MMP-13 Plays a Role in Keratinocyte Migration,Angiogenesis, and Contraction in Mouse Skin Wound Healing

Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-β1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.Wound healing is a complex process that includes an acute inflammatory reaction, regeneration of parenchyma cells, cell migration and proliferation, angiogenesis, extracellular matrix (ECM) synthesis, contraction, and tissue remodeling.^1,2,3 Cutaneous wound healing by second intention is characterized by the following three continuous and overlapping processes: an inflammatory phase, a proliferative phase, and a contraction and remodeling phase.² In the inflammatory phase, tissue injury causes the loss of cells and tissue, disruption of blood vessels, extravasation of blood constituents and infiltration of inflammatory cells, composed mainly of neutrophils and macrophages, and provides a provisional ECM for keratinocyte migration. The major events during the proliferative phase are re-epithelialization and angiogenesis, both of which require cell proliferation and migration of keratinocytes and endothelial cells, respectively. In the contraction and remodeling phase, myofibroblasts differentiated from fibroblasts play a key role in wound contraction and controlled synthesis and degradation of ECM proteins, especially collagens, leading to increased wound strength. All of these events occurring during wound healing require the collaborative efforts of many different tissues and cell types.Accumulated lines of evidence have demonstrated that members of the matrix metalloproteinase (MMP) gene family are essential to the degradation of ECM macromolecules and non-ECM molecules such as growth factors and cytokines under various pathophysiological conditions.^4,5 Enhanced expression of MMPs 1, 2, 3, 8, 9, 10, 13, 14, 19, and 26 in wound tissues has been reported in experimental animals and humans.^{6,7,8,9,10,11,12,13} It is clear that MMP activity is required in wound closure by keratinocyte re-epithelialization and migration and angiogenesis, since broad-spectrum MMP inhibitors inhibit the processes.^{14,15,16,17,18} However, since many MMPs are expressed by re-epithelializing keratinocytes, inflammatory cells, fibroblasts, and endothelial cells, it is uncertain which MMP species plays a central role in the process of wound healing and how these MMPs function in wounded tissues.One of the most powerful methods to directly address these questions is to analyze wound healing in MMP knockout (KO) mice. Indeed, wound healing of the skin has been studied in mice deficient for the MMP-3, MMP-8, MMP-9, MMP-13, or MMP-14 genes.^{13,19,20,21,22} However, all of these KO mice, except for the MMP-8 KO mice, had negative results. In the MMP-8 KO mice, wound closure and re-epithelialization were inhibited mainly due to impaired infiltration of neutrophils,²⁰ which are responsible for MMP-8 production. The study suggested that the effect of MMP-8 on the wound healing is secondary to persistent inflammation at the later time point and alterations in transforming growth factor-β (TGF-β) signaling.²⁰ Keratinocytes at the leading edge of the cutaneous wound express MMP-9 and MMP-13.^13,21 Induction of MMP-9 at the wound site in vitro²³ and in vivo²⁴ promotes migration of keratinocytes probably because of its effect on detachment of basal keratinocytes from the basal membrane. Keratinocyte migration over the wound bed is known to be dependent on the attachment of keratinocytes to fibrillar type I collagen through integrins α1β1 and α2β1 and subsequent degradation of the collagen by collagenolytic MMPs.^25,26 These studies suggest that both MMP-9 and MMP-13 are involved in re-epithelialization in wound healing in mice, a species that lacks the gene for MMP-1.²⁷ However, previous studies on wound healing in MMP-9 KO and MMP-13 KO mice failed to demonstrate significant difference or showed even acceleration of wound closure and re-epithelialization, as compared with that in wild-type mice.^13,21 These studies did not provide the reasonable explanations for the unexpected findings except for redundancy among MMPs.¹³ MMP-9 and MMP-13 synergize with each other during endochondral ossification at the growth plates.²⁸ Thus, MMP-9/13 double KO mice could identify additive effects of MMP-9 and MMP-13 on wound healing, although no such studies have been reported.In the present study, we developed the MMP-9/13 double KO mice by crossing the MMP-9 KO mice with the MMP-13 KO mice, and evaluated the influence of targeted deletion of the MMP-9 and/or MMP-13 genes on healing by secondary intention by generating large skin wounds in MMP-9 KO, MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Our study provides the first evidence that MMP-13 plays a key role in keratinocyte migration, angiogenesis, and contraction in wound healing, and that MMP-9 is implicated in keratinocyte migration.     

Alternative Proteolytic Processing of Hepatocyte Growth Factor during Wound Repair

Wound healing is a crucial regenerative process in all organisms. We examined expression, integrity, and function of the proteins in the hepatocyte growth factor (HGF)/c-Met signaling pathway in normally healing and non-healing human skin wounds. Whereas in normally healing wounds phosphorylation of c-Met was most prominent in keratinocytes and dermal cells, in non-healing wounds phosphorylation of c-Met was barely detectable, suggesting reduced c-Met activation. In wound exudates obtained from non-healing, but not from healing wounds, HGF protein was a target of substantial proteolytic processing that was different from the classical activation by known serine proteases. Western blot analysis and protease inhibitor studies revealed that HGF is a target of neutrophil elastase and plasma kallikrein during skin repair. Proteolytic processing of HGF by each of these proteases significantly attenuated keratinocyte proliferation, wound closure capacity in vitro, and c-Met signal transduction. Our findings reveal a novel pathway of HGF processing during skin repair. Conditions in which proteases are imbalanced and tend toward increased proteolytic activity, as in chronic non-healing wounds, might therefore compromise HGF activity due to the inactivation of the HGF protein and/or the generation of HGF fragments that ultimately mediate a dominant negative effect and limit c-Met activation.Tissue damage in skin induces a complex network of signaling systems such as growth factors, their receptors, extracellular matrix molecules, and different classes of proteases.¹ The stringently regulated interaction of these mediators directs the restoration of the epidermis by epithelialization and of dermal structures by granulation tissue formation and matrix deposition. Many experimental studies have identified the essential functions of specific growth factors, their receptors and downstream signaling components in cutaneous repair^2,3 and based on these studies much insight has been gained into human wound physiology. In contrast, mechanisms leading to impaired of healing are poorly understood and currently no efficient therapy is available for chronic wounds, which are often associated with diabetes or venous insufficiency.⁴ A better understanding of the pathogenic mechanisms leading to impaired healing is fundamental for the development of effective therapies for wound healing disorders.Hepatocyte growth factor (HGF), also named scatter factor, is a pleiotropic growth factor that plays an essential role in cell growth, motility, and morphogenesis in different organ systems.⁵ Genetic deletion in mice demonstrated that HGF is critical for embryonic development.^6,7 Comprehensive experimental evidence suggests that HGF is also a pivotal factor in postnatal processes such as cancer development, tissue repair and regeneration.⁵ HGF is a multidomain protein, synthesized and secreted as a biologically inert 90-kd monomeric precursor, that is converted into its bioactive form in the extracellular environment through a single cleavage at the Arg494-Val495 peptide bond by tightly regulated serine proteases (henceforth named classical HGF activation pathway). This proteolytic cleavage of HGF induces conformational changes to allow productive binding, dimerization, and activation of its receptor, c-Met. The mature HGF molecule is a heterodimer consisting of disulfide-linked α- and β-chains.^8,9 The α-chain (60 kd) is composed of an N-terminal hairpin loop (N), homologous to the plasminogen activation peptide and four kringle domains (K1–K4), triple-looped cystein-rich motives involved in protein-protein interactions.^8,9 The β-chain (36 kd) has strong homology to the protease domain of serine proteases, but it is devoid of any enzymatic activity. Several pro-HGF-converting enzymes and their inhibitors have been identified, including HGF activator,¹⁰ the blood coagulation factors XI¹¹ and XII,¹² the membrane-bound matriptase,¹³ as well as the urokinase-type plasminogen activator.¹⁴ A common feature of all known pro-HGF activators is the requirement for proteolytic conversion into an active enzyme, a process that is mediated by another set of proteases. Thus, the HGF/c-Met pathway is highly regulated by proteases and involves a complex network of activating enzyme and inhibitors. A more detailed characterization of protease activities and their impact on HGF function during tissue remodeling will contribute to a better understanding of the role of the HGF/c-Met axis in wound healing.HGF mediates its biological functions on binding to its receptor c-Met, a tyrosine kinase encoded by the c-Met proto-oncogene.⁵ Due to the complex modular architecture of the HGF protein and different experimental designs, functional studies on ligand-receptor interactions have been complicated and results are contradictory. Progress in structure/function analysis of HGF has been achieved through protein engineering experiments and the determination of crystal structures for different domains.⁸ Earlier studies indicated that HGF mutants containing deletions of the N-, K1-kringle- or β-chain domains are biologically inactive, whereas mutants lacking the K2-, K3- or K4-kringle domains retain varying degrees of biological activity.^15,16 These results have been partially supported by recent findings demonstrating that the K1-, K2- and K4-kringle domains of HGF are sufficient for c-Met activation, whereas the N- and β-chain domains are not essential, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF.^8,9 A NK4 fragment composed of the first 447 residues of the α-chain has been shown to be a potent antagonist of c-Met-mediated HGF effects.¹⁷ Yet, the exact structural basis for c-Met activation remains incompletely understood.HGF is widely expressed in different tissues, predominantly by mesenchymal cells, and acts as a paracrine effector on several cells of epithelial origin,¹⁸ on endothelial cells¹⁹ and on cells of the macrophage/monocyte lineage.²⁰ Due to these properties, HGF has been implicated as a crucial molecule coordinating and facilitating cellular events in tissue repair, including re-epithelialization, angiogenesis, and granulation tissue formation. Circulating HGF levels rise markedly in several types of tissue injury, such as liver damage²¹ or arterial thrombosis.²² Neutralization of HGF in mice leads to retarded cutaneous healing associated with decreased neovascularization and granulation tissue formation.²³ Consistently, vascularization and granulation tissue formation was increased in transgenic mice overexpressing HGF.²⁴ A recent study demonstrated that keratinocytes deficient for c-Met were unable to contribute to the epithelialization of skin wounds and in conditional c-Met deficient mice wound closure was attenuated.²⁵ Furthermore, diabetes impaired healing in mice was accelerated by topical application of recombinant human HGF (rhHGF), pointing to a unique potential of this molecule for the therapy of chronic non-healing wounds.^23,26 To date, the impact of the HGF/c-Met pathway on human skin repair and its failure has not been comprehensively analyzed. Furthermore, detailed knowledge on the proteolytic processing of HGF at the wound site in skin repair is lacking.Several classes of proteinases, as well as their inhibitors, have been implicated in the repair response in skin and genetic evidence obtained in mice indicates their crucial function.²⁷ Cardinal functions of proteases in repair include regulation of cell motility, matrix remodeling, host defense, and modulation of cytokine activation. Dysregulation of wound proteases and their inhibitors is considered to be one of the major pathogenic mechanism underlying chronic non-healing wounds in man.^4,28 The chronic non-healing wound environment of venous stasis ulcers, the most common cause of non-healing wounds at the lower leg in man, is the paradigm pathological condition were disintegrating inflammatory cell subsets release active peptidases and overwhelm local antipeptidase defenses. Substantial experimental evidence indicates that this process leads to uncontrolled destruction of factors promoting wound healing.^29,30,31,32 However, detailed knowledge on the mechanisms by which uncontrolled proteases cause disturbed repair responses is lacking. Here we demonstrate for the first time that during skin repair HGF protein is a target of substantial proteolytic processing, which is different from the classical activation by known serine proteases and has major functional impact on the HGF/c-Met pathway in skin repair or its failure.     

IL-4/IL-13-Dependent Alternative Activation of Macrophages but Not Microglial Cells Is Associated with Uncontrolled Cerebral Cryptococcosis

Both interleukin (IL)-4- and IL-13-dependent Th2-mediated immune mechanisms exacerbate murine Cryptococcus neoformans-induced bronchopulmonary disease. To study the roles of IL-4 and IL-13 in cerebral cryptococcosis, IL-4 receptor α-deficient (IL-4Rα^−/−), IL-4-deficient (IL-4^−/−), IL-13-deficient (IL-13^−/−), IL-13 transgenic (IL-13^T/+), and wild-type mice were infected intranasally. IL-13^T/+ mice displayed a higher fungal brain burden than wild-type mice, whereas the brain burdens of IL-4Rα^−/−, IL-4^−/−, and IL-13^−/− mice were significantly lower as compared with wild-type mice. On infection, 68% of wild-type mice and 88% of IL-13-overexpressing IL-13^T/+ mice developed significant cerebral lesions. In contrast, only a few IL-4Rα^−/−, IL-4^−/−, and IL-13^−/− mice had small lesions in their brains. Furthermore, IL-13^T/+ mice harbored large pseudocystic lesions in the central nervous system parenchyma, bordered by voluminous foamy alternatively activated macrophages (aaMphs) that contained intracellular cryptococci, without significant microglial activation. In wild-type mice, aaMphs tightly bordered pseudocystic lesions as well, and these mice, in addition, showed microglial cell activation. Interestingly, in resistant IL-4^−/−, IL-13^−/−, and IL-4Rα^−/− mice, no aaMphs were discernible. Microglial cells of all mouse genotypes neither internalized cryptococci nor expressed markers of alternative activation, although they displayed similar IL-4Rα expression levels as macrophages. These data provide the first evidence of the development of aaMphs in a central nervous system infectious disease model, pointing to distinct roles of macrophages versus microglial cells in the central nervous system immune response against C. neoformans.The opportunistic pathogenic yeast Cryptococcus neoformans causes life-threatening fungal infections of most internal organs including the central nervous system (CNS), primarily in patients affected by immunodeficiency syndromes such as AIDS.¹ The pathogenesis of cryptococcosis is not fully understood, however, especially in cases of different levels of immunocompetence. It is generally accepted that the fungus first invades the respiratory system, where it leads to relatively mild or asymptomatic bronchopneumonia in the immunocompetent.^2,3,4,5 Fungemia with generalization of the infection may result from reduced immunological control mechanisms.^6,7,8,9 Invasion of the CNS with subsequent development of meningoencephalitis is the major cause of death during cryptococcosis.^10,11The precise reaction pattern of recruited inflammatory cells, especially monocytes/macrophages, due to fungal invasion of the CNS parenchyma has been addressed mainly via analysis of helper T cell (Th)1 responses.¹² In this context, in addition to protective Th1-driven immune responses, the role of Th2 cytokines has gained interest recently.¹³ The major Th2 cytokines interleukin (IL)-4 and IL-13 act via the IL-4Rα chain together with the γ_c chain or the IL-13Rα1/2 chains, and regulate macrophage functional status.¹⁴ IL-4 has been shown to be detrimental in murine models of systemic and pulmonary cryptococcosis,^{6,15,16,17,18} and we have recently illustrated the role of IL-13 in inducing the formation of alternatively activated macrophages (aaMphs) in murine pulmonary cryptococcosis.¹⁹The activation phenotype of macrophages may critically influence the regulatory mechanisms by which inflammation and infection in the CNS are controlled. According to the current paradigm, classically activated macrophages are primed by interferon-γ and produce tumor necrosis factor, IL-1, oxygen and nitrogen radicals,²⁰ thereby producing proinflammatory cytokines that regulate the Th1 immune response. In contrast, aaMph²¹ develop in response to Th2 cytokine stimulation such as IL-4 and IL-13 and are characterized by expression of genes associated with endocytosis and tissue repair such as arginase-1, mannose receptor (CD206), found-in-inflammatory-zone (FIZZ), and chitinase 3-like 3 (YM1) and largely fail to produce nitric oxide (NO) due to their induction of arginase.²² As such, they are thought to be involved in tissue repair and remodeling,^22,23 in protection against diet-induced obesity,^24,25 and schistosomiasis,²⁶ but they may also elicit adverse tissue processes such as pulmonary or liver fibrosis.^{27,28,29,30,31} In particular, their development renders the host vulnerable to infection with pathogens where macrophage activation and killing functions are required.³²In murine models of pulmonary C. neoformans infection, aaMph have been shown to be associated with uncontrolled lung infection.^18,19 The role of aaMph versus classically activated macrophage in the CNS due to pulmonary infection with the neurotropic pathogen C. neoformans has not been defined yet. In this study, we aimed to characterize the morphology and functional status of CNS macrophages in cerebral cryptococcosis following intranasal infection of susceptible wild-type and IL-13-transgenic BALB/c mice. Moreover, using mice unable to produce IL-4 or IL-13 or respond to both (IL-4Rα^−/− mice), we show that abrogation of CNS aaMph development is associated with controlled infection.     

Chemokine CXCL16 Regulates Neutrophil and Macrophage Infiltration into Injured Muscle,Promoting Muscle Regeneration

Only a few specific chemokines that mediate interactions between inflammatory and satellite cells in muscle regeneration have been identified. The chemokine CXCL16 differs from other chemokines because it has both a transmembrane region and active, soluble chemokine forms. Indeed, we found increased expression of CXCL16 and its receptor, CXCR6, in regenerating myofibers. Muscle regeneration in CXCL16-deficient (CXCL16KO) mice was severely impaired compared with regeneration in wild-type mice. In addition, there was decreased MyoD and myogenin expression in regenerating muscle in CXCL16KO mice, indicating impaired satellite cell proliferation and differentiation. After 1 month, new myofibers in CXCL16KO mice remained significantly smaller than those in muscle of wild-type mice. To understand how CXCL16 regulates muscle regeneration, we examined cells infiltrating injured muscle. There were more infiltrating neutrophils and fewer macrophages in injured muscle of CXCL16KO mice compared with events in wild-type mice. Moreover, absence of CXCL16 led to different expression of cytokines/chemokines in injured muscles: mRNAs of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and MIP-2 were increased, whereas regulated on activation normal T cell expressed and secreted, T-cell activation-3, and monocyte chemoattractant protein-1 mRNAs were lower compared with results in muscles of wild-type mice. Impaired muscle regeneration in CXCL16KO mice also resulted in fibrosis, which was linked to transforming growth factor-β1 expression. Thus, CXCL16 expression is a critical mediator of muscle regeneration, and it suppresses the development of fibrosis.Skeletal muscle regeneration following injury involves proliferation and differentiation of satellite cells leading to the formation of new myofibers.¹ The regeneration process initially involves infiltration of inflammatory cells into injured muscle, including neutrophils, monocytes and macrophages; these accumulate in response to cytokines and chemokines.² This is important because the types of infiltrating cells influence the severity of the injury and the regeneration processes. For example, when neutrophils were depleted by administering an antibody, muscle regeneration following lipopolysaccharide-induced muscle fiber damage was accelerated.³ Neutrophil infiltration was emphasized because these cells cause tissue damage by processes that are related to the production of reactive oxygen species.^4,5,6 The respiratory bursts from infiltrating leukocytes produce oxidizing reactions that damage cells during the early inflammatory period. Indeed, neutrophils obtained from humans or rodents were shown to damage cell membranes of C2C12 myotubes.⁷In contrast to the adverse influence of infiltrating neutrophils on injured muscle, infiltration of monocytes/macrophages can be beneficial.^8,9,10,11,12 For example, when macrophage infiltration into injured muscle was suppressed, muscle regeneration was sharply impaired and this was associated with the development of muscle fibrosis.^13,14 Macrophages not only remove necrotic myofibers by phagocytosis, they also release cytokines as well as growth factors including hepatocyte growth factor, insulin-like growth factor-1, fibroblast growth factor, and tumor necrosis factor-α.^8,9,10,12,15 Release of these cytokines and growth factors stimulate satellite cells, which are closely linked to the processes of muscle regeneration.The recruitment of neutrophils and macrophages into injured muscles is at least partially mediated by chemokines, and consequently, their influence has been examined extensively. For example, the reports of Warren et al¹⁵ and Shireman et al¹⁶ provided the critical evidence that the CC chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CCR2, were critical for the regeneration processes occurring in injured muscle. Specifically, knocking out of the CCR2 receptor or blocking the action of MCP-1 significantly delayed the muscle regeneration occurring in injured tissue. There is evidence, however, that changes in the expression of cytokines besides MCP-1 contribute to muscle regeneration.¹⁷Structurally and functionally, CXCL16 differs from MCP-1 and other chemokines.¹⁸ MCP-1 and the majority of other chemokines are small molecules secreted by inflammatory cells, whereas CXCL16 is synthesized as a transmembrane multidomain molecule consisting of a chemokine domain plus a glycosylated mucin-like stalk linked to a single transmembrane helix. There are two forms of CXCL16 resulting from cleavage at the cell surface. The soluble form of CXCL16 is composed of the extracellular stalk and the chemokine domain. It functions as chemoattractant to promote cell migration and changes in the functions of recruited cells.¹⁹ The remaining transmembrane structure of CXCL16 interacts with its receptor, CXCR6, to establish cell to cell adhesion. Indeed, CXCR6 is expressed on several types of inflammatory cells including macrophages.^{18,20,21,22,23,24,25,26} Previously, we found that inhibition of CXCL16 significantly reduces the infiltration of macrophages into the kidney of rats with anti-glomerular basement membrane antibody-associated glomerulonephritis.²⁷ Given the unique features of CXCL16 and the importance of macrophages in the processes of muscle regeneration, we studied the role of CXCL16 in regulating muscle regeneration. We studied CXCL16 knockout (CXCL16KO) mice using a standard model of muscle injury and regeneration, cardiotoxin injection into tibialis anterior (TA) muscles. Our results reveal that CXCL16 is critical for recruitment of macrophages, which are essential for satellite cell proliferation and differentiation in vivo.     

17β-Estradiol Inhibits Wound Healing in Male Mice via Estrogen Receptor-α

Although estrogens have long been known to accelerate healing in females, their roles in males remain to be established. To address this, we have investigated the influence of 17β-estradiol on acute wound repair in castrated male mice. We report that sustained exposure to estrogen markedly delays wound re-epithelialization. Our use of hairless mice revealed this response to be largely independent of hair follicle cycling, whereas other studies demonstrated that estrogen minimally influences wound inflammation in males. Additionally, we report reduced collagen accumulation and increased gelatinase activities in the wounds of estrogen-treated mice. Increased wound matrix metalloproteinase (MMP)-2 activity in these animals may i) contribute to their inability to heal skin wounds optimally and ii) stem, at least in part, from effects on the overall levels and spatial distribution of membrane-type 1-MMP and tissue inhibitor of MMP (TIMP)-3, which respectively facilitate and prevent MMP-2 activation. Using mice rendered null for either the α or β isoform of the estrogen receptor, we identified estrogen receptor-α as the likely effector of estrogen’s inhibitory effects on healing.Although anecdotal evidence has long suggested that differences exist in the abilities of females and males (particularly the elderly) to heal acute wounds, only recently have they been substantiated by published research. Indeed, it was observed sex differences in key parameters such as restoration of the basement membrane¹ and elastin regeneration² that previously encouraged us to make detailed comparisons of healing in males and females. We discovered that, although repair is broadly similar in intact (young) male and female mice, castrated males heal acute skin wounds far better than do their ovariectomized female counterparts.³ Furthermore, males and females differed in their responsiveness to macrophage migration inhibitory factor (MIF): a potent inhibitor of repair in females, in males it has minimal influence.These studies encouraged us to conclude that sex differences in the responses to cutaneous injury do exist but that they are masked in young individuals by the combined actions of gonadal sex steroids. In males, testosterone and its more potent metabolite 5α-dihydrotestosterone inhibit repair^1,4; in females, estrogens such as 17β-estradiol accelerate healing.^5,6Although the effects of estrogens on female cutaneous physiology are well characterized, their roles in males are poorly understood. A handful of studies have sought to address this. In a group of aged males, locally administered 17β-estradiol was shown to reduce macroscopically determined day 7 wound areas in an excisional wounding model.⁶ It was recently shown that an overwhelming majority of genes displaying different wound expression between young and elderly human males are subject to estrogenic control.⁷ In a separate study, thrice-weekly application of 17β-estradiol to sun-protected skin in aged males induced the synthesis of collagen I; increased dermal collagen bundle thickness and density; and stimulated keratinocyte proliferation.⁸ Although these and other studies have provided useful insights, little is yet known about the healing properties of i) systemic and ii) prolonged estrogen treatment.Having previously reported preliminary evidence that systemic 17β-estradiol treatment may impair cutaneous wound healing in castrated male mice,³ we aimed with the present study to fully characterize the effects of 17β-estradiol on the healing of acute wounds in males and to delineate the mechanisms underpinning any identified responses. Because estrogens are well-known to influence the cycling of hair follicles,⁹ which themselves were recently shown to be beneficial to repair,¹⁰ the contribution of hair to estrogen-impaired healing provided our initial focus. We report that estrogen treatment of castrated mice significantly retards wound re-epithelialization in both hairless (hr/hr) mice and strain-matched controls, confirming that systemic estrogen treatment does indeed inhibit repair and suggesting that the presence of cycling hair follicles is not critical to this response. Subsequent studies identified estrogen receptor (ER)-α as the likely effector of estrogenic inhibition and highlighted the potential involvement of increased matrix metalloproteinase (MMP)-2 activity in the reduced wound accumulation of collagen that we observed in estrogen-treated mice.     

IGF-1R Contributes to Stress-Induced Hepatocellular Damage in Experimental Cholestasis

The insulin-like growth factor type 1 receptor (IGF-1R) controls aging and cellular stress, both of which play major roles in liver disease. Stimulation of insulin-like growth factor signaling can generate cell death in vitro. Here, we tested whether IGF-1R contributes to stress insult in the liver. Cholestatic liver injury was induced by bile duct ligation in control and liver-specific IGF-1R knockout (LIGFREKO) mice. LIGFREKO mice displayed less bile duct ligation-induced hepatocyte damage than controls, while no differences in bile acid serum levels or better adaptation to cholestasis by efflux transporters were found. We therefore tested whether stress pathways contributed to this phenomenon; oxidative stress, ascertained by both malondialdehyde content and heme oxygenase-1 expression, was similar in knockout and control animals. However, together with a lower level of eukaryotic initiation factor-2 α phosphorylation, the endoplasmic reticulum stress protein CHOP and its downstream pro-apoptotic target Bax were induced to lesser extents in LIGFREKO mice than in controls. Expression levels of cytokeratin 19, transforming growth factor-β1, α-smooth muscle actin, and collagen α1(I) in LIGFREKO mice were all lower than in controls, indicating reduced ductular and fibrogenic responses and increased cholestasis tolerance in these mutants. This stress resistance phenotype was also evidenced by longer post-bile duct ligation survival in mutants than controls. These results indicate that IGF-1R contributes to cholestatic liver injury, and suggests the involvement of both CHOP and Bax in this process.The family of insulin-like growth factors (IGFs) comprises two ligands, IGF-I and IGF-II, that activate the IGF type 1 receptor (IGF-1R), a transmembrane tyrosine kinase receptor structurally and functionally related to the insulin receptor.¹ It was recently discovered that IGF-1R regulates lifespan and response to oxidative stress. Although complete knockout of IGF-1R is not compatible with normal life, heterozygous knockout mice live significantly longer than control littermates.² IGF-1R-deficient mice also show longer than control survival after exposure to paraquat and increased resistance to oxygen-induced lung injury; also, embryonic fibroblasts from these mutants are more resistant to oxidative stress induced by hydrogen peroxide.^2,3 Consistent with these findings, but in apparent contradiction with pro-survival and pro-mitogenic functions of IGF-1R, transfection experiments indicate that IGF-1R can generate cell death signals in vitro.^4,5 IGF signaling can also provoke apoptosis in cancer cell lines under low oxygen tension.⁶ In this setting, IGF-induced cell death is related to enhanced endoplasmic reticulum (ER) stress and requires CCAAT/enhancer binding protein homologous protein (CHOP).⁶ CHOP, also known as growth arrest- and DNA damage-inducible gene 153, is a key factor in ER stress-mediated apoptosis.⁷ Recent studies implicate ER stress in neurodegenerative, cardiovascular and liver diseases,^8,9 and CHOP-deficient mice display resistance to apoptosis in animal models of these diseases.^10,11 In particular, CHOP deficiency attenuates cell death induced by alcohol or cholestasis in the liver.^12,13 Various lines of evidence, including the re-expression of fetal IGF-II, and overexpression of IGF-I, IGF-1R and its downstream signaling molecules indicate that the IGF-1R pathway is activated in liver disease.^14,15,16,17 Indeed, IGF-1R may control regeneration,¹⁸ fibrogenesis^15,19 and carcinogenesis²⁰ in the liver. Hepatocellular injury is the major triggering event of the wound healing response that leads to liver fibrosis and cancer, so we investigated whether IGF-1R influences the hepatocellular stress response in the liver. We used a mouse model of liver-specific Igf1r gene inactivation that we established previously,^18,21 and examined cellular stress and fibrogenic responses induced by cholestasis in these animals. We found that IGF-1R deletion confers protection against ER stress and cellular injury induced by cholestasis in the liver.     

CD19, a Response Regulator of B Lymphocytes,Regulates Wound Healing through Hyaluronan-Induced TLR4 Signaling

Immune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-β. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-β through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing.Healing of cutaneous wounds is a complex biological event that results from the interplay of a large number of resident and infiltrating cell types, including leukocytes.¹ Accumulating evidence has revealed a critical role of leukocytes in wound healing. Infiltrating neutrophils form a first line of defense against local infections and are also a source of pro-inflammatory cytokines to activate fibroblasts and keratinocytes.² Macrophages also regulate wound healing by antimicrobial function, wound debridement, and production of various growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor, and TGF-α.^3,4,5,6 These factors stimulate the synthesis of extracellular matrix by local fibroblasts, generate new blood vessels, promote the granulation tissue formation, and enhance re-epithelialization.^4,5 Furthermore, a series of experimental studies have indicated a significant role for T lymphocytes in wound healing as growth factor-producing cells as well as immunological effector cells.^1,7,8,9 Thus, immune cells have an integral function in wound healing beyond their role in inflammation and host defense, mainly through the secretion of signaling molecules, such as cytokines and growth factors.⁶However, little is known regarding a role of B cells in wound healing. Previous studies have revealed that B cells are present within wound tissue^9,10,11 and that B cell count is rapidly increased in the first 4 days after wounding, and thereafter reaches a plateau before falling as the wounds heal.¹¹ Furthermore, recent assessment of the role of B cells in the immune system has indicated that B cells are more than just the precursors of antibody (Ab)-secreting cells.¹² B cells have essential functions in regulating immune responses, including the production of various cytokines and growth factors, antigen presentation, regulation of T cell activation and differentiation, and regulation of lymphoid organization.¹² Therefore, the increased numbers of B cells within wound tissue may reflect a role for these cells that has hitherto been unrecognized.Both innate and adaptive immune responses contribute to host defense cooperatively. B cells play a principal role in adaptive immune response through B cell antigen receptor (BCR). BCR-induced signals are further modified by other cell surface molecules including CD19. CD19, a major positive response regulator, is a critical B cell-specific signal transduction molecule of the immunoglobulin superfamily expressed by early pre-B cells from the time of heavy chain rearrangement until plasma cell differentiation.¹³ B cells also primarily participate in innate immunity; indeed, B cells express toll-like receptors (TLRs) and respond to exogenous innate stimuli such as lipopolysaccharide (LPS), a major component of Gram-negative bacteria. CD19 also regulates LPS signaling: B cells from CD19-deficient (CD19^−/−) mice are hyporesponsive to most transmembrane signals, including BCR ligation and LPS, while B cells from CD19-transgenic (CD19Tg) mice that overexpress CD19 by ∼threefold are hyperresponsive to these signals.^14,15 Thus, CD19 regulates both adaptive and innate immune responses.In the current study, to clarify the roles of B cells in wound healing, we investigated the wound-healing model in CD19^−/− and CD19Tg mice. The results of this study indicate that CD19 controls cytokine and growth factor production by B cells mainly through TLR4 signaling, which was activated by an endogenous TLR4 ligand hyaluronan (HA) increased in the wounded skin, and thereby CD19 regulates the skin wound-healing process.     

Lack of CXC Chemokine Receptor 3 Signaling Leads to Hypertrophic and Hypercellular Scarring

CXC chemokine receptor 3 (CXCR3) signaling promotes keratinocyte migration while terminating fibroblast and endothelial cell immigration into wounds; this signaling also directs epidermal and matrix maturation. Herein, we investigated the long-term effects of failure to activate the “stop-healing” CXCR3 axis. Full-thickness excisional wounds were created on CXCR3 knockout^(−/−) or wild-type mice and examined at up to 180 days after wounding. Grossly, the CXCR3^−/− mice presented a thick keratinized scar compared with the wild-type mice in which the scar was scarcely noticeable; histological examination revealed thickening of both the epidermis and dermis. The dermis was disorganized with thick and long collagen fibrils and contained excessive collagen content in comparison with the wild-type mice. Interestingly, the CXCR3^−/− wounds presented lower tensile/burst strength, which correlates with decreased alignment of collagen fibers, similar to published findings of human scars. Persistent Extracellular matrix turnover and immaturity was shown by the elevated expression of proteins of the immature matrix as well as expression of matrix metallopeptidase-9 MMP-9. Interestingly, the scars in the CXCR3^−/− mice presented evidence of de novo development of a sterile inflammatory response only months after wounding; earlier periods showed resolution of the initial inflammatory stage. These in vivo studies establish that the absence of CXCR3^−/− signaling network results in hypertrophic and hypercellular scarring characterized by on-going wound regeneration, cellular proliferation, and scars in which immature matrix components are undergoing increased turnover resulting in a chronic inflammatory process.Scar formation after excisional wound repair results from a dysfunction in remodeling the two skin compartments, the ectodermally–derived epithelial epidermis and the mesodermally–derived mesenchymal dermis.¹ As a result, there is excessive deposition and misalignment of extracellular matrix proteins. Hypertrophic scar formation, resulting in a thickened skin, which is raised above the unwound tissue, is caused by increased wound cellularity and excessive matrix. Although it is well recognized that a scar results from an imbalance in cellular responses to promotive and inhibitory signals² that signal in a paracrine fashion between the dermis and epidermis,^3,4 the factors that influence this balance between production and degradation are only now being ascertained.Scarring is a challenge to study because the animal models for scarring are such that the hypertrophy occurs in privileged sites, on physiochemical insult, or in a genetically influenced manner.^5,6 In addition, these animal models do not recapitulate the complex situation of human hypertrophic scars or keloids. For instance, hypertrophic scars generated by traction forces being applied during the regenerative phase of healing present a mixed picture of both dermal and epidermal hyperproliferation.² Still, this mixed picture aspect of wound hypertrophy is reminiscent of the early stages of human wound hypertrophic scarring.⁷ Thus, the challenge in these models is to understand the signaling network that under normal conditions limits the wound healing response to prevent human hypertrophic scars. We have taken the approach to determine whether signals that arise late in the regenerative phase act not only to drive wound resolute but also prevent the emergence of hypertrophic scarring.Earlier, we had found that the exuberant cellular responses of wound repair are resolved late in the healing process, at least in part, by a related group of chemokines that appear in the late remodeling and persist into the resolving phase of wound healing; this is the time at which cellularity is reversed and the wound bed matures.^8,9 These chemokines, IP-9/CXCL11 expressed by re-differentiating keratinocytes and IP-10/CXCL10 produced by maturing endothelium deep in the dermis,^4,10,11 both bind to the ubiquitous seven transmembrane G-protein couple chemokine receptor, CXCR3.¹² Signaling through CXCR3 blocks the growth factor induced motility of fibroblasts and endothelial cells by suppressing m-calpain, CAPN2, activation.^13,14 Yet in contrast, these chemokines increase keratinocytes migration via lessened adhesiveness secondary to u-calpain, CAPN1, activation.¹⁵ It is the cellular effects and, most important, the timing of the expression of IP-9 and IP-10 that suggests these chemokines are at least part of the key communication between the dermis and epidermis that signals an end to the remodeling phase and initiation of the resolving phase of wound repair.These CXCR3 ligands signal between the two compartments to stop wound healing. Using mice lacking either the receptor or the CXCL11/IP-9 ligand as in vivo models, we found that in the absence of this signaling axis, excisional wounds matured at a retarded rate with a still weakened and immature dermis even 90 days after wounding.⁹ Furthermore, while there was a delay in re-epithelialization, as predicted by the promotive effects on keratinocytes, the closed wounds presented an epidermis that contained more cell layers than the syngenic wild-type mice. However, in both models the skin had developed normally despite the absence of the receptor or ligand, and the wounds did close and were maturing, reflecting a critical redundancy in wound repair elements.¹ Thus, we expected that at extended time periods the wounds would fully resolve. Surprisingly, we now report that not only does the wound resolution not go to completion, but these wounds appear to revert to an ongoing regenerative-like process that results in a visible scar characterized by hyperkeratinization, excessive but dysfunctional collagen matrix, and an inflammatory infiltrate.     

Protective Role of Interleukin-17 in Murine NKT Cell-Driven Acute Experimental Hepatitis

NKT cells are highly enriched within the liver. On activation NKT cells rapidly release large quantities of different cytokines which subsequently activate, recruit, or modulate cells important for the development of hepatic inflammation. Recently, it has been demonstrated that NKT cells can also produce interleukin-17 (IL-17), a proinflammatory cytokine that is also known to have diverse immunoregulatory effects. The role played by IL-17 in hepatic inflammation is unclear. Here we show that during α-galactosylceramide (αGalCer)-induced hepatitis in mice, a model of hepatitis driven by specific activation of the innate immune system via NKT cells within the liver, NK1.1⁺ and CD4⁺ iNKT cells rapidly produce IL-17 and are the main IL-17-producing cells within the liver. Administration of IL-17 neutralizing monoclonal antibodies before αGalCer injection significantly exacerbated hepatitis, in association with a significant increase in hepatic neutrophil and proinflammatory monocyte (ie, producing IL-12, tumor necrosis factor-α) recruitment, and increased hepatic mRNA and protein expression for the relevant neutrophil and monocyte chemokines CXCL5/LIX and CCL2/MCP-1, respectively. In contrast, administration of exogenous recombinant murine IL-17 before α-GalCer injection ameliorated hepatitis and inhibited the recruitment of inflammatory monocytes into the liver. Our results demonstrate that hepatic iNKT cells specifically activated with α-GalCer rapidly produce IL-17, and IL-17 produced after α-GalCer administration inhibits the development of hepatitis.The cytokine interleukin-17A (IL-17) has been increasingly identified as an important regulator of the inflammatory response.^1,2,3 Initially, a new subset of CD4⁺ T cells were considered to be the source of IL-17 and were classified as Th17 cells.^2,3 IL-17 secreted from Th17 cells was implicated as a proinflammatory mediator in a number of experimental models of inflammation, especially those associated with autoimmunity and an adaptive immune response.^4,5,6 However, more recently IL-17 has also been shown to be able to suppress inflammatory responses, mainly in experimental models which are characterized by a more pronounced innate immune response. Specifically, IL-17 has been shown to suppress inflammation in experimental murine models of asthma,⁷ gastritis,⁸ colitis,^9,10 and atherosclerosis.¹¹ However, the role of IL-17 in regulating hepatic inflammation remains unclear. In patients with viral hepatitis, alcoholic liver disease, and autoimmune liver diseases, numbers of IL-17-producing hepatic T cells are increased.¹² In murine models of liver inflammation the role of IL-17 in regulating the inflammatory response remains controversial. In murine T-cell-mediated hepatitis induced by concanavalin A administration, IL-17 has been shown to be both proinflammatory, as well as without a direct inflammation modulating role.^13,14NKT cells are an important component of the innate immune response and are highly enriched within the liver.¹⁵ NKT cells are activated by glycolipid antigens presented in association with the major histocompatibility complex class I–like molecule CD1d expressed on the surface of antigen presenting cells.¹⁶ Activation of NKT cells in this fashion results in the rapid production and release of large amounts of both Th1; eg, interferon (IFN) γ, tumor necrosis factor (TNF) α, and Th2 (eg, IL-4) cytokines.¹⁶ NKT cells have been implicated in human liver disease and are of critical importance in the initiation and development of hepatitis in numerous murine models.^15,17,18 More recently, NKT cells have also been shown to be capable of rapidly producing IL-17 after activation.^19,20,21 To date IL-17 has been reported to be produced mainly by type II (ie, non-invariant) and NK1.1 negative NKT cells^19,22,23; however, within the murine liver most NKT cells express CD4 and NK1.1 and are classified as invariant (iNKT) or type I NKT cells.^15,16α-Galactosylceramide (αGalCer) is a glycolipid, originally isolated from a marine sponge, which specifically activates iNKT cells in both humans and mice after being presented by antigen presenting cells in the context of CD1d.¹⁶ iNKT cells activated in this fashion can in turn transactivate numerous other cell types within the liver, including other components of the innate immune response such as macrophages and NK cells.^24,25 This property of αGalCer has generated interest in developing this compound as an immune stimulating agent for the treatment of human disease, including liver cancers.²⁴ However, αGalCer treatment also induces hepatitis in mice and therefore has been used as an experimental model to study hepatic immune and inflammatory responses which result from the specific activation of iNKT cells and the subsequent downstream stimulation of the hepatic innate immune system.^26,27Therefore, we undertook this series of experiments to determine first whether hepatic NK1.1 positive iNKT cells could also produce IL-17 after specific activation. In addition, given that the adaptive Th17 response develops more slowly, we wanted to determine the role of IL-17, released as part of the early iNKT cell–driven innate hepatic immune response, in the regulation of hepatitis induced by the administration of αGalCer.     

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2^fl/fl conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2^fl/fl mice but not in CamKII-Cre;ERK2^fl/fl mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.The extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPKs) has been intensely studied for roles in brain development, including control of both axonal^1,2 and dendritic³ growth. Astroglial process extension is also dependent on ERK signaling.⁴ Recent work strongly implicates ERK-mediated signaling in synaptic plasticity and memory formation, and mutations in ERK2 and other ERK pathway components are known to underlie some forms of inherited human cognitive disorders.^5,6,7 ERK MAPK activation is also linked to astroglial activation in several forms of central nervous system (CNS) injury.^8,9,10To directly test the importance of ERK MAPKs in vivo, gene knockout approaches are needed. Whereas ERK1 general knockout (KO) mice are viable and have architecturally normal brains,¹¹ general KO of ERK2 is embryonic lethal, because of defects in mesoderm differentiation and placental development.^12,13,14 Thus, conditional KO (cKO) strategies are necessary to study brain-specific roles of ERK2. Two recent reports^15,16 describe aspects of the developmental phenotype resulting from CNS-specific KO of the mapk1 gene, encoding ERK2. We now present a novel aspect of the adult phenotype of a brain-specific ERK2 cKO driven by Nestin:Cre. Our findings suggest an unexpected and specific role for this classical MAPK member in the regulation of brain collagen deposition.     

Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation

The attenuated S. typhimurium SL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A^d/I-E^d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80⁺ and CD11b⁺ spleen cells.Salmonella infections still pose a serious health hazard worldwide, affecting both humans and animals. Salmonella typhi, the agent of human typhoid fever, is not pathogenic for common laboratory animals. Therefore, natural resistance and acquired immunity to Salmonella are studied mainly in the mouse model by using host-adapted salmonellae which cause systemic infections believed to mimic the human disease.In mice, early bacterial growth in the reticuloendothelial system (RES) is controlled by the innate resistance Nramp (Ity) gene, which is expressed in macrophages (22). In lethal infections, salmonellae rapidly reach large numbers in the tissues and death occurs presumably by endotoxin poisoning when bacterial counts reach levels of ca. 10⁸ CFU per organ (30). In sublethal infections, survival requires a host response that suppresses the exponential growth of the organisms in the RES towards the end of the first week, resulting in a plateau phase (17, 25). The establishment of the plateau phase does not require functional T cells. In fact, nude (T-cell-deficient) mice and mice depleted of T cells by administration of anti-CD4 and anti-CD8 antibodies can still suppress Salmonella growth in infected tissues (17). A bone marrow-dependent influx of radiation-sensitive cells is required for the plateau phase and for the formation of granulomas rich in mononuclear cells (17, 32). Most of the salmonellae in the spleens and livers of the infected animals are localized within the phagocytes present in the focal lesions (38). Tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and nitric oxide (NO) derivatives appear to be required for the suppression of salmonella growth in the RES (27, 28, 32, 36, 37, 48). TNF-α is needed for the recruitment of mononuclear cells in the tissues and for granuloma formation (32); IFN-γ can activate macrophages to kill salmonellae in vitro (20).The establishment of the plateau phase coincides with the development of hypersusceptibility to the toxic and lethal effects of bacterial lipopolysaccharide (LPS) (29, 33). We have previously shown that mice immunized with a live attenuated aromatic-dependent Salmonella vaccine strain show transient hypersusceptibility to LPS, which can be prevented by treatment with anti-TNF-α antibodies (29). The role of other cytokines in this phenomenon is not known.Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine produced by macrophages, B cells, polymorphonuclear leukocytes, and dendritic cells in response to a variety of stimuli including products of bacterial origin (5, 10). IL-12 mediates resistance to intracellular organisms including Listeria, Toxoplasma, Candida, Leishmania, Mycobacterium tuberculosis, and Brucella abortus (8, 13, 18, 23, 39, 46, 50). IL-12 is generally believed to mediate host resistance by inducing IFN-γ production by NK and T cells as well as by contributing to the establishment of protective Th1 antigen-specific responses (5, 6, 9, 10, 12, 13, 24, 34, 39, 43, 47).Evidence for IL-12 induction in salmonellosis has been provided. IL-12 and IL-12-specific mRNA have been detected in vivo and in vitro in response to Salmonella. Elicited peritoneal mouse macrophages stimulated with Salmonella dublin express elevated levels of IL-12 p40-specific mRNA (4, 7). Oral infection with virulent or live attenuated S. dublin induces early (6 and 20 h postinfection) production of IL-12-specific mRNA in Peyer’s patches and mesenteric lymph nodes (3); biologically active IL-12 in lymph node homogenates has been documented 36 h after S. dublin infection (21). We and others previously reported that in vivo IL-12 neutralization reduces the ability of the host to suppress the growth of virulent salmonellae in the tissues and impairs IFN-γ production (21, 31). A recent report indicates that a mutation in the IL-12 receptors renders humans more susceptible to salmonellosis (11). Nevertheless, the mechanisms by which IL-12 mediates host resistance to Salmonella are still unclear.In the present study, we attempted to clarify the mechanisms by which IL-12 contributes to host resistance in mice infected with Salmonella. We investigated the role of IL-12 in survival, granuloma formation, and macrophage activation in mice infected with an attenuated Salmonella strain that normally causes very mild infections in BALB/c mice. We also investigated the involvement of IL-12 in the toxic and lethal effects of high bacterial loads in the tissues as well as in the expression of hypersusceptibility to LPS normally seen in mice infected with salmonellae. We also wished to clarify the involvement of IFN-γ in IL-12-mediated resistance to salmonellosis.     

Interleukin-17 Promotes Early Allograft Inflammation

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-γ-producing cells. As interferon-γ is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2^a) cardiac allografts placed into wild-type BALB/c (H-2^d) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17^−/− recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17^−/− and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17^−/− recipients. In contrast, IL-17^−/− mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donor-reactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17^−/− recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.Despite the increasing quality of immunosuppression, acute cellular rejection episodes occur in more that half of solid organ transplants. Acute allograft rejection is initiated and executed by alloreactive T cells primed in peripheral lymphoid organs and recruited to the graft. Donor-specific T cell responses following transplantation are typically dominated by interferon (IFN)γ-producing cells.^1,2,3 However, IFNγ is dispensable for graft destruction, indicating that other cytokines may contribute to the inflammation cascade and facilitate rejection.^4,5,6Interleukin (IL)-17 (also called IL-17A) is a pleiotropic cytokine with multiple pro-inflammatory functions. Due to the ubiquitous expression of IL-17R, IL-17 can target many different cell types including epithelial and endothelial cells, fibroblasts, and macrophages, and orchestrate tissue inflammation.^7,8 A signature downstream effect of IL-17 is the recruitment and activation of neutrophils and monocytes. IL-17 plays a critical role in host defense against bacterial and fungal pathogens including Klebsiella pneumoniae, Listeria monocytogenes, and Candida albicans.^7,9,10,11 Increased expression of IL-17 is observed in patients with autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease.^12,13 Rodent studies have confirmed the involvement of IL-17 in the pathogenesis of autoimmune diseases that were traditionally thought to be IFNγ- and Th1-dependent, including experimental autoimmune encephalomyelitis and collagen-induced arthritis.^14,15,16,17Recent data from clinical and experimental transplantation suggest the involvement of IL-17 in allograft rejection. For example, IL-17 mRNA and protein expression are elevated in human renal and lung allografts during acute rejection episodes.^18,19,20 In experimental transplantation, increased intragraft IL-17 levels have been observed in animal models of heart and renal allograft rejection.¹⁹ In addition, two groups have reported that IL-17-producing cells mediate cardiac allograft rejection in mice unable to mount Th1 alloimmune responses.^21,22,23 The potential significance of IL-17 in transplantation is further underscored by findings that a neutralizing IL-17R-Ig fusion protein reduced intragraft production of IFNγ and prolonged survival of heart and aorta transplants in rodent models.^24,25 However, the temporal appearance and the role of IL-17 in allograft rejection by wild-type recipients under normal physiological conditions as well as the nature of cooperation between donor-specific CD4 and CD8 T cells producing IL-17 and IFNγ remain largely undefined.In this study, we evaluated the contribution of IL-17 to the induction and amplification of inflammation in class I and class II major histocompatibility complex (MHC)-mismatched murine heart allografts. Our findings indicate that IL-17 up-regulates neutrophil chemoattractant molecules and enhances early neutrophil influx into allografts thus facilitating further recruitment of pathogenic IFNγ-producing T cells into the graft.     

Circulating Monocytes Expressing CD31 : Implications for Acute and Chronic Angiogenesis

To identify the roles of various circulating cells (eg, endothelial and/or stem and progenitor cells) in angiogenesis, we parabiosed a wild-type syngeneic mouse with a transgenic syngeneic green fluorescent protein mouse. Following the establishment of a common circulation between these parabionts, we investigated acute (7 to 10 days), subacute (2 to 3 weeks), and chronic (4 to 6 weeks) phases of angiogenesis in wild-type mice using wound healing, implanted gel foam fragments, and subcutaneous tumor assays, respectively. We found that under in vitro conditions, circulating murine monocytes expressed F4/80, CD31, and vascular endothelial growth factor receptor 2, but neither CD133 nor von Willebrand factor, whereas murine endothelial cells expressed CD31, vascular endothelial growth factor receptor 2, and von Willebrand factor, but neither CD133 nor F4/80. Immunofluorescence analysis revealed that green fluorescent protein-positive cells in the walls of new vessels in wounds, gel foam blocks, and tumors expressed both F4/80 and CD31, that is, macrophages. Pericytes, cells that express both CD31 and desmin, were found both in the walls of tumor-associated vessels and within tumors. Collectively, these data demonstrate that monocytes (ie, cells that express both CD31 and F4/80) may be recruited to the site of tissue injury and directly contribute to angiogenesis, reaffirming the close relationships between various cell types within the reticuloendothelial system and suggesting possible targets for anticancer treatments.The progressive growth of neoplasms and establishment of cancer metastasis depend on the development of adequate vasculature, ie, angiogenesis.^1,2,3,4 The identification of critical factors that contribute to angiogenesis is a major goal of antivascular therapy.^5,6 Whether postnatal neovascularization results from the proliferation and migration of endothelial cells of pre-existing blood vessels^7,8,9 or from circulating stem and progenitor cells that are mobilized from the bone marrow and differentiate into mature endothelial cells^{10,11,12,13,14,15} has been controversial. Circulating endothelial cells (CD31+) have been reported to participate in blood vessel formation occurring during physiological and pathological processes, such as inflammation, wound healing, cardiovascular diseases, and cancer,^{9,10,11,12,13,14,15,16,17,18} and these circulating cells have been targets of cancer therapy.^19,20,21,22 While several investigators have concluded that tumor-associated blood vessels consist of 50% bone marrow-derived endothelial cells,^23,24,25 others have reported that the contribution of circulating bone marrow-derived endothelial cells was either low or undetectable.^26,27 One possibility to account for these differences could be the mice under study and their general state of health. In several studies reporting the participation of circulating CD31+ cells in angiogenesis, mice were given lethal x-irradiation or a high dose of chemotherapy leading to myeloablation and then reconstituted with green fluorescence protein (GFP)-labeled bone marrow cells. Whether the same pattern of angiogenesis occurs in physiological conditions is unclear.To directly investigate the contribution of circulating endothelial cells to the establishment of neovasculature in normal mice, we used the same technique that ruled out the assumption that ovulated oocytes in adult mice are derived from circulating germ cells,²⁸ ie, we examined the formation of blood vessels in wounds, implanted gel-foam sponges, and subcutaneous tumors in mice surgically joined by parabiosis.^29,30 Specifically, we joined a genetically marked GFP mouse with a normal mouse. The parabiosed mice developed a common circulation,^28,29,30,31 which allowed us to track genetically marked cells passing from one parabiont to the other and to determine whether these circulating cells contributed to the establishment of blood vessels in healing wounds, gel foam sponges, and subcutaneous tumors.     

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1^−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1^−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1^−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium.Intercellular adhesion molecule-1 (ICAM-1, CD54)¹ is a conserved member of the immunoglobulin supergene family² and is expressed by many cell types in response to stimuli such as cytokines,^3,4 and oxidative and physical stress.^5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium⁷ and epithelium,^8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.^9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),¹² and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.^7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.⁷ This capability influences functions such as leukocyte transendothelial migration⁷ and vascular permeability.¹³Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.^{14,15,16,17,18,19,20,21} It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.¹⁴ Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,^22,23 it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.^24,25As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,^14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,²⁷ and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,^20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.