Search

The complement activation product, C5a, is a key factor for regulation of inflammatory responses. C5a and C5adesArg bind to their receptors, C5a R and C5 L2, but the functional roles of C5 L2 remain controversial. We screened the patterns of 23 inflammatory mediators in cultures of LPS-activated mouse peritoneal elicited macrophages ( PEMs) in the presence or absence of recombinant mouse C5a. Production of most mediators studied was suppressed by C5a, whereas G- CSF production was enhanced. G- CSF gene expression and secretion from PEMs was amplified two- to threefold by C5a in a dose- and time-dependent fashion. The degradation product C5adesArg promoted lower levels of G- CSF. The effects of C5a on G- CSF were associated with activation of PI3 K/ Akt and MEK1/2 signaling pathways. C5a did not enhance G- CSF production in cultures of PEMs from either C5a R- or C5 L2-deficient mice, indicating that both C5a receptors are indispensable for mediating the effects of C5a in the production of G- CSF. Finally, G- CSF levels in plasma during polymicrobial sepsis after cecal ligation and puncture were substantially lower in C5a R- or C5 L2-deficient mice as compared with that in C57 BL/6 J WT mice. These findings elucidate the functional characteristics of the C5 L2 receptor during the acute inflammatory response. [ABSTRACT FROM AUTHOR]

These studies were undertaken to extend emerging evidence that β2 adrenergic receptor (β2AR) agonists, in addition to their bronchorelaxing effects, may have broad anti-inflammatory effects in the lung following onset of experimental acute lung injury (ALI). Young male C57BL/6 mice (25 g) developed ALI following airway deposition of bacterial LPS or IgG immune complexes in the absence or presence of appropriate stereo-isomers (enantiomers) of β2AR agonists, albuterol or formoterol. Endpoints included albumin leak into lung and buildup of polymorphonuclear neutrophils and cytokines/chemokines in bronchoalveolar fluids. Both β2AR agonists suppressed lung inflammatory parameters (IC50 = 10-7 M). Similar effects of β2AR agonists on mediator release were found when mouse macrophages were stimulated in vitro with LPS. The protective effects were associated with reduced activation (phosphorylation) of JNK but not of other signaling proteins. Collectively, these data suggest that2AR agonists have broad anti-inflammatory effects in the setting of ALI. While β2AR agonists suppress JNK activation, the extent to which this can explain the blunted lung inflammatory responses in the ALI models remains to be determined. [ABSTRACT FROM AUTHOR]

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 (IC50, 50-100 nM C5a) and IL-23 (IC50, 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. [ABSTRACT FROM AUTHOR]

The interleukin-17 (ID17) family of cytokines plays important roles in innate immune defenses against bacterial and fungal pathogens. While much is known about IL-17A, much less information is available about the IL-17F isoform. Here, we investigated gene expression and release of IL-17F and its regulation by the complement system. IL-17F was produced in mouse peritoneal elicited macrophages after TLR4 activation by LPS, peaking after 12 h. This effect was completely dependent on the presence of the adaptor protein MyD88. The copresence of the complement activation product, C5a (EC50 = 10 nM), amplified IL-17F production via the receptor C5aR. In vitro signaling studies indicated that LPS or C5a, or the combination, caused phosphorylation of Akt occurring at threonine 308 but not at serine 473. Treatment of macrophages with pharmacologic inhibitors of PI3K-Akt greatly reduced production of IL-17F as well as mRNA for IL-17F. In endotoxemia, C5a levels peaked at 6 h, while IL-17F levels peaked between 6-12 h. Full in vivo production of IL-17F during endotoxemia required C5a. A similar result was found in the cecal ligation and puncture sepsis model. These data suggest that maximal production of IL-17F requires complement activation and presence of C5a. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) in mouse lung occurs after distal airway deposition of IgG immune complexes (IgGICs), resulting in a breakdown of the vascular-airway barrier, causing intrapulmonary edema, hemorrhage, and accumulation of neutrophils [polymorphonuclear leukocytes (PMNs)] in the alveolar compartment, these changes being complement (C5a) and C5a receptor (C5aR) dependent. In this ALI model, C5aR expression (protein) was found to occur on upper (bronchial) and lower (alveolar) airway epithelial cells. An adenovirus construct (siRNA) was used to silence mRNA for C5aR in the lung. Under such conditions, C5aR protein was markedly reduced on lung epithelial cells, resulting in much reduced leakage of albumin into the lung, diminished buildup of PMNs, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluids. These studies indicate that bronchial and alveolar epithelial cell CSaR is up-regulated and greatly contributes to inflammation and injury in the lung. The use of siRNA administered into the airways avoids systemic suppression of C5aR, which might compromise innate immunity. It is possible that such an intervention might be employed in humans with ALI or acute respiratory distress syndrome as well as in upper-airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, where there is evidence for complement activation and buildup of PMNs. [ABSTRACT FROM AUTHOR]

Defective cardiac function during sepsis has been referred to as "cardiomyopathy of sepsis." It is known that sepsis leads to intensive activation of the complement system. In the current study, cardiac function and cardiomyocyte contractility have been evaluated in rats after cecal ligation and puncture (CLP). Significant reductions in left ventricular pressures occurred in vivo and in cardiomyocyte contractility in vitro. These defects were prevented in CLP rats given blocking antibody to C5a. Both mRNA and protein for the C5a receptor (C5aR) were constitutively expressed on cardiomyocytes; both increased as a function of time after CLP. In vitro addition of recombinant rat C5a induced dramatic contractile dysfunction in both sham and CLP cardiomyocytes, but to a consistently greater degree in cells from CLP animals. These data suggest that CLP induces C5aR on cardiomyocytes and that in vivo generation of C5a causes C5a-C5aR interaction, causing dysfunction of cardiomyocytes, resulting in compromise of cardiac performance. [ABSTRACT FROM AUTHOR]

Presents findings of a study which investigated the presence and regulation of the second C5a receptor on neutrophils and various organs during sepsis. Evaluation the role for C5L2 nucleotide sequence in balancing the biological responses to C5a; Comparison of 5′-RACE polymerase chain reaction, cyclic DNA cloning and sequence in the rat C5L2 receptor; Contrast between C5L2 and C5aR expression in neutrophils during sepsis; Expression of nRNA for C5L2 during sepsis; Effects of αC5aR and αC5L2 on in vitro and in vivo production of interleukin-6.

Presents a study on the effects of C5a on lipopolysaccharide(LPS)-induced interleukin(IL)-6 generation in neutrophils in vitro and on in vivo IL-6 production in septic rodents. Effects of C5a on LPS-induced IL-6 production and gene transcription; Effects of blockade of C5a/C5a receptor activation on serum IL-6 levels during sepsis; Effects of signaling pathway inhibitors on IL-6 production in neutrophils; Conclusions of the study.

Signaling in neutrophils involving the powerful complement anaphylatoxin (C5a) and its receptor (C5aR) plays an essential role in innate immunity. We have previously shown in rodents that blockade of either C5a or C5aR dramatically improves survival rates in experimental sepsis. We have also shown that defensive functions of neutrophils are compromised during sepsis in a C5a-dependent manner. The main aim of the current study was to elucidate the mechanisms for C5aR alteration on neutrophils during sepsis and to evaluate the relationship between C5aR levels on neutrophils and survival after the onset of sepsis. [ABSTRACT FROM AUTHOR]

Endothelial glycocalyx degradation is thought to facilitate the development of sepsis. Histone is a significant mediator in sepsis. Unfractionated heparin (UFH) possessed beneficial effects on sepsis. Thereby, this study aims to figure out whether histone can disrupt glycocalyx and to investigate the protective effect and mechanism of UFH. Male mice (C57BL/6, 8–10 weeks old, weighing 20–25 g) were randomly divided into five groups including control group, histone group, histone + UFH group, histone + heparinase (HPA) inhibitor group, and histone + UFH + HPA inhibitor group. The mice were treated with histone (50 mg/kg) via tail vein immediately after HPA (20 mg/kg) injection. UFH (400 U/kg) was injected 1h after histone administration. The other groups were injected with equal volume of sterile saline accordingly. UFH alleviated histone-induced lung injury and pulmonary edema. UFH inhibited histone-induced lung coagulation activation and inflammatory response. UFH treatment markedly inhibited pulmonary glycocalyx degradation by reducing the histone-induced decrease in the levels of lung syndecan-1 mRNA and protein. UFH downregulated histone-induced expression of HPA mRNA and protein, and thus alleviated glycocalyx degradation. UFH protects against histone-induced pulmonary glycocalyx injury partly by heparinase pathway. [ABSTRACT FROM AUTHOR]

The complement system is activated in tuberculous pleural effusion (TPE), with increased levels of the anaphylatoxins stimulating pleural mesothelial cells (PMCs) to secrete chemokines, which recruit nonclassical monocytes to the pleural cavity. The differentiation and recruitment of naive CD4+ T cells are induced by pleural cytokines and PMC-produced chemokines in TPE. However, it is unclear whether anaphylatoxins orchestrate CD4+ T cell response via interactions between PMCs and monocytes in TPE. In this study, CD16+ and CD16- monocytes isolated from TPE patients were cocultured with PMCs pretreated with anaphylatoxins. After removing the PMCs, the conditioned monocytes were cocultured with CD4+ T cells. The levels of the cytokines were measured in PMCs and monocyte subsets treated separately with anaphylatoxins. The costimulatory molecules were assessed in conditioned monocyte subsets. Furthermore, CD4+ T cell response was evaluated in different coculture systems. The results indicated that anaphylatoxins induced PMCs and CD16+ monocytes to secrete abundant cytokines capable of only inducing Th17 expansion, but Th1 was feeble. In addition, costimulatory molecules were more highly expressed in CD16+ than in CD16− monocytes isolated from TPE. The interactions between monocytes and PMCs enhanced the ability of PMCs and monocytes to produce cytokines and that of monocytes to express HLA-DR, CD40, CD80 and CD86, which synergistically induced Th17 expansion. In the above process, anaphylatoxins enhanced the interactions between monocytes and PMCs by increasing the level of the cytokines IL-1β, IL-6, IL-23 and upregulating the phenotype of CD40 and CD80 in CD16+ monocytes. Collectively, these data indicate that anaphylatoxins play a central role in orchestrating Th17 response mainly via interactions between CD16+ monocytes and PMCs in TPE. Author summary: Tuberculous pleural effusion is characterized by intense chronic accumulations of fluid and lymphocyte cells and monocytes/macrophages in the pleural space. Complement mediators play important roles in providing protection against Mycobacterium tuberculosis. Our results demonstrated that Mycobacterium tuberculosis infection induced the amplification of complement activation in TPE. Complement activation produces anaphylatoxins that induce PMCs and CD16+ monocytes to secrete abundant cytokines capable of only inducing Th17 expansion, but Th1 was feeble. In addition, costimulatory molecules were more highly expressed in CD16+ than in CD16− monocytes isolated from TPE. The interactions between monocytes and PMCs enhanced the ability of PMCs and monocytes to produce cytokines and that of monocytes to express HLA-DR, CD40, CD80 and CD86, which synergistically induced Th17 expansion. In the above process, anaphylatoxins enhanced the interactions between monocytes and PMCs by increasing the level of the cytokines IL-1β, IL-6, IL-23 and upregulating the phenotype of CD40 and CD80 in CD16+ monocytes. In summary, these data highlighted the importance of anaphylatoxins and the innate immune system in eliciting pathogenic T cell responses in TPE and suggested that monocytes, especially the CD16+ subset, might be an efficient target for controlling inflammation. [ABSTRACT FROM AUTHOR]