The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost

Chemokines are small cytokines primarily known for their roles in inflammation. More recently, however, they have been implicated in processes involved in development of the granulation tissue of wounds, but little is known about their functions during this process. Fibroblasts play key roles in this phase of healing: some fibroblasts differentiate into myofibroblasts, alpha-smooth muscle actin (SMA)-producing cells that are important in wound closure and contraction. Here we show that the CXC chemokine chicken chemotactic and angiogenic factor (cCAF) 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 alpha-SMA expression resulted in abrogation of cCAF-induced contraction. Furthermore, application of cCAF to wounds in vivo increases the number of myofibroblasts present in the granulation tissue and accelerates wound closure and contraction. We also show that these effects in culture and in vivo can be achieved by a peptide containing the NH2-terminal 15 amino acids of the cCAF protein and that inhibition of alpha-SMA expression also results in inhibition of N-peptide-induced collagen gel contraction. We propose that chemokines are major contributors for the differentiation of fibroblasts into myofibroblasts during formation of the repair tissue. Because myofibroblasts are important in many pathological conditions, and because chemokines and their receptors are amenable to pharmacological manipulations, chemokine stimulation of myofibroblast differentiation may have implications for modulation of functions of these cells in vivo.     

The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue

Background  

During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF), is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM) is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules.     

Interleukin-8 of Cynoglossus semilaevis is a chemoattractant with immunoregulatory property

Interleukin-8 (IL-8), or CXCL8, is a member of the CXC chemokine family that in mammals is known to mediate inflammatory response. In this study, we identified and analyzed an IL-8 orthologue, CsIL8, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsIL8 contains 100 residues and is closely related to the lineage 1 IL-8 of a number of fish species. In silico analysis identified in CsIL8 a CXC chemokine domain that contains four conserved cysteine residues, two of which form the CXC signature motif of CXC chemokines. Expression of CsIL8 as determined by quantitative real time RT-PCR was detected in a wide range of tissues under normal physiological conditions and was upregulated by bacterial challenge and by vaccination with a subunit vaccine. Purified recombinant CsIL8 (rCsIL8) induced migration of peripheral blood leukocytes and head kidney (HK) lymphocytes and stimulated the proliferation of these cells in a dose-dependent manner. When rCsIL8 was added to the cell culture of HK lymphocytes, it upregulated the expression of interleukin-1β and CsIL8 in a time-dependent fashion. These results indicate that CsIL8 is a biologically active CXC chemokine with immunoregulatory activity and that CsIL8 is involved in pathogen-induced inflammatory response.     

Molecular cloning and genomic structure of an interleukin-8 receptor-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Chemokines are small proteins (70-100 amino acids) which play an important role in recruitment and activation of leucocytes to migrate to the site of inflammation. Based on the position of the first two conserved cysteines, chemokines are classified into four subfamilies: C, CC, CXC and CX3C. To date, many members of CC and CXC have been found and studied extensively [1]. Chemokines exert effects on their target cell via chemokine receptors, which are G-protein coupled receptors containing seven transmembrane domains with an extracellular N-terminus and an intracellular C-terminus [2]. Interleukin 8 (IL-8) belongs to the CXC chemokine subfamily. It can activate and attract migratory neutrophils to an inflammation site. Two IL-8 receptors, CXCR1 and CXCR2, have been identified in mammals [3-6]; both of these receptors have high affinity for IL-8 and are expressed on the neutrophil. CXCR1 just binds IL-8; however, CXCR2 binds IL-8 and other structurally related chemokines such as growth-related oncogene (GRO) a, GRObeta, GROgamma, neutrophil-activating peptide-2 (NAP-2) and epithelial cell-derived neutrophil activating peptide-78 (ENA-78) [7, 8]. Several studies on fish chemokine receptors have been reported [9-11]. Thus far, however, IL-8 and CXCR1 and CXCR2 proteins from rainbow trout have not been reported: however, the sequence of a rainbow trout IL-8 has been noted (GenBank Accession No. AJ279069 [12]). Cloning of the IL-8 receptor is important to study the function of IL-8/CXCR1 and (CXCR2) in inflammation and signal transduction in fish. This paper reports the molecular cloning and genomic structure of an IL-8 receptor-like gene from four homozygous clones of rainbow trout: Oregon State University (OSU), Hot Creek (HC), Arlee (AR) and Swanson (SW).     

Secretion of oncostatin M by infiltrating neutrophils: regulation of IL-6 and chemokine expression in human mesothelial cells

Recently, we identified that regulation of leukocyte recruitment by IL-6 requires shedding of the IL-6R from infiltrating neutrophils. In this study, experiments have examined whether other IL-6-related cytokines possess similar properties. Levels of oncostatin M (OSM) and leukemia inhibitory factor were analyzed in patients with overt bacterial peritonitis during the first 5 days of infection. Although no change in leukemia inhibitory factor was observed throughout the duration of infection, OSM was significantly elevated on day 1 and rapidly returned to baseline by days 2-3. The source of OSM was identified as the infiltrating neutrophils, and OSM levels correlated both with leukocyte numbers and i.p. soluble IL-6R (sIL-6R) levels. FACS analysis revealed that OSM receptor beta expression was restricted to human peritoneal mesothelial cells. Stimulation of human peritoneal mesothelial cells with OSM induced phosphorylation of gp130 and OSM receptor beta, which was accompanied by activation of STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant protein-1 and IL-6. Although OSM itself did not modulate CXC chemokine ligand 8/IL-8 release, it effectively suppressed IL-1beta-mediated expression of this neutrophil-activating CXC chemokine. Moreover, OSM synergistically blocked IL-1beta-induced CXC chemokine ligand 8 secretion in combination with the IL-6/sIL-6R complex. Thus suggesting that OSM and sIL-6R release from infiltrating neutrophils may contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

Differential regulation of neutrophil-activating chemokines by IL-6 and its soluble receptor isoforms

Interleukin-6 signaling via its soluble receptor (sIL-6R) differentially regulates inflammatory chemokine expression and leukocyte apoptosis to coordinate transition from neutrophil to mononuclear cell infiltration. sIL-6R activities may, however, be influenced in vivo by the occurrence of two sIL-6R isoforms that are released as a consequence of differential mRNA splicing (DS) or proteolytic cleavage (PC) of the cognate IL-6R (termed DS- and PC-sIL-6R). Using human peritoneal mesothelial cells and a murine model of peritoneal inflammation, studies described in this work have compared the ability of both isoforms to regulate neutrophil recruitment. In this respect, DS- and PC-sIL-6R were comparable in their activities; however, these studies emphasized that IL-6 trans signaling differentially controls neutrophil-activating CXC chemokine expression. In vitro, stimulation of mesothelial cells with IL-6 in combination with either DS-sIL-6R or PC-sIL-6R showed no induction of CXC chemokine ligand (CXCL)1 (GRO alpha) and CXCL8 (IL-8), whereas both isoforms enhanced CXCL5 (ENA-78) and CXCL6 (granulocyte chemotactic protein-2) expression. Moreover, when complexed with IL-6, both isoforms specifically inhibited the IL-1 beta-induced secretion of CXCL8. These findings were paralleled in vivo, in which induction of peritoneal inflammation in IL-6-deficient (IL-6(-/-)) mice resulted in enhanced keratinocyte-derived chemokine and macrophage-inflammatory protein-2 (the murine equivalent of CXCL1 and CXCL8) levels, but reduced LPS-induced CXC chemokine (the murine equivalent of CXCL5) expression. Reconstitution of IL-6 signaling in IL-6(-/-) mice with IL-6 and its soluble receptor isoforms corrected this chemokine imbalance and suppressed overall neutrophil infiltration. These data confirm that sIL-6R-mediated signaling primarily limits neutrophil influx; however, induction of CXCL5 and CXCL6 may regulate other neutrophil responses.     

ELR+ CXC chemokines in human milk

CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif are crucial mediators in neutrophil-dependent acute inflammation. Interestingly, however, Interleukin (IL)-8/CXC ligand (CXCL) 8 is expressed in human milk in biologically significant concentrations, and may play a local maturational role in the developing human intestine. In this chemokine subfamily, there are six other known peptides beside IL-8/CXCL8, all sharing similar effects on neutrophil chemotaxis and angiogenesis. In this study, we measured the concentrations of these chemokines in human milk, sought their presence in human mammary tissue by immunohistochemistry, and confirmed chemokine expression in cultured human mammary epithelial cells (HMECs). Each of the seven ELR(+) CXC chemokines was measurable in milk, and except for NAP-2/CXCL7, these concentrations were higher than serum. The concentrations were higher in colostrum (except for GRO-beta/CXCL2 and NAP-2/CXCL7), and correlated negatively with time elapsed postpartum. IL-8/CXCL8, GRO-gamma/CXCL3, and ENA-78/CXCL5 concentrations were higher in preterm milk. There was intense immunoreactivity in mammary epithelial cells for all ELR(+) CXC chemokines, and the intensity of staining was higher in breast tissue with lactational changes. The supernatants from confluent HMEC cultures also contained measurable concentrations of all the seven ELR(+) CXC chemokines. These results confirm that all ELR(+) CXC chemokines are actively secreted by the mammary epithelial cells into human milk. Further studies are needed to determine if these chemokines share with IL-8/CXCL8 the protective effects on intestinal epithelial cells.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

Differential expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cancer cell lines in response to Clostridium difficile toxin A

Intestinal epithelial cells are the initial sites of host response to Clostridium difficile infection and can play a role in signaling the influx of inflammatory cells. To further explore this role, the regulated expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cells were investigated. An expression of the CXC chemokines, including IL-8 and growth-related oncogene (GRO)-alpha, and the CC chemokine monocyte chemoattractant protein (MCP)-1 from HT-29 cells increased in the 1-6 hr following C. difficile toxin A stimulation, assessed by quantitative RT-PCR. In contrast, the expression of neutrophil activating protein-78 (ENA-78) was delayed for 18 hr. The up-regulated mRNA expression of chemokines was paralleled by the increase of protein levels. However, the expression of macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated on activation normal T cells expressed and secreted), and interferon-gamma-inducible protein-10 (IP-10) was not changed in HT-29 or Caco-2 cells stimulated with toxin A. Upon stimulation of the polarized Caco-2 epithelial cells in a transwell chamber with toxin A, CXC and CC chemokines were released predominantly into the basolateral compartment. Moreover, the addition of IFN-gamma and TNF-alpha to toxin A stimulated Caco-2 cells increased the basolateral release of CC chemokine MCP-1. In contrast, IFN-gamma and TNF-alpha had no effect on the expression of the CXC chemokines IL-8 and GRO-alpha. These results suggest that a CXC and CC chemokine expression from epithelial cells infected with C. difficile may be an important factor in the mucosal inflammatory response.     

Gastrin induces CXC chemokine expression in gastric epithelial cells through activation of NF-kappaB

Although hypergastrinemia is frequently observed in individuals with a chronic Helicobacter pylori infection, its pathophysiological significance in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of CXC chemokines in gastric epithelial cells. Human and rat gastric epithelial cells, transfected with gastrin receptor, were stimulated with gastrin. The expression of mRNAs for human interleukin-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant-1 and release of human IL-8 protein were then determined by Northern blot analysis and ELISA, respectively. Gastrin not only induced the expression of mRNAs for these chemokines but also stimulated IL-8 protein release. A luciferase assay using IL-8 promoter genes showed that nuclear factor (NF)-kappaB is absolutely required and activator protein-1 (AP-1) is partly required for the maximum induction of IL-8 by gastrin. An electrophoretic mobility shift assay revealed that gastrin is capable of activating both NF-kappaB and AP-1. In addition, the inhibition of NF-kappaB abrogated gastrin-induced chemokine expression. These results suggest that gastrin is capable of upregulating CXC chemokines in gastric epithelial cells and therefore may contribute to the progression of the inflammatory process in the stomach.     

The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity

We have previously shown that members of the ELR(+) CXC chemokine family, including IL-8; growth-related oncogenes alpha, beta, and gamma; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR(+) CXC chemokines has not been determined. Because all ELR(+) CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR(+) CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR(+) CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR(+) CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR(+) CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR(+) CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2(-/-) mice. We thus conclude that CXCR2 is the receptor responsible for ELR(+) CXC chemokine-mediated angiogenesis.     

Novel chicken CXC and CC chemokines

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.     

Cxc chemokine receptor expression on human endothelial cells.

CXC chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation. However, recent evidence suggests that these molecules can also regulate endothelial cell functions such as migration, angiogenesis and proliferation. In this study we have investigated CXC chemokine receptor expression in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. We found that both cell types express mRNA for chemokine receptors CXCR1, CXCR2 and CXCR4, but not CXCR3. Flow cytometric analysis revealed low levels of CXCR1 but higher levels of CXCR4 cell surface expression. HUVECs responded to SDF-1alpha with a rapid and robust calcium flux, however no calcium flux was seen with either IL-8 or Gro-alpha. HUVECs and ECV304 cells did not proliferate in response to CXC chemokines, although ECV304 cells did migrate towards SDF-1alpha and IL-8. These data demonstrate that HUVECs and the endothelial cell line, ECV304 express functional CXC chemokine receptors.     

Production, purification, and characterization of human recombinant IL-8 from the eucaryotic vector expression system baculovirus.

The cDNA for the human chemotactic interleukin, IL-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene. Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL-8/liter of postinfection cell culture media. The recombinant interleukin was purified (greater than 600-fold) to homogeneity using preparative HPLC. rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8. rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8. Antibody generated against monocyte-derived IL-8 recognized rhIL-8 isolated during all stages of the purification protocol. rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides. rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin. Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production.     

Histamine induces CD86 expression and chemokine production by human immature dendritic cells

Mast cells and immature dendritic cells (DC) are in close contact in peripheral tissues. Upon activation, mast cells release histamine, a mediator involved in the immediate hypersensitivity reaction. We therefore tested whether histamine could affect human DC activation and maturation. Histamine induces CD86 expression on immature DC in a dose-dependent (significant at 10(-7) M) and transient manner (maximal after 24-h stimulation). Histamine also transiently up-regulates the expression of the costimulatory and accessory molecules, CD40, CD49d, CD54, CD80, and MHC class II. As a consequence, immature DC exposed for 24 h to histamine stimulate memory T cells more efficiently than untreated DC. In addition, histamine induces a potent production of IL-6, IL-8, monocyte chemoattractant protein 1, and macrophage-inflammatory protein 1alpha by immature DC and also up-regulates IL-1beta, RANTES, and macrophage-inflammatory protein 1beta but not TNF-alpha and IL-12 mRNA expression. Histamine activates immature DC through both the H1 and H2 receptors. However, histamine-treated DC do not have a phenotype of fully mature cells, as they do neither show significant changes in the expression of the chemokine receptors, CCR5, CCR7 and CXC chemokine receptor 4, nor expression of CD83 de novo. These data demonstrate that histamine activates immature DC and induces chemokine production, thereby suggesting that histamine, via stimulation of resident DC, may participate locally in T cell stimulation and in the late inflammatory reaction associated with allergic disorders.