The Interleukin-6 Cytokine System Regulates Epidermal Permeability Barrier Homeostasis
2004; Elsevier BV; Volume: 123; Issue: 1 Linguagem: Inglês
10.1111/j.0022-202x.2004.22736.x
ISSN1523-1747
AutoresXuping Wang, Michael Schunck, Karl‐Josef Kallen, Claudia Neumann, Christian Trautwein, Stefan Rose‐John, Ehrhardt Proksch,
Tópico(s)Signaling Pathways in Disease
ResumoInterleukin-6 (IL-6) is involved in the growth and differentiation of numerous cell types. In the skin it is produced primarily by keratinocytes. The transcription factor STAT3 is activated by cytokines of the IL-6 family. In this study, we examined the involvement of IL-6, soluble IL-6-receptor, and STAT3 in epidermal barrier repair after injury to the stratum corneum by tape-stripping. After barrier disruption in wild-type mice we found an increased immunostaining of IL-6 and IL-6R on epidermal keratinocytes at 15 min to 5 h after treatment. The increase in IL-6 and IL-6R was confirmed by western blotting using epidermal homogenates and was partially prevented by occlusion immediately after barrier disruption. In IL-6-deficient mice, epidermal barrier repair was reduced at 3–24 h after treatment. Topical application of IL-6 or Hyper-IL-6, a complex of IL-6 linked to the soluble IL-6 receptor, enhanced epidermal barrier repair in wild-type mice. Application of the fusion protein gp130-FC, a specific inhibitor of the agonist IL-6/sIL-6 receptor complex, delayed barrier repair in wild, but not in IL-6-deficient mice. STAT3 tyrosine phosphorylation was induced after barrier disruption in wild-type, but markedly reduced in IL-6-deficient mice. Our results indicate that the IL-6 cytokine system, particularly transsignalling via the soluble IL-6R, is critically involved in barrier repair after skin injury. Interleukin-6 (IL-6) is involved in the growth and differentiation of numerous cell types. In the skin it is produced primarily by keratinocytes. The transcription factor STAT3 is activated by cytokines of the IL-6 family. In this study, we examined the involvement of IL-6, soluble IL-6-receptor, and STAT3 in epidermal barrier repair after injury to the stratum corneum by tape-stripping. After barrier disruption in wild-type mice we found an increased immunostaining of IL-6 and IL-6R on epidermal keratinocytes at 15 min to 5 h after treatment. The increase in IL-6 and IL-6R was confirmed by western blotting using epidermal homogenates and was partially prevented by occlusion immediately after barrier disruption. In IL-6-deficient mice, epidermal barrier repair was reduced at 3–24 h after treatment. Topical application of IL-6 or Hyper-IL-6, a complex of IL-6 linked to the soluble IL-6 receptor, enhanced epidermal barrier repair in wild-type mice. Application of the fusion protein gp130-FC, a specific inhibitor of the agonist IL-6/sIL-6 receptor complex, delayed barrier repair in wild, but not in IL-6-deficient mice. STAT3 tyrosine phosphorylation was induced after barrier disruption in wild-type, but markedly reduced in IL-6-deficient mice. Our results indicate that the IL-6 cytokine system, particularly transsignalling via the soluble IL-6R, is critically involved in barrier repair after skin injury. IL-6 signal transducer gp130 IL-6+soluble IL-6R interleukin-6 IL-6 receptor stratum corneum signal transducer and activator of transcription 3 transepidermal water loss Cytokines are very important for skin permeability repair. Previously, it has been shown that after experimental barrier disruption in mouse and human skin an increase in TNF, IL-1-α and -β mRNA and protein level occurs (Wood et al., 1992Wood L.C. Jackson S.M. Elias P.M. Grunfeld C. Feingold K.R. Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice.J Clin Invest. 1992; 90: 482-487Crossref PubMed Scopus (387) Google Scholar;Nickoloff and Naidu, 1994Nickoloff B.J. Naidu Y. Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.J Am Acad Dermatol. 1994; 30: 535-546Abstract Full Text PDF PubMed Scopus (408) Google Scholar;Wood et al., 1997Wood L.C. Stalder A.K. Liou A. Campbell I.L. Grunfeld C. Elias P.M. Feingold K.R. Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin.Exp Dermatol. 1997; 6: 98-104Crossref PubMed Scopus (67) Google Scholar). Using mice strains that are defective in TNF-R1 or in IL-1R1, we found a delay in skin permeability repair (Jensen et al., 1998Jensen J.M. Kupper T.S. Proksch E. IL-1/IL-1 receptor overexpression and knockout constructs in permeability barrier repair of transgenic mice.J Invest Dermatol. 1998; 110 (Abstract): 499aGoogle Scholar;Jensen et al., 1999Jensen J.M. Schütze S. Förl M. Krönke M. Proksch E. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier.J Clin Invest. 1999; 104: 1761-1770Crossref PubMed Scopus (153) Google Scholar). In fibroblasts it has been shown that TNF and IL-1 stimulate the production of IL-6 (Xing et al., 1998Xing Z. Gauldie J. Cox G. Baumann H. Jordana M. Lei X.F. Achong M.K. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses.J Clin Invest. 1998; 101: 311-320Crossref PubMed Scopus (1112) Google Scholar). In the skin IL-6 is produced primarily by epidermal keratinocytes, whereas macrophages, Langerhans' cells, and fibroblasts in the dermis represent a minor source of this cytokine (Castella-Rodellas et al., 1992Castella-Rodellas A. Castell J.V. Ramirez-Bosca A. Nicolas J.F. Valcuende-Cavero F. Thivolet J. Interleukin-6 in normal skin and psoriasis.Acta Derm Venerol. 1992; 72: 165-168PubMed Google Scholar). In full thickness skin wounds and after thermal injury of mice IL-6 is produced locally and released within 30 min after injury; enhanced levels have been described still 24 h after treatment (Kawakami et al., 1997Kawakami M. Kaneko N. Anada H. Terai C. Okada Y. Measurement of interleukin-6, interleukin-10, and tumor necrosis factor-alpha levels in tissues and plasma after thermal injury in mice.Surgery. 1997; 121: 440-448Abstract Full Text PDF PubMed Scopus (42) Google Scholar;Grellner et al., 2000Grellner W. Georg T. Wilske J. Quantitative analysis of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) in human skin wounds.Forensic Sci Int. 2000; 113: 251-264Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in the growth and differentiation of numerous cell types (Audet et al., 2001Audet J. Miller C.L. Rose-John S. Piret J.M. Eaves C.J. Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic cells.Proc Natl Acad Sci USA. 2001; 98: 1757-1762Crossref PubMed Scopus (111) Google Scholar). Increased levels of IL-6 have been associated with a number of skin and mucous membrane pathologies, such as psoriasis (Castella-Rodellas et al., 1992Castella-Rodellas A. Castell J.V. Ramirez-Bosca A. Nicolas J.F. Valcuende-Cavero F. Thivolet J. Interleukin-6 in normal skin and psoriasis.Acta Derm Venerol. 1992; 72: 165-168PubMed Google Scholar), lupus erythematosus (Nurnberg et al., 1995Nurnberg W. Haas N. Schadendorf D. Czarnetzki B.M. Interleukin-6 expression in the skin of patients with lupus erythematosus.Exp Dermatol. 1995; 4: 52-57Crossref PubMed Scopus (27) Google Scholar), or Sjogren's Syndrome (Kawasaki et al., 2003Kawasaki S. Kawamoto S. Yokoi N. Connon C. Minesaki Y. Kinoshita S. Okubo K. Up-regulated gene expression in the conjunctival epithelium of patients with Sjogren's syndrome.Exp Eye Res. 2003; 77: 17-26Crossref PubMed Scopus (67) Google Scholar). IL-6 first binds to its membrane bound receptor (IL-6R). Thereupon, the IL-6/IL-6R complex associates with the gp130 receptor subunit, followed by homodimerization of gp130 (Groetzinger et al., 1999Groetzinger J. Kernebeck T. Kallen K.J. Rose-John S. IL-6 type cytokine receptor complexes: Hexamer, tetramer or both?.Biol Chem. 1999; 380: 803-813PubMed Google Scholar). Neither IL-6 nor the IL-6R alone bind or activate gp130. Homodimerization of gp130 causes phosphorylation of gp130 and the transcription factors STAT1 and STAT3 by Janus-Kinases (JAK1, JAK2, TYK2), which are constitutively associated with gp130 (Heinrich et al., 1998Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway.Biochem J. 1998; 334: 297-314Crossref PubMed Scopus (1658) Google Scholar). STAT3-dependent gene expression also causes upregulation of inhibitory proteins of the SOCS family that interfere with JAK activity (Naka et al., 1997Naka T. Narazaki M. Hirata M. Matsumoto et al.Structure and function of a new STAT-induced inhibitor.Nature. 1997; 387: 924-929Crossref PubMed Scopus (1103) Google Scholar;Starr et al., 1997Starr R. Wilson T.A. Viney E.M. et al.A family of cytokine inducible inhibitors of signaling. Nature.. 1997; 387: 917-921Google Scholar;Ernst et al., 2001Ernst M. Inglese M. Waring P. et al.Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation.J Exp Med. 2001; 194: 189-203Crossref PubMed Scopus (202) Google Scholar). Also, STAT3 regulates the migration of cells, in particular keratinocytes (Hirano et al., 2000Hirano T. Ishihara K. Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayted through the IL-6 family of cytokine receptors.Oncogene. 2000; 19: 2548-2556Crossref PubMed Scopus (908) Google Scholar) and is therefore important for wound healing. STAT3-/- mice are not viable, but show early embryonic death. A keratinocyte-specific ablation of STAT3 in mice led to a reduced wound healing (Sano et al., 1999Sano S. Itami S. Takeda K. et al.Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis.EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (412) Google Scholar). Interestingly, IL-6 can also associate with the naturally occurring soluble IL-6R (sIL-6R). The complex of IL-6 and sIL-6R can thereby activate target cells which do not express the membrane bound IL-6R. Such cells in the absence of sIL-6R would not be able to respond to IL-6. This process has been named “transsignaling” (Rose-John and Heinrich, 1994Rose-John S. Heinrich P.C. Soluble receptors for cytokines and growth factors: Generation and biological function.Biochem J. 1994; 300: 281-290Crossref PubMed Scopus (653) Google Scholar). The IL-6/sIL-6R complex may therefore either potentiate the IL-6 activity on cells expressing the transmembrane IL-6R (Mackiewicz et al., 1992Mackiewicz A. Schooltink H. Heinrich P.C. Rose-John S. Complex of soluble human IL-6-receptor/IL-6 up-regulates expression of acute-phase proteins.J Immunol. 1992; 149: 2021-2027PubMed Google Scholar;Peters et al., 1996Peters M. Jacobs S. Ehlers M. et al.The function of the soluble interleukin 6 (IL-6) receptor in vivo: Sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6.J Exp Med. 1996; 183: 1399-1406Crossref PubMed Scopus (228) Google Scholar;Peters et al., 1998Peters M. Müller A.M. Rose John S. Interleukin-6 and soluble interleukin-6 receptor: Direct stimulation of gp130 and hematopoiesis.Blood. 1998; 92: 3495-3504Crossref PubMed Google Scholar) or widen the array of potential IL-6 targets to cells devoid of the transmembrane IL-6R (Jones, 2001Jones R.W. Inflammation and Alzheimers disease.Lancet. 2001; 358: 436-437Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar;Jones and Rose-John, 2002Jones S.A. Rose-John S. The role of soluble receptors in cytokine biology: The agonistic properties of the sIL-6R/IL-6 complex.Biochim Biophys Acta. 2002; 1592: 251-263Crossref PubMed Scopus (211) Google Scholar). Since all human cells express gp130 on the cell surface, the IL-6/sIL-6R complex can principally activate all body cells. In recent years, increasing attention has therefore been directed to the role of the sIL-6R in human diseases (Jones, 2001Jones R.W. Inflammation and Alzheimers disease.Lancet. 2001; 358: 436-437Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar;Jones and Rose-John, 2002Jones S.A. Rose-John S. The role of soluble receptors in cytokine biology: The agonistic properties of the sIL-6R/IL-6 complex.Biochim Biophys Acta. 2002; 1592: 251-263Crossref PubMed Scopus (211) Google Scholar;Kallen, 2002Kallen K.J. The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases.Biochim Biophys Acta. 2002; 1592: 323-343Crossref PubMed Scopus (168) Google Scholar). Two engineered fusion proteins have helped to elucidate the biology of the sIL-6R. The designer cytokine Hyper-IL-6, which covalently links IL-6 with the sIL-6R (Fischer et al., 1997Fischer M. Goldschmitt J. Peschel C. et al.A bioactive designer cytokine with high activity on human hematopoietic progenitor cells expansion.Nature Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (393) Google Scholar), shows a much higher activity on cells than the IL-6/sIL-6R complex. A recently cloned fusion protein of the extracellular domains of gp130 with the constant region of the heavy chain of human IgG1, gp130-Fc, has been shown to specifically inhibit processes mediated via the sIL-6R, but not those relayed by the membrane bound IL-6R (Atreya et al., 2000Atreya R. Mudter J. Finotto S. et al.Blockade of interleukin 6 trans signalling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: Evidence in Crohn disease and experimental colitis in vivo.Nat Medicine. 2000; 6: 583-588Crossref PubMed Scopus (1008) Google Scholar;Hurst et al., 2001Hurst S.M. Wilkinson T.S. McLoughlin R.M. et al.IL-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation.Immunity. 2001; 14: 705-714Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar;Jostock et al., 2001Jostock T. Müllberg J. Ozbek S. et al.Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses.Eur J Biochem. 2001; 268: 160-167Crossref PubMed Scopus (443) Google Scholar). Using IL-6-deficient mice, Hyper-IL-6 and gp130-Fc we have analyzed the role of the IL-6 cytokine system in skin barrier repair. We show that IL-6 accelerates barrier repair and that this effect seems to be mainly mediated via the sIL-6R. The cell-specific localization of cytokines is important for their functions. We investigated the localization of IL-6 and IL-6R in murine skin before and after skin permeability barrier disruption immunohistochemically using specific antibodies (Figure 1). As has been described previously in human skin (reviewed inGallucci et al., 2000Gallucci R.M. Simeonova P.P. Matheson J.M. Kommineni C. Guriel J.L. Sugawara T. Luster M.I. Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.FASEB J. 2000; 14: 2525-2531Crossref PubMed Scopus (306) Google Scholar) we found little staining for IL-6 and IL-6R on fibroblasts or lymphocytes. Instead IL-6 and IL-6R were visible on keratinocytes. In unperturbed epidermis we found faint staining on keratinocytes in the basal, granular, and horny layers, but not in the spinous layers. Fifteen min after skin barrier disruption by tape-stripping staining of basal and stratum granulosum keratinocytes was clearly visible. Also, some suprabasal keratinocytes in the spinous layers were stained. With time, staining intensity increased and reached the highest levels for IL-6 and for IL-6R at 1–3 h after treatment. At these points in time, staining was equally distributed in the entire nucleated epidermis. Thereafter, staining intensity was reduced, at 5 h the basal layer was devoid of cytokine/receptor staining (IL-6: Figure 1a–e, IL-6R: Figure 1g–k). These findings demonstrate that IL-6 and IL-6R are localized to keratinocytes in mouse skin, and that their expression increases after skin barrier disruption. After occlusion by a Latex foil following barrier disruption, an increase in staining for IL-6 and IL-6R was still detectable 3 h after treatment. Staining for IL-6 and especially for IL-6R, however, was reduced and concentrated in the outer nucleated epidermal layers (Figure 1f and l). We carried out additional western blotting of epidermal samples and found nearly 2-fold increased levels for IL-6 at 1 h and for IL-6R at 3 h after barrier disruption (Figure 2a and b). To evaluate the possible involvement of IL-6, we examined barrier repair in IL-6-deficient mice after tape-stripping. As shown in Figure 3, IL-6-deficient mice exhibited a significant delay in barrier repair at 3–24 h after treatment. The delay in barrier repair peaked at 7 h (-25% reduced recovery; p<0.005, n=5). The delay in permeability barrier repair was reversed by topical application of 3 μg of recombinant IL-6 in a volume of 30 μL (barrier recovery at 1h: IL-6 def. 20%, IL-6 def.+IL-6 26%, wild-type 30%; 3h: IL-6 def. 29%, IL-6 def.+IL-6 59%, wild-type 41%; 5h: IL-6 def. 45%, IL-6 def.+IL-6 65%, wild-type 55%; 24h: IL-6 def. 72%, IL-6 def.+IL-6 85%, wild-type 77%). Although this data indicate, that IL-6 plays an important role in barrier repair, it does not clarify whether the IL-6 effect in barrier repair of the skin was mediated via the membrane bound or the soluble IL-6R (see below). Repeated tape-stripping of the skin removed cells from the stratum corneum and resulted in a superficial wound. As a marker of barrier disruption, we determined an increase in TEWL from 1.8±0.2 to 45.0±15.3 g per m2 per h. Immediately after treatment barrier repair commenced, a rapid decrease in TEWL leading to about 60% barrier recovery occurred in hairless mice within 7 h. This was followed by slower kinetics of barrier recovery within 24 h (Figure 4). To further investigate the possible involvement of IL-6 in barrier repair, the cytokine was applied topically immediately after barrier disruption at different concentrations. IL-6 at a concentration of 100 ng per μL (30 μL applied in total) significantly enhanced permeability barrier repair 1–7 h (20% at 7 h) after treatment compared with the vehicle-treated control (p<0.01, n=4). A slight but not significant enhancement in barrier repair also occurred after topical application at a low concentration of 10 ng per μL 3, 5, and 7 h after treatment. The high concentration of 1000 ng per μL led to a significant enhancement at 1h (p<0.01, n=4). These results show that topically applied IL-6 is able to enhance permeability barrier repair in a concentration-dependent manner. Topical application of Hyper-IL-6 also enhanced skin permeability barrier repair. At a concentration of 100 ng per μL, we found a significant enhancement 1–24 h after treatment (Figure 5). A low concentration of 10 ng per μL led to a slight (but not significant) enhancement in barrier repair after 1 h. At a high concentration of 1000 ng per μL, Hyper-IL-6 enhance the barrier at 1 h. We also induced barrier disruption with acetone and applied 100 ng per μL Hyper-IL-6 and got results similar to tape-stripping-induced barrier disruption (not shown). This demonstrates that Hyper-IL-6 also improved permeability barrier repair. Hyper-IL-6, however, did not show higher activity in barrier repair than IL-6. To further clarify whether the IL-6 effect on barrier repair depended on the membrane bound or the soluble IL-6R, the gp130-Fc fusion protein, specific inhibitor of trans-signalling via the sIL-6R, was topically applied to the skin of hairless mice after tape-stripping. In contrast to IL-6 or Hyper-IL-6, gp130 significantly delayed barrier repair at 1–24 h (Figure 6). Topical application of gp130-Fc in IL-6-deficient mice did not significantly delay the already reduced barrier repair in IL-6-deficient mice examined after tape-stripping (barrier recovery at 1h: IL-6 def. 20%, IL-6 def.+gp130 14%; 3h: IL-6 def. 29%, IL-6 def.+gp130 30%; 5h: IL-6 def. 45%, IL-6 def.+gp130 50%; 24h: IL-6 def. 72%, IL-6 def.+gp130 65%). These results shows that gp130 is able to retard the skin repair process in wild-type mice after superficial wounding, indicating an involvement of sIL-6R in the process of skin repair. To further substantiate the role of the IL-6-dependent signaling pathway in barrier repair, we examined whether STAT3 was activated (i.e., phosphorylated) during permeability barrier repair. After skin injury by tape-stripping we examined the phosphorylation of STAT3 in the epidermis using western immunoblots. STAT3 phosphorylation was low in untreated epidermis, but significantly increased 1h after tape-stripping (Figure 7). This shows that the transcription factor STAT3, which is induced by IL-6-mediated gp130 dimerization, is activated during skin barrier repair. In IL-6-deficient mice, STAT3 phosphorylation was markedly reduced, but not completely absent compared with wild-type mice (Figure 7). This may suggest that IL-6 is an important though not sole regulator of STAT3 phosphorylation in the skin. In this study, we found that the IL-6 and IL-6R proteins are preferentially localized in low amounts in the skin of hairless mice under basal conditions to keratinocytes in the basal, granular, and horny layers, but not in the spinous layers of the epidermis (Figure 1a and g). In the dermis we found little staining by IL-6 and the IL-6R antibodies on fibroblasts, and a lack of staining on other cells in the skin. Preferred localization of IL-6 to keratinocytes in the skin has been described previously in human skin (Paquet and Pierard, 1996Paquet P. Pierard G.E. Interleukin-6 and the skin.Int Arch Allery Immunol. 1996; 109: 308-317Crossref PubMed Scopus (103) Google Scholar). Within the epidermal keratinocytes, localization to the basal proliferating compartment, but not to the upper differentiated cell layers in human keratinocyte culture has also been reported (Yoshizaki et al., 1990Yoshizaki K. Nishimoto N. Matsumoto K. et al.Interleukin 6 and expression of its receptor on epidermal keratinocytes.Cytokine. 1990; 2: 381-387Crossref PubMed Scopus (56) Google Scholar;Castella-Rodellas et al., 1992Castella-Rodellas A. Castell J.V. Ramirez-Bosca A. Nicolas J.F. Valcuende-Cavero F. Thivolet J. Interleukin-6 in normal skin and psoriasis.Acta Derm Venerol. 1992; 72: 165-168PubMed Google Scholar). After experimental barrier disruption by tape-stripping, an increase for IL-6 and IL-6R in a time-dependent manner was noted. Fifteen min after treatment, staining of basal and stratum granulosum keratinocytes and some suprabasal keratinocytes in the spinous layers was visible. Staining intensity increased and reached the highest levels for IL-6 and IL-6R at 1–3 h after treatment. IL-6 and IL-6R were equally distributed in the entire nucleated epidermis. Thereafter, staining intensity was reduced. After 5 h the basal layer was devoid of the cytokine/receptor (Figure 1a–e and g–k). We also carried out western blotting and, as already shown by immunohistochemistry, we found increased levels for IL-6 1 h and increased levels for IL-6R 3 h after barrier disruption (Figure 2a and b). These findings are in accordance with published mRNA studies using the sensitive RNase protection assays.Wood et al., 1997Wood L.C. Stalder A.K. Liou A. Campbell I.L. Grunfeld C. Elias P.M. Feingold K.R. Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin.Exp Dermatol. 1997; 6: 98-104Crossref PubMed Scopus (67) Google Scholar reported an increase in IL-6 mRNA after barrier disruption. There are small differences, however, regarding the time curve. Wood et al found an increase in mRNA levels only 2.5 h after barrier disruption. We found that the time curve for barrier repair depends on the degree of barrier disruption. After severe barrier disruption the time curve for barrier regeneration is delayed because of the toxicity to the cells induced by vigorous treatments with acetone, SDS, or tape-stripping. In these studies we carefully disrupted the barrier. In accordance with our results Kawakami et al described increased IL-6 levels in the skin 30 min after thermal injury in mice (Kawakami et al., 1998Kawakami M. Terai C. Okada Y. Changes of the interleukin-6 levels in skin at different sites after thermal injury.J Trauma. 1998; 44: 1056-1063Crossref PubMed Scopus (12) Google Scholar). Elevated levels for IL-6 and IL-6R still occurred after occlusion by a Latex foil following barrier disruption. Compared with unoccluded skin staining was slightly reduced (1f and l). Similarly, Wood et al reported that occlusion did not significantly modify the increased expression of TNF and IL-1 after barrier disruption (Wood et al., 1997Wood L.C. Stalder A.K. Liou A. Campbell I.L. Grunfeld C. Elias P.M. Feingold K.R. Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin.Exp Dermatol. 1997; 6: 98-104Crossref PubMed Scopus (67) Google Scholar). Warner et al very recently described that hyper-hydration by application of an occlusive patch test chamber in human skin in vivo disrupts human stratum corneum ultrastructure already 4 h after treatment (Warner et al., 2003Warner R.R. Stone K.J. Boissy Y.L. Hydration disrupts human stratum corneum ultrastructure.J Invest Dermatol. 2003; 120: 275-284Crossref PubMed Scopus (231) Google Scholar). Also, it is common knowledge in clinical dermatology that application of occlusive foils in acute dermatitis does not reduce, but may rather enhance inflammation, because occlusion hinders heat emission. Therefore, it is reasonable that occlusion after barrier disruption may reduce but not totally prevent the increase in inflammation-related cytokines TNFα, IL-1, and IL-6. In IL-6-deficient mice we determined a significant delay in permeability barrier repair after 1–24 h (Figure 3). Previously, it has been published that TNFα and IL-1β stimulate the production of IL-6 fibroblasts (Xing et al., 1998Xing Z. Gauldie J. Cox G. Baumann H. Jordana M. Lei X.F. Achong M.K. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses.J Clin Invest. 1998; 101: 311-320Crossref PubMed Scopus (1112) Google Scholar). It is of interest to compare the effects of TNFα and IL-1β with the effects of IL-6 on barrier repair in the mice in vivo. We previously examined skin permeability barrier repair in TNF-R1 and in IL-1R-1-deficient mice. In TNF-R1-deficient mice we found a significant delay in the repair process 1, 3, and 5 h after tape-stripping (Jensen et al., 1999Jensen J.M. Schütze S. Förl M. Krönke M. Proksch E. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier.J Clin Invest. 1999; 104: 1761-1770Crossref PubMed Scopus (153) Google Scholar). In IL-1R1-deficient mice the repair was only slightly reduced and did not reach statistical significance (Jensen et al., 1998Jensen J.M. Kupper T.S. Proksch E. IL-1/IL-1 receptor overexpression and knockout constructs in permeability barrier repair of transgenic mice.J Invest Dermatol. 1998; 110 (Abstract): 499aGoogle Scholar). As shown in this study, a deficiency in IL-6 caused a more pronounced reduction in barrier repair than a deficiency in either TNFα or IL-1β. This is in line with other studies, which suggest that IL-6 appears to act as a downstream effector of TNFα and IL-1β in many experimental systems (Kallen et al., 1999aKallen K.J. Galle P.R. Rose-John S. IL-6 dependent pathology: Where do we stand and what are the options.Exp Opin Invest Drugs. 1999; 8: 1327-1349Crossref PubMed Scopus (12) Google Scholar). Topical application of IL-6 to barrier-disrupted skin enhanced the skin barrier repair in a concentration-dependent manner. A significant effect was found with a concentration of 100 ng per μL, where enhanced repair was found 1, 3, 5, and 7 h after treatment. At a low concentration of 10 ng per μL there was a tendency to increased barrier repair only. The high concentration of 1000 ng per μL IL-6-enhanced barrier repair only at 1 h after treatment (Figure 4). A bell-shaped curve of IL-6 concentration-dependency with decreased activity at higher doses has been described in the past (Aarden, 1989Aarden L.A. Hybridoma growth factor.Ann NY Acad Sci. 1989; 557 (discussion 198–199): 192-198Crossref PubMed Scopus (28) Google Scholar;van Dam et al., 1993van Dam M. Müllberg J. Schooltink H. et al.Structure-function analysis of interleukin-6 utilizing human/murine chimeric molecules. Involvement of two separate domains in receptor binding.J Biol Chem. 1993; 268: 15285-15290Abstract Full Text PDF PubMed Google Scholar) and has been explained by a transition from an active tetrameric to an inactive hexameric receptor complex (Groetzinger et al., 1999Groetzinger J. Kernebeck T. Kallen K.J. Rose-John S. IL-6 type cytokine receptor complexes: Hexamer, tetramer or both?.Biol Chem. 1999; 380: 803-813PubMed Google Scholar). This mechanism could be the basis of the dose effects observed here. Again, we were interested in comparing the effects of IL-6 with TNFα and IL-1β in the same system in vivo. We previously performed topical application of TNFα and IL-1β to mouse skin after barrier disruption. TNFα as well as IL-1β led to an increase in barrier repair 1, 3, and 5 h after tape-stripping. Topical application of IL-6, however, was more effective than either TNFα or IL-1β. Topical application of IL-6 was also effective in IL-6-deficient mice. The delay in permeability barrier repair was reversed by topical application of the cytokine down to levels similar to vehicle treated wild-type mice. Like IL-6, Hyper-IL-6-enhanced permeability barrier repair. As with IL-6 the effect was highly significant with a concentration of 100 ng per μL, whereas, with a low (10 ng per μL) concentration there was only a tendency (n.s.) for an increased barrier repair. At a high concentration (1000 ng per μL) Hyper-IL-6-enhanced permeability barrier repair only 1 h after treatm
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