Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo. Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti‐angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor‐α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.     

Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation

It has not previously been possible to live image the earliest interactions between the host environment and oncogene-transformed cells as they initiate formation of cancers within an organism. Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin. Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden. Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages. We show that a major component of the signal drawing inflammatory cells to oncogenic HRAS(G12V)-transformed cells is H(2)O(2), which is also a key damage cue responsible for recruiting neutrophils to a wound. Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair. These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble non-healing wounds.     

Progranulin is a mediator of the wound response

Annually, 1.25 million individuals suffer burns in the United States and 6.5 million experience chronic skin ulcers, often from diabetes, pressure or venous stasis. Growth factors are essential mediators of wound repair, but their success as therapeutics in wound treatment has, so far, been limited. Therefore, there is a need to identify new wound-response regulatory factors, but few have appeared in recent years. Progranulin (also called granulin or epithelin precursor, acrogranin or PC-derived growth factor) is a growth factor involved in tumorigenesis and development. Peptides derived from progranulin have been isolated from inflammatory cells, which led to suggestions that progranulin gene products are involved in the wound response, but this remains undemonstrated. We report that in murine transcutaneous puncture wounds, progranulin mRNA is expressed in the inflammatory infiltrate and is highly induced in dermal fibroblasts and endothelia following injury. When applied to a cutaneous wound, progranulin increased the accumulation of neutrophils, macrophages, blood vessels and fibroblasts in the wound. It acts directly on isolated dermal fibroblasts and endothelial cells to promote division, migration and the formation of capillary-like tubule structures. Progranulin is, therefore, a probable wound-related growth factor.     

Wound healing and inflammation: embryos reveal the way to perfect repair

Tissue repair in embryos is rapid, efficient and perfect and does not leave a scar, an ability that is lost as development proceeds. Whereas adult wound keratinocytes crawl forwards over the exposed substratum to close the gap, a wound in the embryonic epidermis is closed by contraction of a rapidly assembled actin purse string. Blocking assembly of this cable in chick and mouse embryos, by drugs or by inactivation of the small GTPase Rho, severely hinders the re-epithelialization process. Live studies of epithelial repair in GFP-actin-expressing Drosophila embryos reveal actin-rich filopodia associated with the cable, and although these protrusions from leading edge cells appear to play little role in epithelial migration, they are essential for final zippering of the wound edges together-inactivation of Cdc42 prevents their assembly and blocks the final adhesion step. This wound re-epithelialization machinery appears to recapitulate that used during naturally occurring morphogenetic episodes as typified by Drosophila dorsal closure. One key difference between embryonic and adult repair, which may explain why one heals perfectly and the other scars, is the presence of an inflammatory response at sites of adult repair where there is none in the embryo. Our studies of repair in the PU. 1 null mouse, which is genetically incapable of raising an inflammatory response, show that inflammation may indeed be partly responsible for scarring, and our genetic studies of inflammation in zebrafish (Danio rerio) larvae suggest routes to identifying gene targets for therapeutically modulating the recruitment of inflammatory cells and thus improving adult healing.     

Immunohistochemical study on the expression of c-Fos and c-Jun in human skin wounds

An immunohistochemical study on the temporal expression of c-Fos and c-Jun, both of which designate proto-oncogene products, was performed on 60 human skin wounds with different post-infliction intervals. In unwounded skin, c-Fos or c-Jun was immunolocalized at the nuclei of the epidermal cells in the basal layer, hair follicle cells and sweat gland cells. During the early inflammatory phase of wound healing, the nuclei of polymorphonuclear cells (probably neutrophils), mainly infiltrating at the wound site, were labeled with anti-c-Fos or -c-Jun antibody. As the wound age increased, the neutrophils had disappeared at the wound site, and both mononuclear cells (probably macrophages) and spindle-shaped fibroblastic cells, which expressed a c-Fos or c-Jun positive reaction in the nuclei, were mainly observed. Morphometrically, the distribution of the c-Fos-positive ratio was very similar to that of the c-Jun-positive ratio; the positive ratio was considerably increased in wound specimens with a post-infliction interval of 1 day, thus indicating the late inflammatory or proliferative phase. This study showed that c-Fos and c-Jun were closely involved in the inflammatory phase as well as the proliferative phase of the wound healing process.     

Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract.

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants such as hydrogen peroxide mostly contributed by neutrophils and macrophages. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. This study provides first evidence showing that natural extracts such as grape seed proanthocyanidin extract containing 5000 ppm resveratrol (GSPE) facilitates oxidant-induced VEGF expression in keratinocytes. Using a ribonuclease protection assay (RPA), the ability of GSPE to regulate oxidant-induced changes in several angiogenesis-related genes were studied. While mRNA responses were studied using RPA, VEGF protein release from cells to the culture medium was studied using ELISA. Pretreatment of HaCaT keratinocytes with GSPE upregulated both hydrogen peroxide as well as TNF-alpha-induced VEGF expression and release. The current results suggest that GSPE may have beneficial therapeutic effects in promoting dermal wound healing and other related skin disorders.     

Wound Administration of M2-Polarized Macrophages Does Not Improve Murine Cutaneous Healing Responses

Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.     

IL-5-overexpressing mice exhibit eosinophilia and altered wound healing through mechanisms involving prolonged inflammation

Leucocytes are essential in healing wounds and are predominantly involved in the inflammatory and granulation stages of wound repair. Eosinophils are granulocytic leucocytes and are specifically regulated by interleukin-5 (IL-5), a cytokine produced by T helper 2 (Th2) cells. To characterize more clearly the role of the IL-5 and eosinophils in the wound healing process, IL-5-overexpressing and IL-5-deficient mice were used as models of eosinophilia and eosinophil depletion, respectively. Our results reveal a significantly altered inflammatory response between IL-5-overexpressing and IL-5 knockout mice post-wounding. Healing was significantly delayed in IL-5-overexpressing mice with wounds gaping wider and exhibiting impaired re-epithelialization. A delay in collagen deposition was observed suggesting a direct effect on matrix synthesis. A significant increase in inflammatory cell infiltration, particularly eosinophils and CD4(+) cells, one of the main cell types which secrete IL-5, was observed in IL-5-overexpressing mice wounds suggesting that one of the main roles of IL-5 in wound repair may be to promote the infiltration of eosinophils into healing wounds. Healing is delayed in IL-5-overexpressing mice and this corresponds to significantly increased levels of eosinophils and CD4(+) cells within the wound site that may contribute to and exacerbate the inflammatory response, resulting in detrimental wound repair.     

Inflammatory cells during wound repair: the good, the bad and the ugly

Damage to any tissue triggers a cascade of events that leads to rapid repair of the wound - if the tissue is skin, then repair involves re-epithelialization, formation of granulation tissue and contraction of underlying wound connective tissues. This concerted effort by the wounded cell layers is accompanied by, and might also be partially regulated by, a robust inflammatory response, in which first neutrophils and then macrophages and mast cells emigrate from nearby tissues and from the circulation. Clearly, this inflammatory response is crucial for fighting infection and must have been selected for during the course of evolution so that tissue damage did not inevitably lead to death through septicemia. But, aside from this role, exactly what are the functions of the various leukocyte lineages that are recruited with overlapping time courses to the wound site, and might they do more harm than good? Recent knockout and knockdown studies suggest that depletion of one or more of the inflammatory cell lineages can even enhance healing, and we discuss new views on how regulation of the migration of inflammatory cells to sites of tissue damage might guide therapeutic strategies for modulating the inflammatory response.     

Immunohistochemical localization of growth factors in fetal wound healing

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.     

Regulatory role of endogenous interleukin-10 in cutaneous inflammatory response of murine wound healing.

This study investigated the role of endogenous interleukin (IL)-10 in cutaneous wound healing. Both IL-10 mRNA and protein were detectable in murine incised wounds for 10 days after injury. The IL-10 protein level peaked 3 h after incision, returned to the normal level by 24 h, but increased again to another peak at 72 h. In situ hybridization studies and immunostaining revealed that epidermal cells and infiltrating mononuclear cells were the major source of IL-10. Neutralizing antibody studies demonstrated that IL-10 inhibited the infiltration of neutrophils and macrophages toward the site of injury. IL-10 also inhibited overexpression of C-C chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha) and proinflammatory cytokines (IL-1beta, IL-6, tumor necrosis factor-alpha) in vivo. These results suggest that IL-10 may play an important regulatory role in the phase-specific infiltration of neutrophils and macrophages as well as the cytokine production in the inflammatory response of cutaneous wound healing.     

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

Background

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Methodology/Principal Findings

Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.

Conclusions/Significance

Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.     

Apoptotic Neutrophils Augment the Inflammatory Response to Mycobacterium tuberculosis Infection in Human Macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.     

Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.     

Nod-Like Receptor Protein-3 Inflammasome Plays an Important Role during Early Stages of Wound Healing

The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.     

Single-cell analysis of skin immune cells reveals an Angptl4-ifi20b axis that regulates monocyte differentiation during wound healing

The persistent inflammatory response at the wound site is a cardinal feature of nonhealing wounds. Prolonged neutrophil presence in the wound site due to failed clearance by reduced monocyte-derived macrophages delays the transition from the inflammatory to the proliferative phase of wound healing. Angiopoietin-like 4 protein (Angptl4) is a matricellular protein that has been implicated in many inflammatory diseases. However, its precise role in the immune cell response during wound healing remains unclear. Therefore, we performed flow cytometry and single-cell RNA sequencing to examine the immune cell landscape of excisional wounds from Angptl4^+/+ and Angptl4^−/− mice. Chemotactic immune cell recruitment and infiltration were not compromised due to Angptl4 deficiency. However, as wound healing progresses, Angptl4^−/− wounds have a prolonged neutrophil presence and fewer monocyte-derived macrophages than Angptl4^+/+ and Angptl4^LysM−/− wounds. The underlying mechanism involves a novel Angptl4-interferon activated gene 202B (ifi202b) axis that regulates monocyte differentiation to macrophages, coordinating neutrophil removal and inflammation resolution. An unbiased kinase inhibitor screen revealed an Angptl4-mediated kinome signaling network involving S6K, JAK, and CDK, among others, that modulates the expression of ifi202b. Silencing ifi202b in Angptl4^−/− monocytes, whose endogenous expression was elevated, rescued the impaired monocyte-to-macrophage transition in the in vitro reconstituted wound microenvironment using wound exudate. GSEA and IPA functional analyses revealed that ifi202b-associated canonical pathways and functions involved in the inflammatory response and monocyte cell fate were enriched. Together, we identified ifi202b as a key gatekeeper of monocyte differentiation. By modulating ifi202b expression, Angptl4 orchestrates the inflammatory state, innate immune landscape, and wound healing process.Subject terms: Acute inflammation, Acute inflammation     

Inflammatory processes in muscle injury and repair

Modified muscle use or injury can produce a stereotypic inflammatory response in which neutrophils rapidly invade, followed by macrophages. This inflammatory response coincides with muscle repair, regeneration, and growth, which involve activation and proliferation of satellite cells, followed by their terminal differentiation. Recent investigations have begun to explore the relationship between inflammatory cell functions and skeletal muscle injury and repair by using genetically modified animal models, antibody depletions of specific inflammatory cell populations, or expression profiling of inflamed muscle after injury. These studies have contributed to a complex picture in which inflammatory cells promote both injury and repair, through the combined actions of free radicals, growth factors, and chemokines. In this review, recent discoveries concerning the interactions between skeletal muscle and inflammatory cells are presented. New findings clearly show a role for neutrophils in promoting muscle damage soon after muscle injury or modified use. No direct evidence is yet available to show that neutrophils play a beneficial role in muscle repair or regeneration. Macrophages have also been shown capable of promoting muscle damage in vivo and in vitro through the release of free radicals, although other findings indicate that they may also play a role in muscle repair and regeneration through growth factors and cytokine-mediated signaling. However, this role for macrophages in muscle regeneration is still not definitive; other cells present in muscle can also produce the potentially regenerative factors, and it remains to be proven whether macrophage-derived factors are essential for muscle repair or regeneration in vivo. New evidence also shows that muscle cells can release positive and negative regulators of inflammatory cell invasion, and thereby play an active role in modulating the inflammatory process. In particular, muscle-derived nitric oxide can inhibit inflammatory cell invasion of healthy muscle and protect muscle from lysis by inflammatory cells in vivo and in vitro. On the other hand, muscle-derived cytokines can signal for inflammatory cell invasion, at least in vitro. The immediate challenge for advancing our current understanding of the relationships between muscle and inflammatory cells during muscle injury and repair is to place what has been learned in vitro into the complex and dynamic in vivo environment.