The International Journal of Biochemistry & Cell Biology
Volume 44, Issue 6, June 2012, Pages 838-841
Molecules in focus
CXCL12: Role in neuroinflammation
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Abstract
CXCL12, also known as SDF-1 (stromal cell derived factor-1) is a small protein that belongs to the chemokine family, whose members have a crucial role in directing cell migration. CXCL12 has an essential role in neural and vascular development, hematopoiesis and in immunity. It acts through two receptors, CXCR4 and CXCR7. While the former is a classic G protein-coupled transmembrane chemokine receptor, the latter primarily function as a scavenger of CXCL12. CXCL12 has been considered as a standard pro-inflammatory molecule for a long time, as it attracts leukocytes to inflammatory sites and contributes to their activation. However, recent findings indicate that this chemokine has the opposite role in neuroinflammation. In this review, basic data about molecular and functional properties of CXCL12 are presented, while its role in CNS autoimmunity is addressed in details.
CXCL12 is a CXC chemokine that traditionally has been classified as a homeostatic chemokine. It contributes to physiological processes such as embryogenesis, hematopoiesis and angiogenesis. In contrast to these homeostatic functions, increased expression of CXCL12 in general, or of a specific CXCL12 splicing variant has been demonstrated in various pathologies. In addition to this increased or differential transcription of CXCL12, also upregulation of its receptors CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) contributes to the onset or progression of diseases. Moreover, posttranslational modification of CXCL12 during disease progression, through interaction with locally produced molecules or enzymes, also affects CXCL12 activity, adding further complexity. As CXCL12, CXCR4 and ACKR3 are broadly expressed, the number of pathologies wherein CXCL12 is involved is growing. In this review, the role of the CXCL12/CXCR4/ACKR3 axis will be discussed for the most prevalent pathologies. Administration of CXCL12-neutralizing antibodies or small-molecule antagonists of CXCR4 or ACKR3 delays disease onset or prevents disease progression in cancer, viral infections, inflammatory bowel diseases, rheumatoid arthritis and osteoarthritis, asthma and acute lung injury, amyotrophic lateral sclerosis and WHIM syndrome. On the other hand, CXCL12 has protective properties in Alzheimer's disease and multiple sclerosis, has a beneficial role in wound healing and has crucial homeostatic properties in general.
The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1α), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1β and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1β. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.
Classical chemotherapeutic anti-cancer treatments induce cell death through DNA damage by taking advantage of the proliferative behaviour of cancer cells. The more recent approach of targeted therapy (usually protein-targeted) has led to many treatments that are currently available or are under development, all of which are designed to strike at the critical driving forces of cancer cells. The interaction of the cancer cells with their microenvironment is one of these fundamental features of neoplasms that could be targeted in such cancer treatments. Haematological and solid tumour cells interact with their microenvironment through membrane chemokine receptors and their corresponding ligands, which are expressed in the tumour microenvironment. Important representatives of this system are the chemokine ligand CXCL12 and its receptor chemokine receptor 4 (CXCR4). This interaction can be disrupted by CXCR4 antagonists, and this concept is being used clinically to harvest haematopoietic stem/progenitor cells from bone marrow. CXCR4 and CXCL12 also have roles in tumour growth and metastasis, and more recently their roles in cancer cell-tumour microenvironment interaction and angiogenesis have been studied. Our review focuses on these roles and summarises strategies for treating cancer by disrupting this interaction with special emphasis on the CXCR4/CXCL12 axis. Finally, we discuss ongoing clinical trials with several classes of CXCR4 inhibitors, and their potential additive value for patients with a (therapy resistant) malignancy by sensitising cancer cells to conventional therapy.
The subventricular zone (SVZ) of the adult mammalian brain hosts full potential neural stem cells (NSCs). NSCs are able to respond to extracellular signals in the brain, amplifying the pool of progenitor cells and giving rise to neuroblasts that show ability to migrate towards an injury site. These signals can come from vascular system, cerebrospinal fluid, glial cells, or projections of neurons in adjoining regions. CXCL12, a chemokine secreted after brain injury, reaches the SVZ in a gradient manner and drives neuroblasts towards the lesion area. Among many other molecules, matrix metalloproteinase 2 and 9 (MMP-2/9) are also released during brain injury. MMP-2/9 can cleave CXCL12 generating a new molecule, CXCL12(5-67), and its effects on NSCs viability is not well described. Here we produced recombinant CXCL12 and CXCL12(5-67) and evaluated their effect in murine adult NSCs migration and survival in vitro. We showed CXCL12(5-67) does not promote NSCs migration, but does induce cell death. The NSC death induced by CXCL12(5-67) involves caspases 9 and 3/7 activation, implying the intrinsic apoptotic pathway in this phenomenon. Our evidences in vitro make CXCL12(5-67) and its receptor potential candidates for brain injuries and neurodegeneration studies.
Burn wound healing is a complex process consisting of an inflammatory phase, the formation of granulation tissue, and remodeling. The role of the CXCL12/CXCR4 pathway in the recovery of skin following burns is unknown. We found that CXCL12 is similarly expressed in human, swine, and rat skin by pericyte and endothelial cells, fibrous sheet, fibroblasts, and axons. Following burns, the levels of CXCL12 were markedly increased in human burn blister fluids. One day after injury, there was a gradual increase in the expression of CXCL12 in the hair follicles and in blood vessel endothelium surrounding the burn. Three to 11 days following burns, an increased number of fibroblasts expressing CXCL12 were observed in the recovering dermis of rat, swine, and human skin. In contrast to CXCL12, CXCR4 expression was detected in proliferating epithelial cells as well as in eosinophils and mononuclear cells infiltrating the skin. In vitro, CXCL12 was expressed by primary human skin fibroblasts, but not by keratinocytes, and was stimulated by wounding a confluent cell layer of these fibroblasts. Blocking the CXCR4/CXCL12 axis resulted in the significant reduction in eosinophil accumulation in the dermis and improved epithelialization. Thus, blocking CXCR4/CXCL12 interaction may significantly improve skin recovery after burns.
INTRODUCTION
Skin integrity is of importance for the protection and separation of body tissues from the surrounding environment. The loss of skin due to burns or trauma exposes the body to severe stress, impairing or even eliminating the many vital functions this organ performs (Clark, 1988; Cotran et al., 1999). Full-thickness skin tissue is comprised of keratinocytes lined on a basement membrane, produced by fibroblasts and keratinocytes. Deeper layers of the skin include, in addition to fibroblasts, fat cells and multiple subsets of immune cells such as dendritic cells, lymphocytes, and polymorphonuclear cells. The complex organization of normal skin is designed to support the numerous functions of this organ as both an immunologic and a physical barrier. Nevertheless, not much is known about the factors responsible for the complex architecture of this organ under physiologic and pathologic conditions.
Stromal-derived factor-1 (CXCL12) controls many aspects of stem cell function. CXCL12 has been identified as a powerful chemoattractant for immature hematopoietic stem cells (Aiuti et al., 1997). Mice that lack either CXCL12 or its receptor CXCR4 exhibit many defects, including impaired hematopoiesis in the fetal bone marrow (Nagasawa et al., 1996; Ma et al., 1998; Zou et al., 1998; McGrath et al., 1999). Recently, it was shown that mobilization, homing, and engraftment of hematopoietic stem cells as well as the trafficking of neuronal and primordial germ cells are dependent on the expression of CXCL12 and CXCR4 (Peled et al., 1999; Doitsidou et al., 2002). Furthermore, it was also shown that the expression of CXCL12 is upregulated following irradiation and hypoxia and that CXCL12 can induce the recruitment of endothelial progenitor cells in a regeneration model for myocardial infarction (Ponomaryov et al., 2000; Askari et al., 2003; Ceradini et al., 2004). The regulation of CXCL12 and its physiological role in peripheral tissue repair remain incompletely understood. A recent study showed that CXCL12 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 in endothelial cells, resulting in the selective in vivo expression of CXCL12 in ischemic tissue in direct proportion to reduced oxygen tension (Hitchon et al., 2002; Schioppa et al., 2003). Hypoxia-inducible factor-1-induced CXCL12 expression was suggested to increase the adhesion, migration, and homing of circulating CXCR4-positive progenitor cells to ischemic tissue.
Thus, CXCL12 plays an important role in the organization of tissues during development and following damage. CXCL12 is expressed by dendritic cells, fibroblasts, and endothelial cells in human skin (Pablos et al., 1999). Here, we show that following burns, the levels of CXCL12 is markedly increased first in the burn blister and then in the junction tissues surrounding the burn, hair follicles, endothelium blood vessels and fibroblasts in the recovering dermis. Treatment of partial thickness burns in a rat model with antibodies to CXCR4 or the small peptide CXCR4 antagonist, 4F-benzoyl-TN14003 (Tamamura et al., 2003), resulted in improved epithelialization and reduced eosinophilia. These observations suggest a role for eosinophils and the CXCL12/CXCR4 pathway in wound healing and in the recovery of burn skin.
RESULTS
CXCL12 is similarly expressed in human, swine, and rat normal skin
The expression of CXCL12 in normal skin was examined by immunohistochemical staining using monoclonal antibody (mAB) (MAB 350) (R&D Systems Inc., Minneapolis, Minnesota) against the chemokine CXCL12. We first examined the antibody crossreactivity of CXCL12 staining on liver sections of mouse, human, and rat, since previous studies have shown that CXCL12 is specifically expressed in the bile ducts and blood vessels of human liver (Wald et al., 2004). In mouse, human, and rat, liver bile ducts were specifically stained with MAB 350 for CXCL12 (data not shown). Control stain without the primary Ab showed no staining. Using the same mAB staining for CXCL12 in human, swine, and rat, normal skin showed similar expression patterns. CXCL12 in human normal skin (Figure 1) was detected in the basal layer of the epidermis (Figure 1a), on scattered cells in the papillary dermis (Figure 1b), in pericytes, and in the endothelial layer of blood vessels (Figure 1c). The fibrous sheet of hair follicles (Figure 1d), sweat glands (not uniformly) (Figure 1e), axons, and small blood vessels in the nerve tissue (Figure 1f) also expressed CXCL12. No staining was detected with a control antibody used to stain identical skin sections (Figure 1g–h). CXCL12 was detected in rat normal skin on the basal layer of the epidermis (Figure 2a), on scattered cells in the papillary dermis (Figure 2b), and on pericytes and the endothelial layer of blood vessels (Figure 2c). The chemokine CXCL12 was also expressed by axons and small blood vessels in the nerve tissue (Figure 2d) and by fibrous sheet of hair follicles (Figure 2e). No staining was detected with control antibody used to stain the same skin sections (Figure 2f). The chemokine CXCL12 was similarly expressed by swine normal skin cells in the basal layer of the epidermis (Figure 2g), by scattered cells in the papillary dermis (Figure 2h), in pericytes, and by the endothelial layer of blood vessels (Figure 2i). The chemokine CXCL12 was also expressed by fibrous sheets of hair follicles (Figure 2j) and by sweat glands (Figure 2k). No staining was detected with control antibody used to stain the same skin sections (Figure 2l). This unique and conserved expression pattern of CXCL12 may suggest a role for CXCR4/CXCL12 axis in the organization of skin tissue.

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Figure 1. Expression of CXCL12 in normal human skin. Immunohistochemistry staining results using a monoclonal antibody against the chemokine CXCL12 on human normal skin section. (a) Stained cells in the basal layer of the epidermis. (b) Scattered cells stained in the papillary dermis. (c) Endothelial cells and pericytes stained in blood vessel. (d) Fibrous sheet stained in the hair follicle. (e) Sweat glands not uniformly stained. (f) Axons and blood vessels stained in nerve tissue. (g) Sections of epidermis and papillary dermis were stained without the primary antibody ensuring that no background staining was received from the second antibody. (h) Sections of epidermis and papillary dermis were stained with the primary antibody after incubation with CXCR4 ligands CXCL12α and CXCL12β ensuring that the staining is specific for CXCL12. (Original magnification × 400).

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Figure 2. Expression of CXCL12 in rat and swine normal skin. (a–f) Immunohistochemical staining results using monoclonal antibody against the chemokine CXCL12 on rat normal skin sections. (a) Cells stained in the basal layer of the epidermis. (b) Scattered cells stained in the papillary dermis. (c) Endothelial cells and pericytes stained in blood vessel. (d) Axons and blood vessels stained in nerve tissue. (e) Fibrous sheet stained in the hair follicle. (f) Epidermis and papillary dermis control staining, without the primary antibody (original magnification of × 400). (g–l) Immunohistochemical staining results using monoclonal antibody against the chemokine CXCL12 on swine normal skin sections. (g) Cells stained in the basal layer of the epidermis and the papillary dermis. (h) Scattered cells stained in the papillary dermis. (i) Endothelial cells and pericytes stained in blood vessel. (j) Fibrous sheet stained in the hair follicle. (k) Sweat glands staining. (l) control staining, without the primary antibody. (Original magnifications × 200 or × 400).
Following burns, the level of CXCL12 was markedly increased in human burn blister fluids, hair follicles, blood vessels endothelium, and fibroblasts in the recovering dermis of rat, swine, and human skin.
In order to study the effect of burn injury on CXCL12 expression in the skin, we first collected burn wound fluids, and CXCL12 levels were measured by ELISA assay and compared to the levels of IL-8 (Figure 3a and b). The results indicate a unique pattern of the chemokine CXCL12 expression compared to the IL-8. IL-8 appeared first in the burn fluid a few hours after injury, reached a plateau level after 1 day, and remained at the same level for the next 4 days. CXCL12 appeared a few hours after injury, reached a plateau level after 1 day, and remained at the same level for an additional 2 days, and then the level of CXCL12 decreased exponentially. The consistent overexpression of IL-8 in burn wound fluids and skin tissue has also been reported by others (Iocono et al., 2000). These authors suggested that IL-8 has a role in stimulating neutrophils migration and accelerating the angiogenic process within the burn wound.

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Figure 3. (a) IL-8 and (b) CXCL12 mean levels (pg/ml) in human burn wound fluid collected from blisters of patients with second degree burn. Fluids were collected 0–5 days after burn as a medical treatment protocol. Samples were measured for the chemokines by ELISA assays. Each point represents one patient.
The expression of the chemokine CXCL12 following burn infliction was further examined by immunohistochemical staining of rat skin sections. The results shown in Figure 5 indicate accumulation of CXCL12 in the rat burned skin in correlation with time. Six days and 1 day after injury, CXCL12 was not detected in the burned tissue. Three days postburn, CXCL12 was detected in endothelium blood vessels, in the hair follicles, and also in scattered cells accumulated in the dermis. Five and 7 days postburn, a higher expression of the chemokine was detected in blood vessels and in fibroblast-like cells accumulated in the dermis. As was shown before for human normal skin, CXCR4 was detected in normal and proliferating rat epithelial cells and endothelial cells after burn injury (Figure 4). In the dermis of injured skin, CXCR4 expression was also detected in mononuclear cells as well as infiltrating eosinophils.
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Figure 4. The involvement of CXCL12 in rat and swine burn wound healing. Expression of CXCL12 in rat burn wound healing. (a) Immunohistochemical staining of CXCL12 of rat epidermis burned skin sections, at 6 hours, 1 day, 3 days, 5 days, and 7 days after the burn. Staining for CXCR4 is shown at 7 days after the burn (original magnifications × 100, × 200). (b) Immunohistochemical staining of CXCL12 of rat dermis burned skin sections, at 6, 72, and 120 hours after the burn (original magnifications × 100, × 200). (c) Expression of CXCL12 in swine skin after second-degree burn. Immunohistochemical staining of CXCL12 at 4 days, 10 days after the burn, and at time 0 in normal skin. (Original magnifications × 100, × 400).
The pattern of CXCL12 expression in swine skin postburn is similar. Four days postburn, CXCL12 was present in endothelium blood vessels and in scattered cells that accumulated in the papillary dermis. Ten days after injury, a strong expression of the chemokine was detected in blood vessels and in the accumulating fibroblast-like cell population in the papillary dermis of normal skin stained for CXCL12 is shown in Figure 5c.

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Figure 5. Coexpression of vimentin and CXCL12 in rat burned skin. (a) Immunohistochemical staining for vimentin of rat burned skin dermis 5 days after burn. (b) CXCL12 immunohistochemical staining of consecutive tissue section of rat burned skin section 5 days after burn (Original magnification × 400; arrow indicates the staining for CXCL12 and vimentin). Coexpression of GPF and CXCL12 in heterozygous mice bearing a GFP reporter knocked-in to the CX3CR1 locus burned skin. (c and d) Accumulation of GFP+ monocyte/dendritic cells in the dermis of injured skin. (e–h) Coexpression of GFP+ CXCL12 in monocyte/dendritic cells in parallel sections from dermis of injured skin; arrow indicates the staining for CXCL12 and GFP.
In order to determine the cell types that expressed high levels of CXCL12, we stained parallel sections from burned skin for vimentin and CXCL12. The majority of fibroblast-like cells were stained for both CXCL12 and vimentin indicating that fibroblasts were expressing CXCL12 in the skin following burn injury (Figure 5a and b). However, part of the cells that expressed CXCL12 did not express vimentin. CXCL12 was shown to be expressed by human dendritic cells localized to the epidermis and the dermis (Pablos et al., 1999). An excellent means to track monocyte subsets in the skin was through the use of mice bearing a green fluorescent protein (GFP) reporter knocked-in to the CX3CR1 chemokine receptor locus (Qu et al., 2004). Indeed, we found that following injury, monocyte with a dendritic-like shape accumulated in the dermis and epidermis (Figure 5c and d). Part of the monocyte/dendritic cells that expressed the GFP also expressed CXCL12 (Figure 5e–h).
To further study the expression of CXCL12 and CXCR4 in the skin, we used primary skin fibroblast and keratinocyte cultures. In agreement with our in vivo results, we found that while the fibroblasts expressed the chemokine CXCL12 in the mRNA level, the keratinocytes did not. In contrast to CXCL12, keratinocytes, but not the fibroblasts, expressed the receptor CXCR4. In order to verify our finding, we used ELISA assay to check the production of CXCL12 by keratinocytes and fibroblasts. The results demonstrated that while keratinocytes did not express the chemokine CXCL12 at the protein level, fibroblasts did express and secrete CXCL12 (Figure 6a), especially during the recovery of skin fibroblasts migrating into the wound area and accumulating in the dermis. In order to study the effect of wounding on CXCL12 expression by skin fibroblasts, a "scratching" assay was performed on confluent layers of human skin fibroblasts in vitro. Immunohistochemical staining of confluent human skin fibroblasts showed moderate CXCL12 expression. Two days following scratching, an increase in CXCL12 expression by cells adjacent to the affected area was detected (Figure 6d). Fibroblast monolayers were negatively stained with control antibody against cytokeratin.

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Figure 6. CXCL12 is expressed in primary cultures of fibroblasts, but not in keratinocytes. (a) In vitro expression of CXCL12 and CXCR4 in keratinocytes and fibroblasts. Expression of CXCL12 and CXCR4 measured by RT-PCR in primary human keratinocytes and fibroblasts. Expression of CXCL12 in keratinocytes- and fibroblasts-conditioned medium as measured by ELISA assay. (b) Immunostaining of human primary fibroblasts with anti-cytokeratin antibodies as control. (c) Immunostaining of human primary fibroblasts with anti CXCL12 antibodies. (d) Immunostaining of human primary fibroblasts with anti-CXCL12 of human primary fibroblast 2 days after wounding the fibroblasts monolayer. (Original magnifications × 40 and × 200). Fib.=fibroblasts; Ker.=keratin.
Inhibition of the CXCL12/CXCR4 pathway resulted in reduced eosinophil accumulation and improved epithelialization
In order to evaluate the effect of the CXCR4 antagonist, 4F-benzoyl-TN14003, and neutralizing antibodies to the receptor on the recovery of rat skin, we first tested their ability to inhibit the migration of rat lymphocytes in response to CXCL12. Migration assay was carried out on total rat lymphocytes separated by Ficoll gradient, and their migrating ability to medium containing CXCL12 was examined. Lymphocytes were incubated with the CXCR4 antagonist, 4F-benzoyl-TN14003, and an antibody against CXCR4. Treatment of cells with 4F-benzoyl-TN14003 exerted a strong inhibitory effect, whereas treatment of cells with neutralizing antibodies to CXCR4 exerted moderate effect on the migration of cells in response to CXCL12 (Figure 7a).

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Figure 7. Effect of neutralizing antibodies to CXCR4 and the small peptide inhibitor of CXCR4 on inflammation and regeneration of skin following burns. (a) Migration of rat lymphocytes in response to CXCL12 (100 ng/ml) was tested in the absence and presence of neutralizing antibodies to CXCR4 (NA), or the CXCR4 antagonist – 4F-benzoyl-TN14003. (b) The effect of CXCR4 antagonists on re-epithelialization. (c) Depicts the epithelialization of burned skin in mice treated with phosphate-buffered saline (PBS) (asterisk indicates the sites of novel epithelialization). (d) Depicts the epithelialization of burned skin in mice treated with 4F-benzoyl-TN14003 (asterisk indicates the sites of novel epithelialization). The number of lymphocytes in the dermis is shown in (e). The number of polymorponuclear cells (PMN) in the epidermis and eosinophils in the dermis 5 days after burn is shown in (f). (g) Depicts the eosinophils in the dermis of mice treated with PBS (arrow indicates the site of eosinophilia). (h) Depicts the eosinophils in the dermis of mice treated with 4F-benzoyl-TN14003 (arrow indicates the site of eosinophilia). The results are the average of two experiments; for each experiment at least five rats were tested (P<0.05).
Next, we examined the inhibitory effect of CXCR4 antagonists on burn wound healing (Figure 7b–d). Inhibitors were injected subcutaneously to the burned area at 0, 1 day, and 3 days, and animals were killed 5 days postburn. Animals injected with the CXCR4 inhibitor, 4F-benzoyl-TN14003, showed an increased epithelialization (Figure 7b). A small but not significant decrease in the polymorphonuclear cell population in the dermis was observed (Figure 7c). However, a strong and significant inhibition in eosinophil accumulation in the dermis was found in the 4F-benzoyl-TN14003 and antibodies to the CXCR4-treated groups (Figure 7d). In contrast to eosinophil accumulation, the accumulation of polymorphonuclear cell population in the epidermis was not affected (Figure 7d). These results suggest a role for CXCR4/CXCL12 interaction in the migration of eosinophils to the skin in the process of epithelialization following burn inflection.
DISCUSSION
Burn wound healing is a complex process consisting of an early phase of energy depletion and necrosis, followed by a two-stage inflammatory phase, formation of granulation tissue, matrix formation, and remodeling (Clark, 1988; Cotran et al., 1999; Spies et al., 2002). The numerous cellular and humoral interactions during these phases of thermal wound healing are complex and not well understood. Partial skin burn wounds could be more effectively treated sooner if the blister wall was maintained intact (Ono et al., 1995). Burn wound fluids from blisters contain relatively large amounts of cytokines such as platelet-derived growth factor, IL-6, transforming growth factor-β, and IL-8 thought to stimulate the wound healing process by regulating epithelialization (Ono et al., 1995; Struzyna et al., 1995). The increased CXCL12 levels in human burn fluid during the first 3 days following burn injury (Figure 3b) and the expression of CXCR4 by human keratinocytes (Figure 4a) may support the survival and tissue organization of these cells. This concept is supported by studies showing that CXCR4 is expressed by skin keratinocytes and is essential for keratinocytes that participate in maintaining skin integrity (Smith et al., 2004). The restricted presence of functional CXCL12 (24–48 hours following burn) may suggest a protective role for CXCL12 in the maintenance of skin tissue following burn. In contrast to the presence of CXCL12 in human burn fluid during the first 3 days following injury, an increased number of fibroblasts and dendritic cells that expressed CXCL12 are observed in the regenerating skin in the first 2 weeks following damage. This difference may be the result of increased levels of proteolytic activity in the burn fluid. Indeed, a variety of proteolytic enzymes such as cathepsin G, elastase, and matric metalloproteinase-9 were recently shown to degrade CXCL12 (Petit et al., 2002).
In partial-thickness burns, the epidermis and the superficial dermis are destroyed and undergo necrosis (Clark, 1988; Cotran et al., 1999; Singer and Clark, 1999). Twenty-four hours to 2 days following burns, the affected area lost CXCL12 expression. However, the expression of CXCL12 in the area adjacent to the burn wound was intensed. During this time period, a massive influx of neutrophils into the wound area was observed. The accumulation of neutrophils could not be blocked by CXCR4 antagonists, suggesting that CXCR4/CXCL12 axes have no detectable role in this process. The accumulation of neutrophils in the wound area was associated with an increased production of the neutrophil chemoattractants neutrophil activating protein-2 (NAP-2), Growth-Regulated Oncogene alpha (GRO-α), and Epithelial neutrophil activating peptide-78 (ENA-78), as well as with the sustained production of IL-8 in human burn blisters in human (Figure 3a) (Faunce et al., 1999; Piccolo et al., 1999; Gillitzer and Goebeler, 2001). In partial-thickness burns, the proliferating and migrating epithelium arose from the wound border as well as from hair follicles. The rate of epithelial cover was modulated by growth factors that stimulated the proliferation and chemotaxsis of epithelial cells (Clark, 1988; Cotran et al., 1999; Singer and Clark, 1999). During the granulation phase, beginning 2–3 days following damage, fibroblasts attracted by macrophages migrated into the wound area; these fibroblasts from swine and rat origin secreted high levels of CXCL12 (Figures 4 and 5). The process of granulation is associated with intense angiogenesis (Cotran et al., 1999; Singer and Clark, 1999). In parallel to migration of fibroblasts expressing high levels of CXCL12, novel and resident endothelial cells lining the blood vessels also expressed CXCL12. During this phase of wound healing, a second wave of immune cells entered the epidermis underlying the burn. These cells include macrophages, lymphocytes, and eosinophils. The chemokines CCL2, CXCL10, CXCL9, and CCL22 were found to be spatially associated with lymphocyte and monocyte accumulation (Gibran et al., 1997; Gillitzer and Goebeler, 2001). We found a minor effect of CXCR4 antagonists on the recruitment of macrophages and lymphocytes, whereas the recruitment of eosinophils was totally blocked (Figure 7).
A fine balance between fibrotic tissue deposition and neovascularization on the one hand and fibrotic tissue degradation and epithelization on the other should be maintained in order to assure successful wound healing. Immune cell subpopulation recruited to the burned site is involved in orchestrating these events. Unbalanced proliferation and activation of fibroblasts may lead to inadequate granulation and the formation of a fibrotic tissue. However, reduced angiogenesis and blood flow into the burn wound can prevent successful epithelialization and wound repair. With regard to the CXCL12/CXCR4 axis, we have found that the most dramatic effect of CXCR4 antagonists was on the number of infiltrating eosinophils. The decrease in eosinophil migration into the wounded tissue and the increased epithelialization observed in mice treated with CXCR4 antagonist indicate that CXCL12/CXCR4 interactions are involved in shaping the balance between fibrosis and epithelialization. Moreover, these data may suggest that eosinophils are linked to the regulation of epithelialization.
Indeed, it was reported by Yang et al. (1997) that anti-interleukin-5 mAB (TRFK-5) treatment can deplete eosinophils in healing of cutaneous wounds and that wound closure by re-epithelialization in the treated animals was 4 days faster than in the control group. This study suggests a role for eosinophils in negatively affecting wound re-epithelialization. Neutralizing antibodies to CXCR4 and AMD3100, an antagonist of CXCR4/CXCL12 interaction, were shown to reduce lung eosinophilia, indicating that CXCR4-mediated signals contribute to lung inflammation in a mouse model of allergic airway disease (Gonzalo et al., 2000; Lukacs et al., 2002). Eosinophils constitutively express CC chemokine receptor 3 and, to a lesser extent, CC chemokine receptor 1. CC chemokine receptor 3 is mainly responsible for migration of resting eosinophils, and its specific ligand, eotaxin, represents the most potent chemoattractant for eosinophils (Nagase et al., 2001b). However, eosinophils in inflamed tissue sites exhibited a decreased CC chemokine receptor 3 and an increased CXCR4 expression (Nagase et al., 2001a). Surface CXCR4 protein was hardly detectable in the peripheral blood or freshly isolated eosinophils. Similarly to the phenomenon observed with eosinophils in inflamed tissues, surface expression of CXCR4 became gradually apparent during in vitro incubation of cells. CXCL12, the natural ligand of CXCR4, elicited an apparent Ca2+ influx in these cells and induced a strong migratory response comparable to that by eotaxin (Nagase et al., 2000)
In summary, we suggest that the presence of CXCL12 in burn blisters is involved in protecting the skin during a short period of time following skin burn injury. Thereafter, CXCL12 expressed by fibroblasts and endothelial cells may induce the accumulation of eosinophils, which in turn slow the epithelialization. Our data suggest that CXCL12 is more predominantly supporting fibrosis than epithelialization. Indeed, we and others have recently shown that during liver fibrosis, the levels of CXCL12 expression by endothelial cells and fibroblasts are dramatically increased (Wald et al., 2004). It is therefore possible that by using inhibitors against CXCR4, the balance between fibrosis and epithelialization can be changed, thereby leading to a better and faster recovery of skin following damage.
MATERIALS AND METHODS
Human cell lines
Human skin fibroblasts and keratinocytes were obtained from skin biopsies. Fibroblasts were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum, and keratinocytes were cultured in H. Green keratinocyte-specific medium (Green et al., 1979). Keratinocytes and fibroblasts were kindly provided by Professor Ben-Basst Laboratory, Hadassah University Hospital, Jerusalem, Israel. Cells were passaged weekly by trypsinization. Bone marrow endothelial cells are microvascular endothelial cells isolated from human bone marrow aspirates. The bone marrow endothelial cell-1 cell line was kindly provided by S Rafii. This cell line was generated by introducing the SV40-large T antigen into an early passage of primary bone marrow endothelial cell, and it has retained the morphology, phenotype, and function of the primary bone marrow endothelial cell. The bone marrow endothelial cell-1 cells were cultured in a complete DMEM and were passaged weekly by trypsinization.
Immunohistochemistry and in vitro scratched assay
Skin tissue samples were routinely fixed with formalin and embedded in paraffin. Antigen retrieval was performed in ethylenediaminetetra-acetic acid buffer for 15 minutes in microwave, and sections were stained with mAB (MAB 350) (R&D Systems Inc., Minneapolis, MN) for CXCL12 (1:100) mAB′ against cytokeratins 1, 5, 15, and 10 (M0630) (DAKO, Glostrup, Denmark) and mAB anti-vimentin (M7020) (DAKO), mAB against rat CXCR4 (Torrey Pines Biolabs, Houston, TX), using biotinylated secondary polymer (87–9,963) (Zymed) based on standard indirect avidin–biotin horseradish peroxidase method, according to the manufacturer's instructions. 3-amino-9-ethylcarbazole was used for color development and sections were counterstained with hematoxylin. Cell immunohistochemistry staining: keratinocyte and fibroblast monolayers were grown in a tissue culture 6 mm plates. Then, cells were scratched using a 200 μl pipette, washed three times with phosphate-buffered saline (PBS), and grown in DMEM with 1% fetal calf serum. After 2 days, cells were fixed using 4% paraformaldehyde and stained for the chemokine CXCL12 as described previously.
ELISA assay and RT-PCR
ELISA assays for CXCL12 and IL-8 in burn fluids of fibroblast and keratinocyte medium were performed using the Quantikine kit (R&D Systems Inc., Minneapolis, Minnesota), according to the manufacturer's instructions. The expression levels of the chemokine CXCL12 and the chemokine receptor CXCR4 were determined by RT-PCR analysis. Total RNA was isolated from primary fibroblast and keratinocyte cultures. Each RNA sample was subjected to cDNA synthesis, and then semiquantative PCR was performed with specific primers at appropriate annealing temperatures. The resulting PCR products were separated on 1% agarose gel.
Transwell migration assays
Rat peripheral blood cells were loaded on Ficoll (Histopaque-1077-1, Sigma), and the peripheral blood mononuclear cells were isolated. Rat peripheral blood mononuclear cell migration was assessed in 24-well chemotaxis chambers (6.5-mm diameter, 5-mm pore polycarbonate transwell culture insert; Costar, Cambridge, Massachusetts). RPMI 1640 (600 μl) with 1% BSA (migration buffer) with or without 100 ng/ml of CXCL12α (Peprotech, Rehovot, Israel) were added to the lower wells, and 2 × 105 cells suspended in 100 μl of RPMI 1640 with 1% BSA were added to the upper wells. After 3 hours incubation, the membrane was removed and migrating cells were counted for 1 minute using fluorescence activated cell sorter.
Tissue collection, histological evaluation of the burn lesion
Human skin tissue samples were obtained from the Plastic Surgery Department, Souraski Medical Center, Tel-Aviv. In order to examine the different phases in burn wound healing and the involvement of the chemokine CXCL12 and the receptor CXCR4, the following experiments were carried out. Wistar female rats were anesthetized and their back was shaved. Burns (1 cm2) were inflicted with a metal rod that has been immersed in a hot boiling water bath and laid on the posterior part of the hip and back for 2–3 seconds. All experiments were approved by the Animal care committee of the Medical Center, Tel-Aviv and the Hebrew University. All the material collected from human specimens was approved by Tel-Aviv Sourasky Medical Center Institutional Committee and in adherence to the Declaration of Helsinki Principles. Heterozygous mice bearing a GFP reporter knocked-in allele to the CX3CR1 locus (Qu et al., 2004) were maintained at the Weizmann Institute of Science Animal Facility. Each group of three rats was injected subcutaneously with either PBS, anti-CXCR4, or with 4F-benzoyl-TN14003 to the burned area. Animals were killed at the indicated times after burn infliction: 0, 6 hours, 1 day, 3 days, 5 days, and 7 days. Histopathological diagnosis was confirmed for each specimen. Histological sections were prepared from formalin-fixed, paraffin-embedded tissues stained with hematoxylin and eosin. The evaluation to the level of epithelialization and white blood cells in the epidermis and dermis was made to each section by a scale from 1 to 5. The sections were scored by two independent pathologists. Treated rats were injected subcutaneously to the burned area with one of the following: PBS, mAB against rat CXCR4, the small peptide CXCR4 inhibitor, or 4F-benzoyl-TN14003. All animals were killed 120 hours after injury. Histopathological diagnosis was confirmed for each specimen. Histological sections were prepared from formalin-fixed, paraffin-embedded tissues stained with hematoxylin and eosin. The evaluation to the level of epithelialization and white blood cells in the dermis was made to each section by a scale from 1 to 5. The grading scale was as follows: 0=no inflammation or epithelialization; 1=low inflammation or epithelialization; 2=low to moderate inflammation or epithelialization; 3=moderate inflammation or epithelialization; 4=high inflammation or epithelialization; and 5=very high inflammation or epithelialization.
Swine burned skin paraffin-embedded sections were provided by the Laboratory of Experimental Surgery, Hadassah University Hospital, Jerusalem, Israel. All experiments were approved by the Animal Care Committee of the Hebrew University. Burn blister fluid collection was collected at the Plastic Surgery Department, Souraski Medical Center, Tel-Aviv, Israel as a medical procedure. The blister fluid was examined for the chemokine CXCL12 levels by ELISA assay, and for the levels of IL-8.
Statistical analysis
Results are expressed as mean±SD. Statistical differences were determined by an analysis of two-tailed Student's t-test.
ACKNOWLEDGMENTS
We thank Mery Clausen (Gene Therapy Institute, Hadassah Hospital) for technical assistance. This study was supported by the Horwitz Foundation, the Israeli Ministry of Science – the Knowledge Center for Gene Therapy, the Blum Foundation, and the Grinspoon Foundation.
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The authors state no conflict of interest.
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