Yu S. et al.Expression of interleukin-18 in murine contact hypersensitivity.J Interferon Cytokine Res. 1998; 18: 653-659Crossref PubMed Scopus (23) Google Scholar). Unexpectedly, we observed highest amounts of IL-18 during repair, when T cells have not yet infiltrated the injured area. T lymphocyte numbers peak at day 7 of repair (Ross and Benditt, 1962Ross R. Benditt E.P. Wound healing and collagen formation: fine structure in experimental scurvy.J Cell Biol. 1962; 12: 533-550Crossref PubMed Scopus (84) Google Scholar;Fishel et al., 1987Fishel R.S. Barbul A. Beschorner W.E. Wasserkrug H.L. Efron G. Lymphocyte participation in wound healing: Morphologic assessment using monoclonal antibodies.Ann Surg. 1987; 206: 25-29Crossref PubMed Scopus (82) Google Scholar). Thus, the slightly elevated levels of IL-18 observed at this stage of the healing process might contribute to T helper type 1-derived IFN-γ and cytokine release during the late inflammatory phase of repair. It is consistent with this finding to suggest a restriction of the IFN-γ-inducing properties of IL-18 to the late inflammatory phase, as T cells which require the IL-18 costimulus to produce IFN-γ are not present in early wounds (Ross and Benditt, 1962Ross R. Benditt E.P. Wound healing and collagen formation: fine structure in experimental scurvy.J Cell Biol. 1962; 12: 533-550Crossref PubMed Scopus (84) Google Scholar;Fishel et al., 1987Fishel R.S. Barbul A. Beschorner W.E. Wasserkrug H.L. Efron G. Lymphocyte participation in wound healing: Morphologic assessment using monoclonal antibodies.Ann Surg. 1987; 206: 25-29Crossref PubMed Scopus (82) Google Scholar). The observation, that IL-18 cannot act as IFN-γ inducing factor due to the absence of T cells in early wounds corresponds to the well characterized deleterious effects of IFN-γ for the healing process: IFN-γ inhibits keratinocyte proliferation, decreases connective tissue formation, increases deposition of mucopolysaccharides, inhibits neovascularization, and finally leads to a delayed skin closure (Hancock et al., 1988Hancock G.E. Kaplan G. Cohn Z.A. Keratinocyte growth regulation by the products of immune cells.J Exp Med. 1988; 168: 1395-1402Crossref PubMed Scopus (147) Google Scholar;Granstein et al., 1989Granstein R.D. Deak M.R. Jacques S.L. et al.The systemic administration of gamma interferon inhibits collagen synthesis and acute inflammation in a murine skin wounding model.J Invest Dermatol. 1989; 93: 18-27Abstract Full Text PDF PubMed Google Scholar,Granstein et al., 1990Granstein R.D. Flotte T.J. Amento E.P. Interferons and collagen production.J Invest Dermatol. 1990; 95: 75S-80SAbstract Full Text PDF PubMed Google Scholar;Miles et al., 1994Miles R.H. Paxton T.P. Zacheis D. Dries D.J. Gamelli R.L. Systemic administration of interferon-gamma impairs wound healing.J Surg Res. 1994; 56: 288-294Abstract Full Text PDF PubMed Scopus (38) Google Scholar). For these reasons, the proinflammatory properties of IL-18 have to be discussed to be important for the regulation of early wound repair. IL-18 is reported to induce directly TNF-α, IL-1β, the CXC chemokine IL-8, and the CC chemokines macrophage-inflammatory protein-1α and macrophage chemoattractant protein-1 in peripheral blood mononuclear cells, and, thus, act as proinflammatory cytokine (Puren et al., 1998Puren A.J. Fantuzzi G. Gu Y. Su M.S. Dinarello C.A. Interleukin-18 (IFN gamma-inducing factor) induces IL-8 and IL-1beta via TNF alpha production from non-CD14+ human blood mononuclear cells.J Cin Invest. 1998; 101: 711-721Crossref PubMed Scopus (508) Google Scholar). Therefore, by inducing the release of neutrophil and monocyte chemoattractant proteins, we suggest a part for early induced IL-18 to be involved in the amplification of signals guiding increasing numbers of immune cells into the wound at the onset of wound repair (Kunstfeld et al., 1998Kunstfeld R. Lechleitner S. Wolff K. Petzelbauer P. MCP-1 and MIP-1alpha are most efficient in recruiting T cells into the skin in vivo.J Invest Dermatol. 1998; 111: 1040-1044Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 553), and by grants of the Commission of the European Communities (Biomed 2, PL 90979) and the Paul and Ursula Klein-Stiftung. We thank Nicole Kolb for her excellent technical assistance. We also gratefully acknowledge Dr. Martin Kock for his help regarding the animal experiments.