In mammals, several gene families encode peptides with antibacterial activity, such as the beta-defensins and cathelicidins. These peptides are expressed on epithelial surfaces and in neutrophils, and have been proposed to provide a first line of defence against infection by acting as 'natural antibiotics'. The protective effect of antimicrobial peptides is brought into question by observations that several of these peptides are easily inactivated and have diverse cellular effects that are distinct from antimicrobial activity demonstrated in vitro. To investigate the function of a specific antimicrobial peptide in a mouse model of cutaneous infection, we applied a combined mammalian and bacterial genetic approach to the cathelicidin antimicrobial gene family. The mature human (LL-37) and mouse (CRAMP) peptides are encoded by similar genes (CAMP and Cnlp, respectively), and have similar alpha-helical structures, spectra of antimicrobial activity and tissue distribution. Here we show that cathelicidins are an important native component of innate host defence in mice and provide protection against necrotic skin infection caused by Group A Streptococcus (GAS).

Cathelicidins are a family of peptides thought to provide an innate defensive barrier against a variety of potential microbial pathogens. The human and mouse cathelicidins (LL-37 and CRAMP, respectively) are expressed at select epithelial interfaces where they have been proposed to kill a number of gram-negative and gram-positive bacteria. To determine if these peptides play a part in the protection of skin against wound infections, the anti-microbial activity of LL-37 and CRAMP was determined against the common wound pathogen group A Streptococcus, and their expression was examined after cutaneous injury. We observed a large increase in the expression of cathelicidins in human and murine skin after sterile incision, or in mouse following infection by group A Streptococcus. The appearance of cathelicidins in skin was due to both synthesis within epidermal keratinocytes and deposition from granulocyctes that migrate to the site of injury. Synthesis and deposition in the wound was accompanied by processing from the inactive prostorage form to the mature C-terminal peptide. Analysis of anti-microbial activity of this C-terminal peptide against group A Streptococcus revealed that both LL-37 and CRAMP potently inhibited bacterial growth. Action against group A Streptococcus occurred in conditions that typically abolish the activity of anti-microbial peptides against other organisms. Thus, cathelicidins are well suited to provide defense against infections due to group A Streptococcus, and represent an important element of cutaneous innate immunity.

Tissues must quickly recognize injury to respond to the rapid pace of microbial growth. In skin, dermal microvascular endothelial cells must also react to danger signals from the surrounding tissue and immediately participate by initiating the wound repair process. Components of the extracellular matrix such as hyaluronan are rapidly broken down into smaller molecular weight oligosaccharides in a wound, and these can activate a variety of biological processes. This study set out to determine if hyaluronan fragments released following injury can stimulate endothelial cells and what mechanism is responsible for this response. Using genechip microarray analysis, a response to hyaluronan fragments was detected in endothelial cells with the most significant increase observed for the chemokine IL-8. This observation was verified with qualitative reverse transcriptase-PCR and ELISA in human endothelial cell culture, and in a mouse model by observing serum levels of MIP-2 and KC following hyaluronan fragment administration in vivo. Activation was TLR4-dependent, as shown by use of TLR4 blocking antibody and TLR4-deficient mice, but not due to the presence of undetected contaminants as shown by inactivation following digestion with the hyaluronan-degrading enzyme chondroitinase ABC or incubation with the hyaluronan-specific blocking peptide Pep-1. Inactivation of LPS activity failed to diminish the action of hyaluronan fragments. These observations suggest that endogenous components of the extracellular matrix can stimulate endothelia to trigger recognition of injury in the initial stages of the wound defense and repair response.

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2-and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.Components of the extracellular matrix are broken down following injury or inflammation. These extracellular matrix products in turn participate in various phases of the wound repair process. Following injury, proteoglycans (PG) 1 and their glycosaminoglycan (GAG) chains are released and become soluble and abundant in fluids collected from wounds (1). Dermatan sulfate (DS) is the most common GAG in the skin and is estimated to comprise between 36 and 78% of the total sulfated GAG in wound fluid samples (1). DS consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid (GlcA)-linked ␤134 and ␤133, respectively. Similar to modifications in heparin and heparan sulfate (HS) but distinct from the other chondroitin sulfates (CS), the GlcA of DS undergoes epimerization of the C-5 carbon, resulting in iduronic acid (IdoUA). In addition, sulfation modifications can occur at the 2-position of the uronic acid and at the 4-or 6-position of the N-acetylgalactosamine residue. GAGs and their PG core proteins participate in a variety of functions during wound healing, including binding multiple growth factors and promoting their activities (1-8). Of these activities, the fibroblast growth factor ...

Cathelicidin antimicrobial peptides are effectors of innate immune defense in mammals. Humans and mice have only one cathelicidin gene, whereas domesticated mammals such as the pig, cow, and horse have multiple cathelicidin genes. We hypothesized that the evolution of multiple cathelicidin genes provides these animals with enhanced resistance to infection. To test this, we investigated the effects of the addition of cathelicidins by combining synthetic cathelicidin peptides in vitro, by producing human keratinocytes that overexpress cathelicidins in culture, or by producing transgenic mice that constitutively overexpress cathelicidins in vivo. The porcine cathelicidin peptide PR-39 acted additively with human cathelicidin LL-37 to kill group A Streptococcus (GAS). Lentiviral delivery of PR-39 enhanced killing of GAS by human keratinocytes. Finally, transgenic mice expressing PR-39 under the influence of a K14 promoter showed increased resistance to GAS skin infection (50% smaller necrotic ulcers and 60% fewer surviving bacteria). Similarly constructed transgenic mice designed to overexpress their native cathelicidin did not show increased resistance. These findings demonstrate that targeted gene transfer of a xenobiotic cathelicidin confers resistance against infection and suggests the benefit of duplication and divergence in the evolution of antimicrobial peptides.innate immunity ͉ keratinocyte

FGF-7 is induced after injury and induces the proliferation of keratinocytes. Like most members of the FGF family, the activity of FGF-7 is strongly influenced by binding to heparin, but this glycosaminoglycan is absent on keratinocyte cell surfaces and minimally present in the wound environment. In this investigation we compared the relative activity of heparan sulfate and chondroitin sulfate B (dermatan sulfate), glycosaminoglycans that are present in wounds. A lymphoid cell line (BaF/KGFR) containing the FGF-7 receptor (FGFR2 IIIb) was treated with FGF-7 and with various glycosaminoglycans. FGF-7 did not support cell proliferation in the absence of glycosaminoglycan or with addition of heparan sulfate or chondroitin sulfate A/C but did stimulate BaF/KGFR division in the presence of dermatan sulfate or highly sulfated low molecular weight fractions of dermatan. Dermatan sulfate also enabled FGF-7-dependent phosphorylation of mitogen-activated protein kinase and promoted binding of radiolabeled FGF-7 to FGFR2 IIIb. In addition, dermatan sulfate and FGF-7 stimulated growth of normal keratinocytes in culture. Thus, dermatan sulfate, the predominant glycosaminoglycan in skin, is the principle cofactor for FGF-7. Glycosaminoglycans (GAGs)1 and proteoglycans are emerging as key regulators of a variety of cellular behaviors involved in development, homeostasis, and disease. Heparin, heparan sulfate (HS), and hyaluronic acid are GAGs that have been studied extensively in relation to their roles in anticoagulation, growth factor signaling, and connective tissue support. Less well studied, dermatan sulfate (DS) is the predominant GAG expressed in the skin and is released at high concentrations during wound repair, making it a particularly interesting topic for evaluation in relation to growth factors active in wounds. Wound fluid-derived DS, as well as physiologic concentrations of commercially purified DS, has been shown to promote FGF-2 function (1). DS and DS proteoglycans also bind to and influence the activity of other heparin-and HS-binding proteins including hepatocyte growth factor/scatter factor (2), thrombin (3), heparin cofactor II (4, 5), fibronectin (6), platelet factor-4 (7), regulated on activation normal T cell expressed and secreted (RANTES) (8), and interferon-␥ (9, 10). Thus, although less well understood, DS-protein interactions are likely to be important events in control of several cellular behaviors.FGF-7 or keratinocyte growth factor is a polypeptide mitogen that belongs to the family of fibroblast growth factors. Whereas some FGF family members bind to multiple FGF receptors (FGFRs 1-4) (11) and stimulate proliferation in a variety of cell types, FGF-7 binds only to a splice variant of FGFR2 (FGFR2 IIIb) and is a highly specific paracrine growth factor for epithelial cells (12, 13). FGF-7 and its receptor are believed to be important for normal wound healing. The expression of FGF-7 and FGFR2 IIIb are induced in wounds (14, 15), and the application of FGF-7 to wounds has been shown to promote he...

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Antimicrobial peptides are essential to innate host defense as effectors of pathogen clearance and can modify host cell behaviors to promote wound repair. While these two functions appear interrelated, it is unclear whether the ability to aid in wound repair requires inherent antimicrobial function. We hypothesized that the influence of antimicrobial peptides on wound repair is not dependent on antimicrobial function. To explore this, we analyzed the microbial killing activity of peptide fragments and correlated this with the ability to influence wound repair in mice. HB-107, a peptide lacking antimicrobial activity and originally derived from the antimicrobial cecropin B, showed up to 64 percent improvement in wound repair compared to scrambled peptide and vehicle controls, an effect comparable to treatment with recombinant human platelet-derived growth factor-BB (formulated as Regranex). Wounds treated with HB-107 showed keratinocyte hyperplasia and increased leukocyte infiltration. Furthermore, HB-107 stimulated interleukin-8 secretion from cultured endothelial cells, an effect that may explain the increase in leukocyte migration. These findings confirm that antimicrobial peptides can function as effectors of cutaneous wound repair. Moreover, this study furthers our understanding of antimicrobial peptides by showing that their wound repair properties can be independent of antimicrobial function.