Functions of Chicken IL-8 (cCAF) in Wound Healing

Introduction The chicken chemotactic and angiogenic factor (cCAF) is the ortholog of human IL-8. These proteins are small inducible cytokines called chemokines. Chemokines were first identified as proteins that are critical to the process of inflammation. However, more recently, they have been shown to perform a variety of functions in biological processes, such as wound healing, atherosclerosis, and tumorigenesis. This review concentrates on cIL-8 and its role in wound healing and will also refer to other chemokines where appropriate. The author will discuss the biochemical and structural properties of the protein, activators and mechanisms of activation, expression patterns in vivo, and functions during wound healing. The author will also offer a perspective on how cIL-8 may function during the healing process. Chemokines are secreted proteins with extensive sequence homology to each other, are evolutionarily conserved, and most are encoded by early response genes.[1–4] These proteins have four conserved cysteines that form two disulfide bonds, which anchor the tertiary structure of these molecules and are important in their function.[1,5–9] Their molecular weights range between 6 and 10kDa, but multiple forms of these proteins are frequently observed and appear to be related to activation of the molecule.[10–16] The chemokine superfamily is divided into four families based on the position of the first two cysteines (Figure 1).[11] The two major families are the CXC family, in which these cysteines are separated by a single amino acid, and the CC family, in which the cysteines are adjacent. In addition, there are two minor families: The C family in which the first and third cysteines are missing and hence they have only one disulfide bond (the only members known so far are lymphotactin and SCM-1b) and the CX3C family in which the first two cysteines are separated by three amino acids (the only member discovered so far is fractalkine). The CXC family can be further divided into ELR+ and ELR- chemokines depending on the presence or absence of this amino acid motif immediately before the first cysteine. Human and chicken IL-8 both belong to the ELR+ CXC chemokine family, and, according to the new nomenclature based on the family name, IL-8 is called CXCL-8.[11] The molecular structure of chemokines in general has been deduced from that of IL-8. Crystallographic and NMR studies have shown that the structure of chemokines consists of a short, flexible N-terminus followed by a loop, three antiparallel beta-pleated sheets, and a C-terminal alpha-helix.[7,8,17] All of these regions of the protein can represent functional domains. Over the years, investigators have come to realize that chemokines as well as other cytokines perform multiple functions. For a long time, this discovery was perplexing. However, the subsequent realization that these same molecules display multiple forms and/or multiple functional domains, they can interact with more than one receptor, and cells differentially express these receptors provided an explanation for the diversity of functions of chemokines. To date, all the known chemokine receptors are 7-transmembrane domain G-protein-linked receptors in the rhodopsin superfamily. These receptors are divided into four major families, much like their chemokine ligands, and follow a similar nomenclature: Those that bind CC family members are called CCRs, those that bind CXC family members are CXCRs, those that bind C family members are XCRs, and those that bind CX3C family members are CX3CRs.[4] In addition, there is one more type of receptor that binds all chemokines, the Duffy receptor, but to date there are no reports that this receptor is able to transduce signals upon binding to chemokines. Many chemokine receptors are promiscuous, binding more than one protein in the same family, and many chemokines bind to more than one receptor. Many cells express chemokine receptors, but the specific receptor expression depends on the type and activation state of the cell. Furthermore, there is also some evidence that interactions between chemokines and proteoglycans may regulate some cellular responses induced by these proteins.[18–20] Finally, two CC chemokine receptors and one CXC receptor are known to be cofactors for HIV entry into leukocytes.[21–24] The cDNA for cIL-8 was the first to be isolated and was called 9E3[25] or CEF4[26] after the clones that contained the gene. This is one of very few currently known nonmammalian members of the chemokine superfamily.[27,28] It was discovered when studying Rous sarcoma virus-associated changes in gene expression to identify genes that were either overexpressed or underexpressed in transformed cells. Shortly after the discovery of 9E3/CEF4 gene, the cDNA for the closely related human neutrophil activating protein (NAP-1/IL-8), a protein that is highly expressed in activated monocytes,29,30 was cloned and characterized.[31] The 9E3/CEF4 gene, like the hIL-8 gene, contains four exons and three introns. The regulatory sequences and exons 2 and 3 are virtually identical to those of hIL-8.[32] Therefore, henceforth the author will refer to the 9E3/CEF4 gene and its product as cIL-8. Biochemical and Structural Properties of cIL-8 cIL-8 is secreted as a 9kDa form in both normal and transformed cells. However, when cells are cultured on collagen or matrigel, a smaller form of the protein (7kDa) is seen in association with the matrix. The 7kDa protein is produced by post-secretory cleavage at the N-terminus, and the only processing agent identified so far is plasmin, an enzyme produced abundantly during wound healing. The smaller form binds to interstitial collagen and tenascin, two extracellular matrix (ECM) molecules abundantly expressed during wound healing, but does not bind to fibronectin or hyaluronic acid. In addition, the 7kDa form also binds to basement membrane components, such as laminin, and to a lesser extent to complex proteoglycans but does not bind to collagen IV.[14] Moreover, this chemokine is also stimulated in normal cells when they are cultured on interstitial collagen or basement membrane components and, in vivo, is found in areas rich in interstitial collagen and tenascin. Therefore, it is possible that interactions with the matrix are of fundamental importance for the function of the protein and/or that binding to the matrix may prevent degradation of the protein, more efficient presentation to its receptors, and/or dimerization/multimerization.[33] Pulse-chase experiments show that cIL-8 is synthesized and secreted rapidly (less than 10 minutes),[14] suggesting that, given the appropriate stimulus, the level of chemokine can be rapidly elevated in vivo, resulting in similarly rapid biological responses. Indeed, much like heat-shock proteins are considered cellular stress-response proteins, chemokines can be considered the stress-response proteins of tissues and organs. The cIL-8 was modeled after the hIL-8 structure by replacing the amino acids of IL-8 with those of cIL-8 using the Insight Builder module and optimized through energy minimization and molecular dynamics calculations using the Discover module. The major difference between the two molecules occurs at the N-terminus. cIL-8 has 10 extra amino acids that are not present in the shorter form of hIL-8. This peptide assumes the shape of a short a-helix that bends toward the body of the molecule and lies very close to the C-terminus a-helix (Figure 2). Otherwise, the core of the protein is very similar to that of hIL-8 with the loops and the b-pleated sheets in the same configuration. The two a-helices may be sites for intermolecular interactions, because one has a very hydrophobic region, which could serve as a binding site to other hydrophobic regions of the receptors, and the other is very highly positively charged serving as a potential binding site for highly negatively charged regions of the receptors. Activation of the Chemokine by Wound Factors Some chemokines are expressed constitutively and are usually involved in hematopoiesis. However, most chemokines are expressed in cells activated by inflammatory and stress-inducing agents, such as factors released upon wounding. In addition, chemokines can be stimulated by a variety of oncogenes, tumor promoters, cigarette smoke, and pharmacological agents. cIL-8 is stimulated by a variety of factors released upon wounding from platelet a granules, such as TGF-alpha, factors that are produced and/or released locally after wounding (e.g., alphaFGF and betaFGF), and the clotting enzyme thrombin. Although all three growth factors stimulate cIL-8 to a certain extent, the most potent activator is thrombin.[34] The stimulation by thrombin occurs via its proteolytically activated seven-transmembrane receptor, involves transactivation of the EGF receptor tyrosine kinase, and is followed by MAPK cascade activation, which, in turn, activates the Elk1 transcription factor (Figure 3).[34,35] Our most recent results show that Elk1 interacts with the coactivator p300 in an inactive complex and that phosphorylation by MAPK leads to conformational changes that result in alterations in Elk1/p300 interactions, leading to activation of histone acetyl transferase, remodeling of the nucleosome, and initiation of transcription.36 In spite of the fact that thrombin stimulation of cIL-8 occurs through MAPK, this stimulation is independent of mitogenesis; gene activation occurs in a matter of minutes after thrombin interacts with its receptor. In spite of the strong transcription activation of cIL-8 by these agents, this chemokine is also regulated at the level of mRNA stability.[37] Cell- and Tissue-Specific Expression of cIL-8 In quiescent cells, cIL-8 is expressed at very low levels, but upon the addition of an appropriate stress-inducing agent, expression levels rise rapidly.[14,25,34,37–39] Some cells, such as leukocytes, endothelial cells, keratinocytes, and fibroblasts, can be induced to express this and other chemokines.[33] In normal adult tissues, cIL-8 also is expressed at very low levels, primarily in connective tissue, tendon, and bone but not in muscle, bone marrow stroma, or endothelial cells of fully differentiated blood vessels.[40,41] However, if these differentiated blood vessels are exposed to agents that stimulate the expression of cIL-8, the endothelial cells upregulate the expression of this chemokine. This observation may have implications for atherogenesis, because the formation of atherosclerotic plaques is initiated by an injury to the endothelium. This injury may be caused by exposure to stress-inducing agents (e.g., cigarette smoke components) that then stimulate cIL-8 production that in turn chemoattracts leukocytes, such as monocytes, that are known to participate in initiation of plaque formation. Very little is known about the expression of this chemokine gene during embryogenesis. It is expressed primarily in epidermal cells and in the endothelial cells of young blood vessels, but as development proceeds and the blood vessels mature, cIL-8 is no longer expressed by endothelial cells.[15] However, not much is known about the expression of this chemokine in organ tissues during the various stages of embryonic development. Expression of the Chemokines During Wound Repair The first evidence that chemokines are associated with healing came from studies performed in 1990.[40] The author has shown that cIL-8 is overexpressed during wound healing and that the expression patterns varied over time. The mRNA levels for this chemokine rise quickly after wounding, and the protein begins to be produced, establishing a steep gradient. These gradients are known to be important for the chemoattraction of leukocytes to the sites of wounding. As healing progresses, the protein begins to accumulate primarily in areas of the wound that are rich in ECM molecules, especially in association with interstitial collagen.[41] mRNA levels decline after 36 to 48 hours but remain slightly elevated throughout granulation tissue development.[40,41] The very high levels of gene expression found shortly after wounding potentially can be explained by the fact that in culture, cIL-8 expression is stimulated to high levels after treatment of fibroblasts with thrombin.[34] cIL-8 is initially expressed primarily by the fibroblasts and the basal keratinocytes at the edges of the wound. As healing progresses, the monocytes and the endothelial cells of the microvessels in the healing tissue also express it, and expression falls off progressively with distance from the wound. Other CXC chemokines are also expressed shortly after wounding, although the type of chemokine expressed and the kinetics of expression vary with the type of wound. Most of what is known about chemokines other than cIL-8 and expression during wound healing relates to the inflammatory phase of healing. For example, hIL-8 and hgroa (CXCL1) reach a peak of expression in skin wounds around 24 hours, much like cIL-8, and subsequently decrease during the next three days as the wound closes.[42] Groa and its receptor, CXCR2, are expressed by the keratinocytes at the margins of the wound and by the fibroblasts in the granulation tissue.[43] This pattern of expression is also found in nonhuman chemokines, such as murine KC. Expression increases significantly at four hours after burn injury and remains elevated for at least three days.[44] Similar findings were also obtained with the CC chemokines MCP-1 (CCL2) and MIP-1 (CCL3).[45–47] Functions of cIL-8 in Processes Involved in Wound Healing in vivo Wounding causes release of blood proteins (e.g., fibronectin, prothrombin), the contents of platelet a-granules (e.g. PDGF, TGF-alpha, TGF-beta, PF-4, thromboplastin), and tissue factors (e.g., FGFs) into the area of the wound. Some of these proteins work in concert to form the clot, which is composed primarily of a fibrin-fibronectin meshwork with adherent platelets, whereas others initiate the inflammatory stage of healing. During inflammation, the fibrin-fibronectin meshwork serves as a provisional matrix for the subsequent immigration of monocyte/macrophages, fibroblasts, and epithelial cells. Simultaneously, plasminogen activator is expressed and converts the plasminogen present in the tissues to plasmin, which then digests the blood clot. As healing progresses, the leukocytes and other attracted cells, along with the extracellular matrix molecules produced by fibroblasts, contribute to formation of the granulation tissue, which also contains myofibroblasts, cells important in wound contraction and closure, and newly developing blood vessels that bring nutrients and oxygenate the developing repair tissue.[48] Finally, a healthy granulation tissue leads to formation of a healthy scar by remodeling of the ECM and elimination of excess cells by apoptosis. The author has shown that cIL-8 functions in many of the processes involved in this grand scheme of healing. cIL-8 is chemotactic for leukocytes and fibroblasts, stimulates fibroblast differentiation into myofibroblasts, and is angiogenic. Chemotaxis for leukocytes occurs during the inflammatory phase of healing, which corresponds to the time in which the level of cIL-8 rises rapidly, creating a steep gradient. At later times, accumulation of the protein correlates with the early stages of granulation tissue formation that involves angiogenesis, mitogenesis, and differentiation of fibroblasts into myofibroblasts. Using the chorioallantoic membrane assay (CAM), the author found that cIL-8 is chemotactic for monocytes/macrophages. Chemotaxis for these leukocytes is followed by a wave of lymphocytes and formation of a granulation-like tissue that contains numerous microvessels. The full-length protein can only accomplish the chemotaxis for leukocytes. Our work in the CAM assay also showed that cIL-8 can stimulate blood vessel sprouting.[15] This effect also can be obtained by using the C-terminal 28aa a-helix alone, suggesting that the functional domain for the angiogenic properties is localized to this region of the molecule.[49] More recently, we have shown that hIL-8 is able to cause the same angiogenic effect in the CAM,[50] strongly suggesting that the chicken chemokine functions much like the human chemokine. In support of these findings are observations that hIL-8 binds the chicken CXCR1,[51] the receptor homologue for hCXCR1,[52] which specifically binds hIL-8.[53] Mechanistically, hIL-8 stimulates angiogenesis in two steps: 1) low levels of the chemokine induce endothelial cell de-adhesion and 2) higher levels stimulate production of enzymes that are important in basal lamina degradation.[50] Both of these processes are critical for sprouting angiogenesis.[54] Fibroblasts play key roles in the formation of the repair tissue. Some fibroblasts differentiate into myofibroblasts, cells that produce alpha-smooth muscle actin (alpha-SMA) and are important in wound contraction and closure. The author has shown that cIL-8 stimulates fibroblasts to produce high levels of alpha-SMA and to contract collagen gels more effectively than do normal fibroblasts, both characteristic properties of myofibroblasts. Specific inhibition of a-SMA expression results in abrogation of cIL-8-induced contraction. In addition, application of cIL-8 to wounds in vivo increases the number of myofibroblasts present in the granulation tissue and accelerates wound contraction and closure. Of potential importance for clinical application is the finding that the differentiation-inducing function of cIL-8 is also achieved by a peptide containing the N-terminal 15aa of cIL-8 and that inhibition of alpha-SMA expression results in inhibition of N-peptide-induced collagen gel contraction.[16] The author has also shown that hIL-8 is able to stimulate a-SMA production in human fibroblasts; when applied to excision wounds in chickens, it causes the wounds to contract and close more rapidly.[55] Furthermore, cIL-8 is chemotactic for fibroblasts and accelerates their migration.[56] In addition to the functions described above, cIL-8 also stimulates the production of specific ECM molecules. This chemokine can stimulate precocious deposition of tenascin, fibronectin, and collagen I, but not collagen III, during wound healing in vivo.[56] Studies in vitro and in vivo showed that tenascin expression is stimulated by cIL-8 and that this effect can also be achieved by the N-terminal 15aas. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition to the author’s observations, several other studies have shown that modulating the levels or activity of chemokines during wound healing can affect the overall healing process. For example, transgenic mice that constitutively express IP-10 (CXCL10) in keratinocytes had an abnormal wound healing response characterized by a more intense inflammatory phase and the prolonged presence of disorganized granulation tissue with impaired blood vessel formation.[57] Inactivation studies using neutralizing antibodies against MCP-1 or MIP-2 indicated that sustained expression of these chemokines participated in a prolonged presence of neutrophils and macrophages at the wound site during diabetic repair.[58] In addition, wounds of mice treated with MIP-1a antibodies had significantly fewer macrophages than control, resulting in decreased angiogenic activity and collagen synthesis.[45] Proposed Mechanistic Model of cIL-8 Functions in Wound Healing in vivo As presented above, cIL-8 is highly stimulated by thrombin and is secreted as a 9kDa protein that is then processed by plasmin at the N-terminus giving rise to a 7kDa protein and potentially releasing the N-terminal peptide. The 7kDa protein binds to tenascin, laminin, and interstitial collagen. This collagen, in turn, can stimulate the production of the chemokine. In vivo, cIL-8 can perform numerous functions (Figure 4): 1) It is chemotactic for monocytes, and this requires the full-length molecule; 2) it is angiogenic, and this function can be accomplished by the C-terminus of the protein alone; 3) it stimulates differentiation of fibroblasts into myofibroblasts, and this function can be accomplished by the N-terminus of the protein alone; 4) it stimulates expression of tenascin, and this function can also be accomplished by the N-terminus of the protein; and 5) it stimulates accumulation of fibronectin and collagen I, and these require the full-length protein. Based on these findings, the author hypothesizes that cIL-8 participates in wound healing in the following way (Figure 5): Wound repair is initiated by thrombin. At the same time that thrombin stimulates the clotting cascade, it also stimulates local wound fibroblasts to express cIL-8, leading to secretion of the full-length form of the protein, which contributes to the inflammatory phase of wound healing via chemotaxis for monocyte/macrophages. As inflammation proceeds, endothelial cells and macrophages produce plasminogen activator, which processes plasminogen into plasmin that, in turn, cleaves cIL-8, giving rise to the 7kDa form of the molecule and releasing the N-terminal peptide. This peptide stimulates the production and accumulation of tenascin, a matrix molecule known to facilitate cell migration, especially in conjunction with fibronectin, and stimulates fibroblasts to differentiate into myofibroblasts, which aid in wound contraction and closure. The 7kDa protein binds to tenascin and to interstitial collagen to give rise to a hapoptatic gradient that may be instrumental in migration of fibroblasts and endothelial cells. Furthermore, binding of the smaller form to the matrix provides a way to keep the chemokine localized, thereby favoring blood vessel sprouting to form the microvasculature of the wound bed. Sprout formation requires destabilization of the endothelium followed by basal lamina degradation and endothelial cell migration/proliferation. Based on recent results,51 the author proposes that the initial lower levels of IL-8 that are efficient in chemoattracting leukocytes also stimulate endothelial cell de-adhesion. As the concentration of the chemokine increases, it stimulates localized production of matrix-degrading proteinases that in turn degrade the basal lamina to permit endothelial cell emigration from the existing blood vessel to form the sprout. Summary These previously unknown functions for chemokines suggest that cIL-8 enhances healing by rapidly chemoattracting leukocytes and fibroblasts into the wound site, stimulating the latter to differentiate into myofibroblasts, which are critical for wound contraction and closure, and for the production of ECM molecules, which leads to precocious development of granulation tissue. In addition, stimulation of angiogenesis provides the granulation tissue with healthy vasculature for oxygenation and transport of nutrients. This acceleration of the repair process may have important application to healing of impaired wounds. A potentially very important characteristic of cIL-8 is that it is inducible and, therefore, can be more safely targeted than constitutively expressed molecules, such as many of the better-studied wound factors. In addition, it is a small protein that has no modifications other than two disulfide bonds; therefore, it can be produced and manipulated easily. It also signals through seven-transmembrane receptors, which are prime targets for drug development. Acknowledgments The author is in debt to her postdoctoral and graduate student colleagues as well as to the technicians and undergraduates who have worked on this project over the years. NCI, NIGMS, University of California CRCC, and the University of California, Riverside, supported this work.