Anaphylatoxin Receptor Research Articles

The Membrane Attack Complex of complement is required for the development of murine Experimental Cerebral Malaria (112.2)

Abstract Cerebral malaria (CM) is a severe complication of P. falciparum malaria with a 15-30% fatality and 10% of CM survivors have permanent neurological sequelae. Since inflammation is key to CM development, we examined the role complement in experimental cerebral malaria (ECM) development using the P. berghei model. Previous studies indicated that C5-/- mice were protected in ECM. To determine the role of complement activation, we examined mice deficient in pathway specific components, C4 and Factor B, as well as C3, which forms the C5 convertase. We found no differences in survival between wild type (WT) and C4 or Factor B deficient mice. We observed moderate protection in C3-/- mice, suggesting that blocking C5 cleavage is important in ECM development. We found that complement anaphylatoxin receptor deficient mice were not protected, suggesting that the mechanism of protection in C5-/- mice is not mediated through C5a-induced inflammation. We next examined C5b indirectly by treating WT mice with anti-C9 antibody and found significant increase in survival. We also treated C3-/- mice with anti-C9 antibody to determine if blocking MAC in these mice could abrogate disease, and found significant protection in treated mice compared to controls. Our results suggest that pathological effects of complement in ECM are primarily mediated at C5 through the terminal pathway and that this is occurring even in the absence of C3 suggesting an alternative mechanism for C5 cleavage in C3-/- mice.

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.

Immune receptors and adhesion molecules in human pulmonary leptospirosis

Pulmonary involvement in leptospirosis has been increasingly reported in the last 20 years, being related to the severity and mortality of the disease. The pathogenesis of pulmonary hemorrhage in leptospirosis is not understood. Lung endothelial cells have been proposed as targets in the pathogenesis of lung involvement in leptospirosis through the activation of Toll-like receptor 2 or the complement system, which stimulates the release of cytokines that lead to the activation of adhesion molecules. The aim of this study was to investigate the involvement of immune pathways and of the intercellular and vascular cell adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule, respectively) in the lungs of patients with pulmonary involvement of leptospirosis. We studied the lungs of 18 patients who died of leptospirosis and compared them with 2 groups of controls: normal and noninfectious hemorrhagic lungs. Using immunohistochemistry and image analysis, we quantified the expression of the C3a anaphylatoxin receptor, intercellular adhesion molecule, vascular cell adhesion molecule, and Toll-like receptor 2 in small pulmonary vessels and in the alveolar septa. There was an increased expression of intercellular adhesion molecule (P < .03) and C3a anaphylatoxin receptor (P < .008) in alveolar septa in the leptospirosis group compared with the normal and hemorrhagic controls. In the vessels of the leptospirosis group, there was an increased expression of intercellular adhesion molecule (P = .004), vascular cell adhesion molecule (P = .030), and Toll-like receptor 2 (P = .042) compared with the normal group. Vascular cell adhesion molecule expression in vessels was higher in the leptospirosis group compared with the hemorrhagic group (P = .015). Our results indicate that immune receptors and adhesion molecules participate in the phenomena leading to pulmonary hemorrhage in leptospirosis.

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.

The anaphylatoxin receptor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro.

There is evidence that complement components regulate cytokine production in osteoblastic cells, induce cell migration in mesenchymal stem cells, and play a regulatory role in normal enchondral bone formation. We proved the hypothesis that complement might be involved in bone healing after fracture. We investigated the expression of the key anaphylatoxin receptor C5aR during fracture healing in rats by immunostaining after 1, 3, 7, 14, and 28 days. C5aR expression was additionally analyzed in human mesenchymal stem cells (hMSC) during osteogenic differentiation, in human primary osteoblasts, and osteoclasts by reverse transcriptase polymerase chain reaction and immunostaining. Receptor functionality was proven by the migratory response of cells to C5a in a Boyden chamber. C5aR was expressed in a distinct spatial and temporal pattern in the fracture callus by differentiated osteoblast, chondroblast-like cells in cartilaginous regions, and osteoclasts. In vitro C5aR was expressed by osteoblasts, osteoclasts, and hMSC undergoing osteogenic differentiation. C5aR was barely expressed by undifferentiated hMSC but was significantly induced after osteogenic differentiation. C5aR activation by C5a induced strong chemotactic activity in osteoblasts, and in hMSC, which had undergone osteogenic differentiation, being abolished by a specific C5aR antagonist. In hMSC, C5a induced less migration reflecting their low level of C5aR expression. Our in vitro and in vivo results demonstrated the presence of C5aR in bone forming osteoblasts and bone resorbing osteoclasts. It is suggested that C5aR might play a regulatory role in fracture healing in intramembranous and in enchondral ossification, one possible function being the regulation of cell recruitment.

Cutting Edge: The Membrane Attack Complex of Complement Is Required for the Development of Murine Experimental Cerebral Malaria

Cerebral malaria is the most severe complication of Plasmodium falciparum infection and accounts for a large number of malaria fatalities worldwide. Recent studies demonstrated that C5(-/-) mice are resistant to experimental cerebral malaria (ECM) and suggested that protection was due to loss of C5a-induced inflammation. Surprisingly, we observed that C5aR(-/-) mice were fully susceptible to disease, indicating that C5a is not required for ECM. C3aR(-/-) and C3aR(-/-) × C5aR(-/-) mice were equally susceptible to ECM as were wild-type mice, indicating that neither complement anaphylatoxin receptor is critical for ECM development. In contrast, C9 deposition in the brains of mice with ECM suggested an important role for the terminal complement pathway. Treatment with anti-C9 Ab significantly increased survival time and reduced mortality in ECM. Our data indicate that protection from ECM in C5(-/-) mice is mediated through inhibition of membrane attack complex formation and not through C5a-induced inflammation.

Requirements for Membrane Attack Complex Formation and Anaphylatoxins Binding to Collagen-Activated Platelets

BackgroundThe activation of complement during platelet activation is incompletely understood. Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets.MethodsC5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation.ResultsC5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg2+-EGTA and significantly reduced by the thrombin inhibitor hirudin (−37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg2+-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (−71% and −44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface.ConclusionsC5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.

The C3a anaphylatoxin receptor is critical in the pathophysiology of RSV infection (49.2)

Abstract Respiratory syncytial virus (RSV) is linked to severe lung disease and may lead to wheezing in infants. RSV infection in mice demonstrates infiltration of inflammatory cells, mucus cell metaplasia, and airway hyperresonsiveness (AHR) to methacholine. Previous studies demonstrated activation of complement by RSV. Other studies showed a requirement for C3 or C3a in AHR associated with antigen sensitization as well as inhalation of particulate matter. We hypothesized, therefore, that C3a is a critical component for the AHR associated with RSV infection. We found that mice lacking the C3aR (C3aR1-/-) do not develop AHR secondary to acute RSV infection in contrast to wild type mice. Further, viral clearance was accelerated in C3aR1-/-mice. The AHR phenotype was restored to wild type when C3aR1-/- mice were transplanted with wild type bone marrow cells,supporting a role for a hematopoetic cell type. T cells were the dominant cell type increased in the lungs of both wild type and C3aR1-/-mice following RSV infection, however, the increases in lung IL-23 and IL-17 were significantly lower in C3aR1-/-mice compared with wild type animals. Additionally the increased levels of the transcription factor RORγT, responsible for regulating IL17 synthesis, seen in wild type mice were greatly reduced in C3aR1-/- mice. Our findings identify a key role for activation of complement anaphylatoxin C3aR in the pathophysiology of RSV disease, and may help identify a therapeutic target in humans.

Complement Inhibition Alleviates Paraquat-Induced Acute Lung Injury

The widely used herbicide, paraquat (PQ), is highly toxic and claims thousands of lives from both accidental and voluntary ingestion. The pathological mechanisms of PQ poisoning-induced acute lung injury (ALI) are not well understood, and the role of complement in PQ-induced ALI has not been elucidated. We developed and characterized a mouse model of PQ-induced ALI and studied the role of complement in the pathogenesis of PQ poisoning. Intraperitoneal administration of PQ caused dose- and time-dependent lung damage and mortality, with associated inflammatory response. Within 24 hours of PQ-induced ALI, there was significantly increased expression of the complement proteins, C1q and C3, in the lung. Expression of the anaphylatoxin receptors, C3aR and C5aR, was also increased. Compared with wild-type mice, C3-deficient mice survived significantly longer and displayed significantly reduced lung inflammation and pathology after PQ treatment. Similar reductions in PQ-induced inflammation, pathology, and mortality were recorded in mice treated with the C3 inhibitors, CR2-Crry, and alternative pathway specific CR2-fH. A similar therapeutic effect was also observed by treatment with either C3a receptor antagonist or a blocking C5a receptor monoclonal antibody. Together, these studies indicate that PQ-induced ALI is mediated through receptor signaling by the C3a and C5a complement activation products that are generated via the alternative complement pathway, and that complement inhibition may be an effective clinical intervention for postexposure treatment of PQ-induced ALI.

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.

Immune Evasion Strategies of Porphyromonas gingivalis

Porphyromonas gingivalis is strongly correlated with chronic periodontitis. Its chronic persistence in the periodontium depends on its ability to evade host immunity without inhibiting the overall inflammatory response, which is actually beneficial for this and other periodontal bacteria. Indeed, the inflammatory exudate (gingival crevicular fluid) is a source of essential nutrients, such as peptides and hemin-derived iron. In this review, I discuss how P. gingivalis can promote its adaptive fitness through instigation of subversive crosstalk signaling. These interactions involve Toll-like receptor-2, complement receptor 3, C5a anaphylatoxin receptor, and CXC-chemokine receptor 4. Their exploitation by P. gingivalis allows the pathogen to escape elimination, obtain nutrients, and collaterally inflict periodontal tissue injury.

The role of complement anaphylatoxin receptors in mesenteric ischemia/reperfusion-induced injury

Inflammation is a major driver of pathology in ischemia reperfusion (IR) injuries. The complement activation products complement anaphylatoxin 3a (C3a) and complement anaphylatoxin (C5a), are powerful pro-inflammatory mediators which activate inflammatory cells and the release of pro-inflammatory substances involved in the pathology of IR. We are currently exploring the role of the anaphylatoxin receptors using mice deficient in these receptors (CD88, C5L2, and C3aR). This study demonstrates a role for complement activation, C5a generation and CD88 activation in the pathogenesis of intestinal IR injury in mice.

The anaphylatoxin receptor C5AR is expressed in bone cells in vitro and during fracture healing in vivo

The anaphylatoxin receptor C5AR is expressed in bone cells in vitro and during fracture healing in vivo

The Complement Split Products C3a and C5a are potent Activators of Mesenchymal Stem Cells

It is known that mesenchymal stem cells (MSC) are recruited to sites of tissue injury to fulfill their repair function. It is also known that there is complement activation, wherever there is tissue injury. We therefore determined the effect of the complement split products C3a and C5a on MSC. FACS analysis indicated that human bone marrow‐derived MSCs express cell‐surface C3a receptors (C3aR) and C5a receptors (C5aR). Both C3a and C5a induced chemotaxis of MSCs in a dose dependent fashion. Specific C3aR and C5aR inhibitors blocked the chemotactic response as did pertussis toxin, indicating that the response was mediated by the known anaphylatoxin receptors in a Gi activation‐dependent fashion.While C5a causes prolonged activation of many cell types, the response to C3a is generally transient and weak. However, in MSCs both C3a and C5a caused prolonged ERK1/2 and Akt phosphorylation. Phospho‐Akt and phospho‐ERK1/2 were translocated to the nucleus with both stimuli, which was associated with subsequent phosphorylation of the transcription factor Elk. More surprisingly, the C3aR itself was translocated to the nucleus in C3a‐stimulated MSCs. Since nuclear translocation of G‐protein coupled receptors has been shown to induce long‐term effects including transcriptional activation, this novel observation implies that C3a exerts far reaching consequences on MSC biology. The prolonged activation of C3aRs and to a lesser degree of C5aRs was associated with increased expression of several angiogenic factors in MSC including VEGF, bFGF and IL‐8. These results suggest that C3a and C5a present in injured tissues contribute to the recruitment of MSC and that means of activating the C3aR or C5aR on MSC could improve their ischemic repair potential, which would be more attractive for C3a, which lacks the inflammatory action of C5a.

Both FcγRIV and FcγRIII are essential receptors mediating type II and type III autoimmune responses via FcRγ‐LAT‐dependent generation of C5a

FcgammaRIV is a relatively new IgG Fc receptor (FcgammaR) that is reported to contribute to the pathogenesis of autoimmune diseases, although its specific role in relation to FcgammaRIII, complement and IgG2 subclasses remains uncertain. Here we define FcgammaRIV on macrophages as a receptor for soluble IgG2a/b complexes but not for cellular bound IgG2a and show that simultaneous activation of FcgammaRIV and FcgammaRIII is critical to mediate certain type II/III autoimmune responses. FcgammaRIII-deficient mice display compensatory enhanced FcgammaRIV expression, are protected from lung inflammation after deposition of IgG complexes, and show reduced sensitivity to IgG2a/b-mediated hemolytic anemia, indicating that increased FcgammaRIV alone is not sufficient to trigger these diseases in the absence of FcgammaRIII. Importantly, however, blockade of FcgammaRIV is also effective in inhibiting phagocytosis and cytokine production in IgG2b-induced anemia and acute lung injury, processes that display a further dependence on C5a anaphylatoxin receptor. Using gene deletion and functional inhibition studies, we found that FcgammaRIII and FcgammaRIV are each essential to trigger an FcRgamma-linker for activation of T-cell-dependent signal that drives C5a production in the Arthus reaction. Together, the results demonstrate a combined requirement for FcgammaRIII and FcgammaRIV in autoimmune injury, and identify the linker for activation of T cells adaptor as an integral component of linked FcgammaR and C5a anaphylatoxin receptor activation to generate inflammation.

Deletion of both the C3a and C5a receptors fails to protect against experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease in which inflammation, leukocyte infiltration, and ultimately, demyelination occur as a result of innate and adaptive immune-mediated mechanisms. The pathophysiological role of the complement system, a major component of innate immunity, in the development and progression of experimental autoimmune encephalomyelitis (EAE), the animal model for MS has been extensively examined. Previous studies from our lab have shown that the complement receptor for the anaphylatoxin C3a, but not for C5a plays an important role in EAE. Based on the important contributions of the complement anaphylatoxin receptors to other inflammatory conditions in the CNS, we reasoned that deletion of both receptors may reveal underlying interactions between them that are important to EAE pathology. We performed EAE in C3aR/C5aR double knockout mice (C3aR/C5aR −/−) and observed delayed onset of disease but no attenuation of disease severity compared to wild type mice. Interestingly there was trend toward greater infiltration of CD4 +, but not CD8 + T cells, in C3aR/C5aR −/− mice with EAE, suggesting altered trafficking of these cells. Antigen-specific T cells isolated from C3aR/C5aR −/− mice during acute EAE produced elevated levels of TNF-α, but markedly reduced levels of IFN-γ and IL-12 compared to wild type mice. It remains unclear how the changes in these disease parameters contribute to the loss of the protective effect seen in C3aR −/− mice, however our data indicate a level of cross-modulation between the C3aR and C5aR during EAE.

Role of the C5a receptor (C5aR) in acute and chronic dextran sulfate-induced models of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a critical public health issue; more and more people are affected, but treatment options remain limited. Complement activation and the anaphylatoxin C5a have been shown to play a role in IBD. In this study, mouse models of acute and chronic dextran sulfate-induced colitis were used to further elucidate the impact of C5a and its receptor (C5aR) on disease development. In C57BL/6J wildtype and C5aR(-/-) mice the extent of complement activation, changes in weight, and water/food consumption were determined. Disease severity was evaluated via a clinical score, histology, cytokine- and myeloperoxidase-determination as well as real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of anaphylatoxin receptors and inflammatory mediators. C5aR(-/-) mice showed milder disease symptoms, less histological damage, and a lower expression of inflammatory mediators in acute colitis, a setting where complement was activated. In chronic colitis the knockout mice exhibited aggravated weight loss, a higher degree of histological damage and granulocyte infiltration. Intriguingly, increases in C3a-receptor and C5L2 mRNA were dependent on C5aR. Compared to wildtype mice, C5aR(-/-) animals displayed smaller lymph nodes in acute colitis, but extensive swelling and diminished IL-4 and IFN-γ responses in the chronic disease, demonstrating that C5aR modifies T-helper cell polarization. C5aR exerts detrimental functions in acute colitis, strongly supporting the idea that a C5aR-antagonist might be useful for IBD treatment. However, since the absence of C5aR was no longer protective and in some regards disadvantageous in chronic IBD, future studies should address the efficacy and the possible side effects of a sustained antagonist treatment.

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVESignificant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.RESEARCH DESIGN AND METHODSWe therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.RESULTSWe demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.CONCLUSIONSOverall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.

Age-related alterations in innate immune receptor expression and ability of macrophages to respond to pathogen challenge in vitro

The impact of ageing in innate immunity is poorly understood. Studies in the mouse model have described altered innate immune functions in aged macrophages, although these were not generally linked to altered expression of receptors or regulatory molecules. Moreover, the influence of ageing in the expression of these molecules has not been systematically examined. We investigated age-dependent expression differences in selected Toll-like and other pattern-recognition receptors, receptors involved in inflammatory amplification, and in transmembrane and intracellular regulators of inflammatory signaling. Young and aged macrophages were examined under resting conditions or upon activation with Porphyromonas gingivalis, a major pathogen in periodontal disease, the prevalence and severity of which increase in old age. We detected a limited number of age-dependent alterations, involving both reduction and increase of immune activity. Interestingly, surface expression of receptors that amplify inflammation (C5a anaphylatoxin receptor and triggering receptor expressed on myeloid cells [TREM]-1) was elevated in aged macrophages. No significant age-dependent differences were observed regarding the phagocytosis and intracellular killing of P. gingivalis, consistent with lack of significant changes in phagocytic receptor expression and induction of antimicrobial molecules. Therefore, at least at the cellular level, certain aspects of innate immune function may not necessarily decline with age.

C3a and C5a Are Chemotactic Factors for Human Mesenchymal Stem Cells, Which Cause Prolonged ERK1/2 Phosphorylation

Mesenchymal stem cells (MSCs) have a great potential for tissue repair, especially if they can be delivered efficiently to sites of tissue injury. Since complement activation occurs whenever there is tissue damage, the effects of the complement activation products C3a and C5a on MSCs were examined. Both C3a and C5a were chemoattractants for human bone marrow-derived MSCs, which expressed both the C3a receptor (C3aR) and the C5a receptor (C5aR; CD88) on the cell surface. Specific C3aR and C5aR inhibitors blocked the chemotactic response, as did pertussis toxin, indicating that the response was mediated by the known anaphylatoxin receptors in a G(i) activation-dependent fashion. While C5a causes strong and prolonged activation of various signaling pathways in many different cell types, the response observed with C3a is generally transient and weak. However, we show herein that in MSCs both C3a and C5a caused prolonged and robust ERK1/2 and Akt phosphorylation. Phospho-ERK1/2 was translocated to the nucleus in both C3a and C5a-stimulated MSCs, which was associated with subsequent phosphorylation of the transcription factor Elk, which could not be detected in other cell types stimulated with C3a. More surprisingly, the C3aR itself was translocated to the nucleus in C3a-stimulated MSCs, especially at low cell densities. Since nuclear activation/translocation of G protein-coupled receptors has been shown to induce long-term effects, this novel observation implies that C3a exerts far-reaching consequences on MSC biology. These results suggest that the anaphylatoxins C3a and C5a present in injured tissues contribute to the recruitment of MSCs and regulation of their behavior.