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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

2000; Elsevier BV; Volume: 115; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1747.2000.00029.x

1523-1747

Christian Wetzler, Heiko Kämpfer, Birgit Stallmeyer, Josef Pfeilschifter, Stefan L. Frank,

NF-κB Signaling Pathways

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. 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. growth-related oncogene macrophage chemoattractant protein-1 melanoma growth stimulatory activity macrophage inflammatory protein-2 polymorphonuclear neutrophils Wound repair represents a highly dynamic process that is characterized by fibroplasia, angiogenesis, and reepithelialization. These processes involve the proliferation of fibroblasts and endothelial and epithelial cells (Clark and Clark, 1996Clark R.A.F. Wound repair: overview and general considerations.in: Clark R.A.F. The Molecular&Cellular Biology of Wound Repair. New York, Plenum Press1996: 3-50Google Scholar). Injury of the skin is often associated with the extravasation of blood from injured blood vessels. The resultant platelet aggregation and blood coagulation then initiate the healing process, and the inflammatory stage of repair subsequently starts with the migration of immune cells into the wound (Martin, 1997Martin P. Wound healing – aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3730) Google Scholar). Polymorphonuclear neutrophils (PMN) represent the first immune cells that arrive at the wound site. These cells have long been considered to be confined to host defense against a variety of infectious agents, but recent studies have demonstrated that PMN also contribute to the production of inflammatory cytokines (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: 548-556https://doi.org/10.1006/cyto.1996.0074Crossref PubMed Scopus (387) Google Scholar). The functional role of the wound macrophage, which enters the injured area temporally following the PMN, is well documented for dermal repair, as a depletion of wound macrophages resulted in delayed healing (Leibovich and Ross, 1975Leibovich S.J. Ross R. The role of the macrophage in wound repair: a study with hydrocortisone and anti-macrophage serum.Am J Pathol. 1975; 78: 71-91PubMed Google Scholar). Wound macrophages have been shown to be crucial for tissue regeneration, as they promote repair by secretion of a wide range of mediators (DiPietro, 1995DiPietro L.A. Wound healing: the role of the macrophage and other immune cells.Shock. 1995; 4: 233-240Crossref PubMed Scopus (248) Google Scholar). Thus, the regulated attraction of leukocytes is essential to ensure a well-ordered inflammatory process that initiates tissue repair. Important to this process are chemokines that represent chemotactic cytokines. Chemokines share important structural features and the ability to attract leukocytes (Baggiolini, 1998Baggiolini M. Chemokines and leukocyte traffic.Nature. 1998; 392: 565-568Crossref PubMed Scopus (2396) Google Scholar;Luster, 1998Luster A.D. Chemokines – chemotactic cytokines that mediate inflammation.New Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3252) Google Scholar). Two families of chemokines, which contain four cysteines in their amino acid sequence, have been characterized extensively: the α- (or CXC-) chemokines (the first two cysteine residues are separated by one amino acid) and the β- (or CC-) chemokines (the first two cysteine residues are adjacent to each other). α- and β-chemokines exhibit functional diversity, as α-chemokines are chemotactic for neutrophils and lymphocytes, whereas β-chemokines act on monocytes, activated T cells, eosinophils, and basophils (Baggiolini, 1998Baggiolini M. Chemokines and leukocyte traffic.Nature. 1998; 392: 565-568Crossref PubMed Scopus (2396) Google Scholar;Luster, 1998Luster A.D. Chemokines – chemotactic cytokines that mediate inflammation.New Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3252) Google Scholar). The observed cell specificity is mediated by CXC- and CC-chemokine receptors that only recognize the chemokines of the corresponding subfamily (Murphy, 1996Murphy P.M. Chemokine receptors: structure, function and role in microbial pathogenesis.Cytokine Growth Fact Res. 1996; 7: 47-64Abstract Full Text PDF PubMed Scopus (282) Google Scholar). Interestingly, a murine homolog to human interleukin-8 (IL-8) has not yet been identified, and thus all known murine α-chemokines bind to a single receptor equivalent to human CXCR2 (Rollins, 1997Rollins B.J. Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar). Thus, the murine growth-related oncogene (Gro-α)/melanoma growth stimulatory activity (MGSA) homolog macrophage inflammatory protein-2 (MIP-2) represents a functional homolog to human IL-8. According to chemokine function, expression of different members of the α- and β-chemokine family has been demonstrated for normal human and murine wound healing (DiPietro et al., 1995DiPietro L.A. Polverini P.J. Rahbe S.M. Kovacs E.J. Modulation of JE/MCP-1 expression in dermal wound repair.Am J Pathol. 1995; 146: 868-875PubMed Google Scholar,DiPietro et al., 1998DiPietro L.A. Burdick M. Low Q.E. Kunkel S.L. Strieter R.M. MIP-1α as a critical macrophage chemoattractant in murine wound repair.J Clin Invest. 1998; 101: 1693-1698Crossref PubMed Scopus (240) Google Scholar;Gibran et al., 1997Gibran N.S. Ferguson M. Heimbach D.M. Isik F.F. Monocyte chemoattractant protein-1 mRNA expression in the human burn wound.J Surg Res. 1997; 70: 1-6https://doi.org/10.1006/jsre.1997.5017Abstract Full Text PDF PubMed Scopus (56) Google Scholar;Engelhardt et al., 1998Engelhardt E. Toksoy A. Goebeler M. Debus S. Bröcker E.B. Gillitzer R. Chemokines IL-8, GROα MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing.Am J Pathol. 1998; 153: 1849-1860Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Clinical evidence shows that diabetic patients as a group develop wound healing disorders that often lead to a severely impaired repair (Goodson and Hunt, 1979Goodson W.H. Hunt T.K. Wound healing and the diabetic patient.Surg Gyn Obstet. 1979; 149: 600-608PubMed Google Scholar;Bennett and Schultz, 1993Bennett N.T. Schultz G.S. Growth factors and wound healing: Part II. Role in normal and chronic wound healing.Am J Surg. 1993; 166: 74-81Abstract Full Text PDF PubMed Scopus (460) Google Scholar;Knighton and Fiegel, 1993Knighton D.R. Fiegel V. Growth factors and repair of diabetic wounds.in: Levin M.E. O'neal L.W. Bowker J.H. The Diabetic Foot. 5th edn. St. Louis, Mosby Year Book1993: 247-257Google Scholar). In contrast to this well-known clinical situation, however, the mechanisms underlying an impaired healing process are only poorly understood. We have chosen the homozygous genetically diabetic mouse (db/db) as a model system to investigate diabetes-associated wound healing disorders. Recently, the mutation of the diabetes gene (db) localized on chromosome 4 (Coleman, 1982Coleman D.L. Diabetes–obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar) turned out to inactivate the signal transduction domain of the leptin receptor ObR (Chen et al., 1996Chen H. Charlat O. Tartaglia L.A. et al.Evidence that the diabetes gene encodes the leptin receptor. identification of a mutation in the leptin receptor gene in db/db mice.Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar;Lee et al., 1996Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Abnormal splicing of the leptin receptor in diabetic mice.Nature. 1996; 379: 632-635Crossref PubMed Scopus (2109) Google Scholar). The homozygous db/db mice develop obesity, insulin resistance, and a severe diabetes with marked hyperglycemia resembling adult-onset diabetes mellitus (Coleman, 1982Coleman D.L. Diabetes–obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar). Furthermore, these animals are characterized by a markedly delayed wound healing. As a well-ordered temporal expression pattern, which was clearly accompanied by the presence of infiltrating immune cells, has been observed for several chemokines (DiPietro et al., 1995DiPietro L.A. Polverini P.J. Rahbe S.M. Kovacs E.J. Modulation of JE/MCP-1 expression in dermal wound repair.Am J Pathol. 1995; 146: 868-875PubMed Google Scholar,DiPietro et al., 1998DiPietro L.A. Burdick M. Low Q.E. Kunkel S.L. Strieter R.M. MIP-1α as a critical macrophage chemoattractant in murine wound repair.J Clin Invest. 1998; 101: 1693-1698Crossref PubMed Scopus (240) Google Scholar;Engelhardt et al., 1998Engelhardt E. Toksoy A. Goebeler M. Debus S. Bröcker E.B. Gillitzer R. Chemokines IL-8, GROα MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing.Am J Pathol. 1998; 153: 1849-1860Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar), we investigated the expression kinetics of a representative neutrophil chemoattractant MIP-2, and the prototypic monocyte chemoattractant macrophage chemoattractant protein-1 (MCP-1), respectively, in diabetic wound healing. Whereas normal healing was characterized by a temporally confined expression of both chemokines during the first days after injury, we observed a sustained expression of both chemokines until the late phase of repair. The sustained availability of MIP-2 and MCP-1 in db/db mice clearly resulted in elevated numbers of PMN and macrophages in late wound tissues. Our data indicate that chemokine expression and subsequent PMN and macrophage migration is dysregulated during wound healing in these animals. The data provide a possible explanation for the observed inflamed morphology during healing, and furthermore the aberrant expression of chemokines might underlie the delayed wound repair in diabetic animals. Female C57BLKS/J-m+/+Leprdb mice obtained from Jackson Laboratories (Bar Harbor, ME) were chosen because they exhibit characteristics similar to those of human adult-onset diabetes as a result of single autosomal recessive mutation on chromosome 4. This mutation inhibits signal transduction of the leptin receptor ObR (Coleman, 1982Coleman D.L. Diabetes–obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar;Chen et al., 1996Chen H. Charlat O. Tartaglia L.A. et al.Evidence that the diabetes gene encodes the leptin receptor. identification of a mutation in the leptin receptor gene in db/db mice.Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar;Lee et al., 1996Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Abnormal splicing of the leptin receptor in diabetic mice.Nature. 1996; 379: 632-635Crossref PubMed Scopus (2109) Google Scholar). Only the homozygous animals develop diabetes (Coleman, 1982Coleman D.L. Diabetes–obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar). Female C57BLKS/J-m and BALB/C mice were used as controls representing a normal, nondiabetic repair process. All animals were 10 wk of age at the start of the experiment. To examine chemokine expression during the diabetic wound healing process, six full-thickness wounds were created on the backs of female C57BLKS/J-m+/+Leprdb, C57BLKS/J-m, or BALB/C mice. Animals were anesthetized with a single intraperitoneal injection of Ketamin (80 mg per kg body weight)/Xylazin (10 mg per kg body weight). The hair on the back of the 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 the 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 7–8 mm in diameter that 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 nonwounded mice. In every experiment, the wounds from four animals (n = 16 wounds) and the nonwounded back skin from four animals 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. C57BLKS/J-m+/+Leprdb mice were wounded as described above. Twelve days after wounding, wounds of these mice were treated topically with a combination of neutralizing MCP-1- and MIP-2-specific goat IgG (R&D Systems, Wiesbaden, Germany) [2 μg anti-MCP-1 and 2 μg anti-MIP-2 diluted in 30 μl phosphate-buffered saline (PBS)] for three times every 6 h (6 pm, 12 pm, and 6 am the next day). Six hours after the last application (12 am the next day) mice were sacrificed and complete wounds were isolated as described above. C57BLKS/J-m+/+Leprdb mice treated with vehicle only (PBS) were used as a control. In every experiment, the wounds from three animals (n = 12 wounds) were combined, frozen immediately in liquid nitrogen, and stored at -80°C until used for RNA isolation. Mice were wounded as described above. Animals were sacrificed at day 5 and day 13 after injury. Complete wounds were isolated from the middle of the back, bisected, and frozen in tissue-freezing medium. Six micrometer frozen serial sections were fixed with acetone and treated for 10 min at room temperature with 1% H2O2 in PBS to inactivate endogenous peroxidases. They were subsequently incubated for 60 min at room temperature with monoclonal antisera against murine Gr-1 (Ly-6G) (PharMingen, Hamburg, Germany) or murine F4/80 antigen (Serotec, Eching, Germany), and polyclonal antisera against murine MCP-1, MIP-2 (both from R&D Systems), or CXCR2 (Santa Cruz, Heidelberg, Germany) [1:2000 (for Gr-1), 1:50 (for F4/80), 1:10 (for MCP-1 and MIP-2), 1:50 (for CXCR2) diluted in PBS, 0.1% goat serum albumin]. The slides were subsequently stained with the avidin-biotin-peroxidase complex system from Santa Cruz 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 were homogenized in lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM ethylendiamine tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 15 μg per ml leupeptin). The tissue extract was cleared by centrifugation. Fifty micrograms of total protein from these lysates were separated using sodium dodecyl sulfate gel electrophoresis. After transfer to a PVDF membrane, Gr-1 or F4/80 protein was detected using monoclonal antibodies as described under Immunohistochemistry. A secondary antibody coupled to horseradish peroxidase and the ECL detection system were used to visualize Gr-1 or F4/80 protein. Phenylmethylsulfonyl fluoride, aprotinin, and leupeptin were from Sigma, and the ECL detection system was obtained from Amersham (Freiburg, Germany). RNA isolation was performed as described byChomczynski 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 (63149) Google Scholar. Twenty micrograms of total RNA from wounded or nonwounded skin were used for RNase protection assays. RNase protection assays were carried out as described byWerner 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 (533) Google Scholar. Protected fragments were separated on 5% acrylamide per 8 M urea gels and analyzed using a PhosphoImager (Fuji, Straubenhardt, Germany). The murine cDNA probes for IL-1β, tumor necrosis factor α (TNF-α), MIP-2, MCP-1, lipocalin, lysozyme M, CXCR2 (IL-8 receptor B), or CCR2 (MCP-1 receptor) were cloned by reverse transcriptase polymerase chain reaction. The cloned cDNA fragments correspond to nucleotides 481–739 (for IL-1β), nucleotides 541–814 (for TNF-α), nucleotides 181–451 (for MIP-2), nucleotides 580–1924 (for MCP-1), nucleotides 816–1481 (for lipocalin), nucleotides 425–446 (exon 1) and 150–170 (exon 2) (for lysozyme M), nucleotides 961–1210 (for CXCR2), or nucleotides 976–1241 (for CCR2) of the published sequences (Pennica et al., 1985Pennica D. Hayflick J.S. Bringman T.S. Palladino M.A. Goeddel D.V. Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor.Proc Natl Acad Sci USA. 1985; 82: 6060-6064Crossref PubMed Scopus (336) Google Scholar;Gray et al., 1986Gray P.W. Glaister D. Chen E. Goeddel D.V. Pennica D. Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1-beta.J Immunol. 1986; 137: 3644-3648PubMed Google Scholar;Cross et al., 1988Cross M. Mangelsdorf I. Wedel A. Renkawitz R. Mouse lysozyme M gene: isolation, characterization, and expression studies.Proc Natl Acad Sci USA. 1988; 85: 6232-6236Crossref PubMed Scopus (194) Google Scholar;Rollins et al., 1988Rollins B.J. Morrison E.D. Stiles C.D. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties.Proc Natl Acad Sci USA. 1988; 85: 3738-3742Crossref PubMed Scopus (372) Google Scholar;Tekamp-Olson et al., 1990Tekamp-Olson P. Gallegos C. Bauer D. et al.Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues.J Exp Med. 1990; 172: 911-919Crossref PubMed Scopus (248) Google Scholar;Harada et al., 1994Harada A. Kuno K. Nomura H. Mukaida N. Murakami S. Matsushima K. Cloning of a cDNA encoding a mouse homolog of the interleukin-8 receptor.Gene. 1994; 142: 297-300Crossref PubMed Scopus (32) Google Scholar;Garay-Rojas et al., 1996Garay-Rojas E. Harper M. Hraba-Renevey S. Kress M. An apparent autocrine mechanism amplifies the dexamethasone- and retinoic acid-induced expression of mouse lipocalin encoding gene 24p3.Gene. 1996; 170: 173-180https://doi.org/10.1016/0378-1119(95)00896-9Crossref PubMed Scopus (73) Google Scholar;Heesen et al., 1996Heesen M. Tanabe S. Berman M.A. et al.Mouse astrocytes respond to the chemokine MCP-1 and KC, but reverse transcriptase-polymerase chain reaction does not detect mRNA for the KC or new MCP-1 receptor.J Neurosci Res. 1996; 45: 382-391https://doi.org/10.1002/(sici)1097-4547(19960815)45:4<382::aid-jnr7>3.0.co;2-5Crossref PubMed Scopus (0) Google Scholar). Total protein (50 μg diluted in lysis buffer to a final volume of 50 μl) from nonwounded skin lysates and wound lysates was subsequently analyzed for the presence of immunoreactive MCP-1 or MIP-2 by ELISA using the Quantikine murine MCP-1 or MIP-2 kit (R&D Systems), respectively, as described by the manufacturer. To investigate possible differences for the inflammatory phase of repair in normal compared with diabetic healing, we determined mRNA expression of the two prototypic inflammatory cytokines IL-1β and TNF-α. We isolated RNA from excisional wounds of control (C57BLKS, BALB/C) and db/db mice from different intervals after wounding and performed RNase protection assays. For each experimental time point, 16 wounds (n = 16) from four different animals were analyzed. Normal back skin from nonwounded mice was used as a control. As recently published (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: 548-556https://doi.org/10.1006/cyto.1996.0074Crossref PubMed Scopus (387) Google Scholar), we observed a large induction of IL-1β and TNF-α mRNA expression during the inflammatory phase of repair in control animals (Figure 1a, b, upper panels). The large induction of IL-1β and TNF-α mRNA expression could also be observed in diabetic mice (Figure 1a, b, lower panels). From day 5 postwounding, normal healing was characterized by a decline in mRNA levels for both cytokines, and no mRNA for IL-1β or TNF-α could be detected after 13 d of repair. Strikingly, expression levels for IL-1β and TNF-α in wounds isolated from diabetic mice did not significantly decrease after injury-mediated induction. As shown in Figure 1(a, b, lower panels), we detected strongly elevated and sustained levels of IL-1β and TNF-α mRNA also during the late phase of repair in db/db mice, indicating a prolonged inflammatory response at the wound site. As PMN and, subsequently, also wound macrophages represent potent producers of inflammatory cytokines (DiPietro, 1995DiPietro L.A. Wound healing: the role of the macrophage and other immune cells.Shock. 1995; 4: 233-240Crossref PubMed Scopus (248) Google Scholar;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: 548-556https://doi.org/10.1006/cyto.1996.0074Crossref PubMed Scopus (387) Google Scholar), we assessed the availability of leukocyte chemoattractants within the injured area. At this point of research, we hypothesized that the sustained production of inflammatory cytokines might be due to elevated numbers of immune cells during late repair, which might be directly associated with the presence of leukocyte-attracting chemokines. For this reason, we determined expression levels of the neutrophil chemoattractant MIP-2 and the prototypic monocyte chemoattractant MCP-1, as representatives for both the α- and β-chemokine family, respectively, during normal and diabetic healing. As shown in Figure 2 and 3, we observed a large induction of MIP-2 mRNA Figure 2a and protein Figure 3a expression early after injury in control (C57BLKS, BALB/C) and db/db mice. Whereas MIP-2 mRNA and protein expression diminished from day 7 postwounding in wounds from control animals, however, we found strongly increased levels of MIP-2 mRNA and protein until day 13 after injury in db/db mice (Figure 2a, Figure 3a). Accordingly, the monocyte-attracting MCP-1 was also strongly induced at the mRNA and protein level in control and diabetic mice (Figure 2b, Figure 3b). As tissue repair proceeded, MCP-1 mRNA and protein could not be detected in control mice from day 7 postwounding. This is in contrast with the diabetic situation, where MCP-1 expression levels remained markedly increased even 13 d after injury (Figure 2b, Figure 3b).Figure 3Elevated levels of MIP-2 and MCP-1 protein during impaired repair. Total protein (50 μg) from lysates of nonwounded and wounded back skin (day 1, 3, 5, 7, and 13 after injury, as indicated) isolated from control mice (BALB/C, C57BLKS) and db/db mice as indicated were analyzed by ELISA for the presence of MIP-2 (A) or MCP-1 (B) specific proteins. Eight wounds (n = 8) from the backs of four animals were excised for each experimental time point and used for protein isolation. Control skin refers to nonwounded back skin of control (BALB/C, C57BLKS) and db/db mice.View Large Image Figure ViewerDownload (PPT) As we observed high levels of MIP-2 and MCP-1 mRNA and protein expression upon injury, we determined cellular sources of both proteins at the wound site using immunohistochemistry. As shown in Figure 4, MIP-2 was strongly expressed within the outer root sheath of hair follicles located near the wound site Figure 4a, b. Keratinocytes of the developing hyperproliferative epithelia located at the wound margins strongly expressed MCP-1 Figure 4e, f. Interestingly, those keratinocytes that are located directly adjacent to the developing granulation tissue often display the highest MCP-1-specific immunopositive signals Figure 4e. Additionally, we could not detect MIP-2 or MCP-1 immunopositive cells within the granulation tissue. As a next step, we determined whether the observed upregulation of MIP-2 and MCP-1 was accompanied by elevated numbers of PMN and macrophages at the wound site. For this purpose, we investigated the expression kinetics of PMN-specific or macrophage-specific marker proteins, respectively, for the control and diabetic wounds Figure 5a, b. Furthermore, we performed immunohistochemistry from serial sections isolated from control (BALB/C) and db/db mice at days 5 and 13 after wounding to confirm the immunoblot data on a histologic basis (Figure 4c, d,g, h, and Figure 6). Not unexpectedly, we could detect strongly elevated levels for both the 25 kDa PMN-specific marker protein Gr-1 Figure 5a and, additionally, the 160 kDa macrophage-specific F4/80 antigen Figure 5b in wounds from db/db mice at day 7 and even at day 13 postwounding. These data clearly indicate the presence of elevated numbers of PMN and wound macrophages even in those phases of impaired repair that are characterized by decreasing numbers of immune cells during the normal healing situation. The additional band of approximately 90 kDa that occurred in F4/80 immunoblot analysis represents a nonspecific signal recognized by this antibody. This observation was further strengthened by immunohistochemistry data Figure 6, which revealed large numbers of PMN at day 13 in diabetic wounds Figure 6b, whereas no PMN could be detected in reepithelialized wounds from control animals Figure 6a. In line with the immunoblot data against the macrophage-specific F4/80 antigen Figure 5b we observed markedly elevated numbers of wound macrophages in 13 d diabetic wounds, although lower numbers of resting wound macrophages could also be observed in 13 d wounds from control animals Figure 6c, d. It is noteworthy that PMN and macrophages appeared to share only the temporally prolonged presence but not their localization within the wound site. Remarkably, PMN and macrophages were confined to clearly restricted areas in the wound. Whereas PMN localized directly beneath the weakly reformed epithelium, macrophages formed a semicircle around the cluster of PMN that extended deep into the underlying granulation tissue Figure 6e, f. The observed differential distribution of PMN and macrophages within the wound tissue of diabetic mice could not be detected in wounds from healthy animals, as PMN and macrophages indeed colocalized at the wound site during normal repair (Figure 4g, h demonstrate PMN- and macrophage-specific immunostaining in the granulation tissue of directly neighbored 6 μm serial sections).Figure 6Localization of PMN and macrophages during late repair in normal wounds and wounds from diabetic mice. (A) and (C, BALB/C), frozen serial sections from 13 d mouse wounds isolated from BALB