Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

Pharmacological targeting of anaphylatoxin receptors during the effector phase of allergic asthma suppresses airway hyperresponsiveness and airway inflammation

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.     

Effect of periodontopathogen lipopolysaccharides and proinflammatory cytokines on CD46, CD55, and CD59 gene/protein expression by oral epithelial cells

Membrane-anchored complement regulatory proteins (CRPs), including CD46, CD55, and CD59, protect host cells from complement attack. In the present study, we investigated whether periodontopathogen lipopolysaccharide and proinflammatory cytokines modulate CRP gene/protein expression in human oral epithelial cells. The lipopolysaccharide of Treponema denticola and Tannerella forsythia were the most potent for increasing the gene expression of CD55 and CD59, and to a lesser extent CD46, after a 48-h stimulation. An lipopolysaccharide-induced upregulation of epithelial cell-surface CRP was also demonstrated. The stimulation of epithelial cells with lipopolysaccharide was associated with interleukin-6 (IL-6) and IL-8 secretion. Although these two cytokines had no effect on CD46 and CD55 gene expression in epithelial cells, IL-1β and tumor necrosis factor-α induced a significant upregulation. The cell-surface expression of CRP was also increased by the stimulation of epithelial cells with cytokines. The CD46, CD55, and CD59 gene/protein expression was upregulated by periodontopathogen lipopolysaccharide and proinflammatory cytokines. It can be hypothesized that, when faced with bacterial challenges and inflammatory conditions associated with active periodontal sites, oral epithelial cells may respond by increasing CRP gene/protein expression to avoid cell lysis by the complement system, which is activated during periodontitis.     

Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL-1β

There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, mesenchymal stem cells (MSC), and osteoclasts. qRT-PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane-bound regulatory proteins CD46, CD55, and CD59 were expressed in human MSC, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)-6 and IL-8 from osteoblasts. However, co-stimulation with the pro-inflammatory cytokine IL-1β significantly induced IL-6 and IL-8 expression as well as the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro-inflammatory environment as well as osteoblast-osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage-colony stimulating factor (M-CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro-inflammatory environment, for example, during bone healing or in inflammatory bone disorders.     

Human colonic epithelial cells detect and respond to C5a via apically expressed C5aR through the ERK pathway

Intestinal epithelial cells (IECs) exhibit numerous adaptations to maintain barrier function as well as play sentinel roles by expressing receptors for microbial products and antimicrobial peptides. The complement system is another important innate sensing and defense mechanism of the host against bacteria and increasing evidence shows that complement plays a role in colitis. The split component C5a is a potent proinflammatory molecule, and the C5a receptor (C5aR) CD88 has been reported on multiple cell types. Here, we examined the question of whether human colonic cell lines can detect activated complement via C5aR and what signaling pathway is critical in the subsequent responses. T84, HT29, and Caco2 cell lines all possessed mRNA and protein for C5aR and the decoy receptor C5L2. Polarized cells expressed the proteins on the apical cell membrane. C5a binding to the C5aR on human IECs activates the ERK pathway, which proved critical for a subsequent upregulation of IL-8 mRNA, increased permeability of monolayers, and enhanced proliferation of the cells. The fact that human IECs are capable of detecting complement activation in the lumen via this anaphylatoxin receptor highlights the potential for IECs to detect pathogens indirectly through complement activation and be primed to amplify the host response through heightened inflammatory mediator expression to further recruit immune cells.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

The C5a receptor impairs IL-12-dependent clearance of Porphyromonas gingivalis and is required for induction of periodontal bone loss

The C5a anaphylatoxin receptor (C5aR; CD88) is activated as part of the complement cascade and exerts important inflammatory, antimicrobial, and regulatory functions, at least in part, via crosstalk with TLRs. However, the periodontal pathogen Porphyromonas gingivalis can control C5aR activation by generating C5a through its own C5 convertase-like enzymatic activity. In this paper, we show that P. gingivalis uses this mechanism to proactively and selectively inhibit TLR2-induced IL-12p70, whereas the same pathogen-instigated C5aR-TLR2 crosstalk upregulates other inflammatory and bone-resorptive cytokines (IL-1β, IL-6, and TNF-α). In vivo, the ability of P. gingivalis to manipulate TLR2 activation via the C5a-C5aR axis allowed it to escape IL-12p70-dependent immune clearance and to cause inflammatory bone loss in a murine model of experimental periodontitis. In the latter regard, C5aR-deficient or TLR2-deficient mice were both resistant to periodontal bone loss, in stark contrast with wild-type control mice, which is consistent with the interdependent interactions of C5aR and TLR2 in P. gingivalis immune evasion and induction of bone-resorptive cytokines. In conclusion, P. gingivalis targets C5aR to promote its adaptive fitness and cause periodontal disease. Given the current availability of safe and effective C5aR antagonists, pharmacological blockade of C5aR could act therapeutically in human periodontitis and reduce associated systemic risks.     

Evidence for anti-inflammatory effects of C5a on the innate IL-17A/IL-23 axis

There is growing evidence that the complement activation product C5a positively or negatively regulates inflammatory functions. The studies presented here report that C5a exerts anti-inflammatory effects by altering production of the cytokines IL-17A and IL-23 during endotoxic shock in young adult male C57BL/6J mice and has similar effects on macrophages from the same mice. IL-17A and IL-23 both appeared in plasma during endotoxemia, and their neutralization improved survival. The relevant sources of IL-17A during endotoxemia were not CD4(+) cells, γδ T cells, or NK cells but CD11b(+)F4/80(+) macrophages. The addition in vitro of C5a to lipopolysaccharide-activated peritoneal macrophages dose dependently antagonized the production of IL-17A (IC(50), 50-100 nM C5a) and IL-23 (IC(50), 10 nM C5a). This suppression required the receptor C5aR, but was independent of the second C5a receptor, C5L2. Genetic absence of C5aR was associated with much higher levels of IL-17A and IL-23 during endotoxic shock. Mechanistically, C5a mediated its effects on the IL-17A/IL-23 axis in a 2-step process. C5a caused activation of the PI3K-Akt and MEK1/2-ERK1/2 pathways, resulting in induction of IL-10, which powerfully inhibited production of IL-17A and IL-23. These data identify previously unknown mechanisms by which the anaphylatoxin C5a limits acute inflammation and antagonizes the IL-17A/IL-23 axis.     

Distribution of rat C5a anaphylatoxin receptor

The anaphylatoxin, complement 5a (C5a), plays a key role in mediating various inflammatory reactions following complement activation. Several investigators have reported that C5a receptor (C5aR) is expressed in non-myeloid cells under certain conditions or in different cell lines. In our study, the abundance of C5aR-positive myeloid cells in rats depended on the organs examined. C5aR was usually expressed at the site of exposure to pathogens, such as in salivary gland or lung, and was up-regulated in liver in the inflammatory state induced by lipopolysaccharide (LPS) administration. Furthermore, the increased expression of C5aR antigen was not accompanied by an increase in C5aR mRNA in Kupffer cells following LPS challenge.     

Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1(-/-)) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1(-/-) mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1(-/-) mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.     

Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat

Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.     

Expression and function of C5a receptor in mouse microvascular endothelial cells

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.     

Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C(2) stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) M. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat. Furthermore, in isolated chondrocytes, C(2) stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis.     

EGR1 promotes the cartilage degeneration and hypertrophy by activating the Krüppel-like factor 5 and β-catenin signaling

Osteoarthritis is one of the most common orthopedic diseases in elderly people who have lost their mobility. In this study,we observed abnormally high EGR1 expression in the articular cartilage of patients with osteoarthritis. We also found significantly high EGR1 expression in the articular cartilage of mice with destabilized medial meniscus (DMM)-induced osteoarthritis and 20-month-old mice. In vitro experiments indicated that IL-1β could significantly enhance EGR1 expression in primary mouse chondrocytes. EGR1 over-expression in chondrocytes using adenovirus could inhibit COl2A1 expression and enhance MMP9 and MMP13 expression. And silencing EGR1, using RNAi, had the opposite effects. Moreover, EGR1 over-expression accelerated chondrocyte hypertrophy in vitro, and EGR1 knockdown reversed this effect. We then explored the underlying mechanism. EGR1 over-expression increased Kruppel-Like Factor 5 (KLF5) protein level without influencing its synthesis. Enhanced EGR1 expression induced its integration with KLF5, leading to suppressed ubiquitination of KLF5. Moreover, EGR1 prompted β-catenin nuclear transportation to control chondrocyte hypertrophy. Ectopic expression of EGR1 in articular cartilage aggravated the degradation of the cartilage matrix in vivo. The EGR1 inhibitor, ML264, protected chondrocytes from IL-1β-mediated cartilage matrix degradation in vitro and DMM-induced osteoarthritis in vivo. Above all, we demonstrate the effect and mechanisms of EGR1 on osteoarthritis and provide evidence that the ML264 might be a potential drug for treating osteoarthritis in the future.     

In vitro and in vivo neo-cartilage formation by heterotopic chondrocytes seeded on PGA scaffolds

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3 weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12 weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6 weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12 weeks in vivo construct size reductions were higher compared with 6 weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.     

IFN-gamma and IL-17 production in experimental autoimmune encephalomyelitis depends on local APC-T cell complement production

IFN-gamma- and IL-17-producing T cells autoreactive across myelin components are central to the pathogenesis of multiple sclerosis. Using direct in vivo, adoptive transfer, and in vitro systems, we show in this study that the generation of these effectors in myelin oligodendrocyte glycoprotein(35-55)-induced experimental autoimmune encephalomyelitis depends on interactions of locally produced C3a/C5a with APC and T cell C3aR/C5aR. In the absence of the cell surface C3/C5 convertase inhibitor decay-accelerating factor (DAF), but not the combined absence of DAF and C5aR and/or C3aR on APC and T cells, a heightened local autoimmune response occurs in which myelin destruction is markedly augmented in concert with markedly more IFN-gamma(+) and IL-17(+) T cell generation. The augmented T cell response is due to increased IL-12 and IL-23 elaboration by APCs together with increased T cell expression of the receptors for each cytokine. The results apply to initial generation of the IL-17 phenotype because naive CD62L(high) Daf1(-/-) T cells produce 3-fold more IL-17 in response to TGF-beta and IL-6, whereas CD62L(high) Daf1(-/-)C5aR(-/-)C3aR(-/-) T cells produce 4-fold less.