A critical role for C5L2 in the pathogenesis of experimental allergic asthma

The complement fragment C5a plays dual roles in the development of experimental allergic asthma. It protects from pulmonary allergy by a regulatory effect on dendritic cells during allergen sensitization, but is proallergic during the effector phase. C5a can bind to two distinct receptors (i.e., C5a receptor and C5a receptor-like 2 [C5L2]). The functional role of C5L2 in vivo remains enigmatic. In this study, we show in two models of OVA- and house dust mite (HDM)-induced experimental allergic asthma that C5L2-deficient mice are protected from the development of airway hyperresponsiveness, Th2 cytokine production, eosinophilic airway inflammation, serum IgE, or mucus production. Surprisingly, HDM-induced experimental asthma in C5L2-deficient mice was associated with increased pulmonary IL-17A production and increased airway neutrophil numbers. To directly assess the role of C5L2 on myeloid dendritic cells (mDCs) during allergen sensitization, we performed single or repeated adoptive transfers of C5L2-deficient mDCs into wild-type mice. HDM-pulsed C5L2-deficient mDCs induced strong Th2 cytokine production, which was associated with marked IFN-γ and IL-17A production, decreased eosinophil numbers, and reduced IgE production as compared with HDM-pulsed mDCs from wild-type mice. HDM stimulation of C5L2(-/-) mDCs in vitro resulted in production of Th17-promoting cytokine IL-23, which was absent in wild-type mDCs. Our findings suggest that C5L2 acts at the mDC/T cell interface to control the development of Th1 and Th17 cells in response to airway HDM exposure. Furthermore, it drives Th2 immune responses independent of mDCs, suggesting a complex role for C5L2 in the development of experimental allergic asthma.     

C5L2 is required for C5a-triggered receptor internalization and ERK signaling

C5L2 is a receptor that binds to C5a and belongs to the family of G protein-coupled receptors, but its role in physiological C5a-mediated responses remains under debate. Here we show that, like the canonical C5a receptor C5aR, C5L2 plays a pro-inflammatory role in a murine model of acute experimental colitis. We demonstrate that C5L2 physically interacts with C5aR and is required for optimal C5a-mediated C5aR internalization and associated ERK activation. Abrogation of C5a-induced receptor internalization by treatment with the dynamin inhibitor dynasore^TM impaired C5a-induced MEK and ERK signaling. Although the presence of C5aR alone was sufficient to recruit the scaffold protein β-arrestin1 to the cell membrane in response to C5a stimulation, it was inadequate to mediate AP2 recruitment and subsequent C5aR internalization. Expression of C5L2 allowed normal internalization of C5aR in response to C5a stimulation, followed by normal ERK signaling. Thus, our work reveals an essential role for C5L2 in C5a-triggered, AP2-dependent C5aR internalization and downstream ERK signaling.     

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5a(desArg) is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.     

Receptors for complement C5a. The importance of C5aR and the enigmatic role of C5L2

Complement component C5a is one of the most potent inflammatory chemoattractants and has been implicated in the pathogenesis of numerous inflammatory diseases. C5a binds two receptors, C5aR and C5L2. Most of the C5a functional effects occur through C5aR, and the pharmaceutical industry has focused on this receptor for the development of new anti-inflammatory therapies. We used a novel approach to generate and test therapeutics that target C5aR. We created human C5aR knock-in mice, and used neutrophils from these to immunize wild-type mice. This yielded high-affinity blocking mAbs to human C5aR. We tested these anti-human C5aR mAbs in mouse models of inflammation, using the human C5aR knock-in mice. These antibodies completely prevented disease onset and were also able to reverse established disease in the K/B x N arthritis model. The physiological role of the other C5a receptor, C5L2 is still unclear, and our studies with blocking mAbs to human C5L2 have failed to demonstrate a clear functional role in signaling to C5a. The development of effective mAbs to human C5aR is an alternative approach to drug development, for this highly attractive target.     

C5L2 is a functional receptor for acylation-stimulating protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.     

Activation of human RNase L by 2'- and 5'-O-methylphosphonate-modified oligoadenylates

To determine the influence of internucleotide linkage and sugar ring conformation, and the role of 5'-terminal phosphate, on the activation of human RNase L, a series of 2'- and 5'-O-methylphosphonate-modified tetramers were synthesized from appropriate monomeric units and evaluated for their ability to activate human RNase L. Tetramers pAAAp(c)X modified by ribo, arabino or xylo 5'-phosphonate unit p(c)X activated RNase L with efficiency comparable to that of natural activator. Moreover, incorporation of phosphonate linkages ensured the stability against cleavage by nucleases. The substitution of 5'-terminal phosphate for 5'-terminal phosphonate in tetramer p(c)XAAA afforded tetramers with excellent activation efficiency and with complete stability against cleavage by phosphomonoesterases.     

The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and β-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the β-arrestin pathway. Association of C5L2 with β-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the β-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.     

Activation of Neutrophils is Daily Inhibited by Saliva

The circadian release of superoxide anion (O 2 ?) by inflammatory neutrophils was investigated with mice kept under a constant light regime. Neutrophils were elicited to the peritoneal cavity by injection of 12% sodium caseinate-PBS solution, collected and stimulated to release O 2 ? to the culture milieu upon the addition of phorbol 12-myristate 13-acetate (PMA). The night phase neutrophils were able to release three fold more O 2 ? than day phase cells. Furthermore, the addition of human saliva in neutrophils cultures dramatically inhibits O 2 ? generation. Diurnal human saliva inhibits more effectively neutrophils activation than the nocturnal saliva. The PMA stimulation induces the synthesis of 13 kDa and 14 kDa molecular mass proteins detected in autoradiograms, and saliva additions decrease their synthesis. These two proteins are well known to be essential to the NAD(P)H oxidase assembly complex and, therefore, making neutrophils able to exert their main function as phagocytic cells.     

Activation of Neutrophils is Daily Inhibited by Saliva

The circadian release of superoxide anion (O 2 ? ) by inflammatory neutrophils was investigated with mice kept under a constant light regime. Neutrophils were elicited to the peritoneal cavity by injection of 12% sodium caseinate-PBS solution, collected and stimulated to release O 2 fito the culture milieu upon the addition of phorbol 12-myristate 13-acetate (PMA). The night phase neutrophils were able to release three fold more O 2 fithan day phase cells. Furthermore, the addition of human saliva in neutrophils cultures dramatically inhibits O 2 figeneration. Diurnal human saliva inhibits more effectively neutrophils activation than the nocturnal saliva. The PMA stimulation induces the synthesis of 13 kDa and 14 kDa molecular mass proteins detected in autoradiograms, and saliva additions decrease their synthesis. These two proteins are well known to be essential to the NAD(P)H oxidase assembly complex and, therefore, making neutrophils able to exert their main function as phagocytic cells.     

C5L2 and C5aR interaction in adipocytes and macrophages: Insights into adipoimmunology

Obesity is associated with inflammation characterized by increased infiltration of macrophages into adipose tissue. C5aR-like receptor 2 (C5L2) has been identified as a receptor for acylation-stimulating protein (ASP) and the inflammatory factor C5a, which also binds C5aR. The present study examines the effects of ligands ASP and C5a on interactions between the receptors C5L2 and C5aR in 3T3-L1 adipocytes and J774 macrophages.BRET experiments indicate that C5L2 and C5aR form homo- and heterodimers in transfected HEK 293 cells, which were stable in the presence of ligand. Cell surface receptor levels of C5L2 and C5aR increased during 3T3-L1 adipocyte differentiation; both receptors are also highly expressed in J774 macrophages. Using confocal microscopy to evaluate endogenous receptors in adipocytes following stimulation with ASP or C5a, C5L2 is internalized with increasing perinuclear colocalization with C5aR. There is little C5a-dependent colocalization in macrophages. While adipocyte-conditioned medium (ACM) increased C5L2–C5aR colocalization in macrophages, this was blocked by C5a. ASP stimulation increased Akt (Ser⁴⁷³) phosphorylation in both cell types; C5a induced slight Akt phosphorylation in adipocytes with less effect in macrophages. ASP, but not C5a, increased fatty acid uptake/esterification in adipocytes.C5L2–C5aR homodimerization versus heterodimerization may thus contribute to differential responses obtained following ASP vs C5a stimulation of adipocytes and macrophages, providing new insights into the complex interaction between these two cell types within adipose tissue. Studying the mechanisms involved in the differential responses of C5L2–C5aR activation based on cell type will further our understanding of inflammatory processes in obesity.     

Activation and evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA

Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-alpha2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2'-5' oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.     

A Role for Ribosomal Protein L5 in the Nuclear Import of 5S rRNA inXenopusOocytes

Prior to ribosome assembly, 5S ribosomal RNA (5S rRNA) binds to ribosomal protein L5 to form a stable ribonucleoprotein particle (5S RNP). We have analyzed the role of L5 binding in the nuclear targeting of 5S rRNA inXenopusoocytes, and have compared the nuclear import pathway of 5S RNPs with other karyophilic molecules. Nuclear import ofin vitro-generated 5S RNPs was found to be sensitive to three general inhibitors of nuclear pore complex-mediated translocation: ATP depletion, chilling, and wheat germ agglutinin. The initial rate and extent of net nuclear import was threefold greater with preassembled 5S RNPs than with 5S rRNA microinjected alone, suggesting that L5 binding is a prerequisite for nuclear accumulation. Nuclear import of 5S rRNA/5S RNPs is a facilitated process dependent on limiting factors, since nuclear import exhibited saturation kinetics. Not only was nuclear import of labeled 5S rRNA reduced in the presence of excess unlabeled 5S rRNA, but also in the presence of the synthetic karyophilic protein P(lys)-BSA. In contrast, import was not inhibited by U1 small nuclear RNA (snRNA) or U3 small nucleolar RNA (snoRNA). 5S rRNA/5S RNP nuclear import therefore appears to follow a pathway of molecular interactions similar to many karyophilic proteins, but not the methylguanosine cap-dependent U1 snRNA pathway or the cap-independent U3 snoRNA pathway.     

Activation of 5-HT2A/C receptors counteracts 5-HT1A regulation of n-methyl-D-aspartate receptor channels in pyramidal neurons of prefrontal cortex

Abnormal serotonin-glutamate interaction in prefrontal cortex (PFC) is implicated in the pathophysiology of many mental disorders, including schizophrenia and depression. However, the mechanisms by which this interaction occurs remain unclear. Our previous study has shown that activation of 5-HT(1A) receptors inhibits N-methyl-D-aspartate (NMDA) receptor (NMDAR) currents in PFC pyramidal neurons by disrupting microtubule-based transport of NMDARs. Here we found that activation of 5-HT(2A/C) receptors significantly attenuated the effect of 5-HT(1A) on NMDAR currents and microtubule depolymerization. The counteractive effect of 5-HT(2A/C) on 5-HT(1A) regulation of synaptic NMDAR response was also observed in PFC pyramidal neurons from intact animals treated with various 5-HT-related drugs. Moreover, 5-HT(2A/C) stimulation triggered the activation of extracellular signal-regulated kinase (ERK) in dendritic processes. Inhibition of the beta-arrestin/Src/dynamin signaling blocked 5-HT(2A/C) activation of ERK and the counteractive effect of 5-HT(2A/C) on 5-HT(1A) regulation of NMDAR currents. Immunocytochemical studies showed that 5-HT(2A/C) treatment blocked the inhibitory effect of 5-HT(1A) on surface NR2B clusters on dendrites, which was prevented by cellular knockdown of beta-arrestins. Taken together, our study suggests that serotonin, via 5-HT(1A) and 5-HT(2A/C) receptor activation, regulates NMDAR functions in PFC neurons in a counteractive manner. 5-HT(2A/C), by activating ERK via the beta-arrestin-dependent pathway, opposes the 5-HT(1A) disruption of microtubule stability and NMDAR transport. These findings provide a framework for understanding the complex interactions between serotonin and NMDARs in PFC, which could be important for cognitive and emotional control in which both systems are highly involved.     

The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74).

The substantial variations in the responses of cells to the anaphylatoxin C5a and its desarginated form, C5adR(74), suggest that more than one type of cell surface receptor for these ligands might exist. However, only a single receptor for C5a and C5adR(74), CD88, has been characterized to date. Here we report that the orphan receptor C5L2/gpr77, which shares 35% amino acid identity with CD88, binds C5a with high affinity but has a 10-fold higher affinity for C5adR(74) than CD88. C5L2 also has a moderate affinity for anaphylatoxin C3a, but cross-competition studies suggest that C3a binds to a distinct site from C5a. C4a was able to displace C3a, suggesting that C5L2, like the C3a receptor, may have a low binding affinity for this anaphylatoxin. Unlike CD88 and C3a receptor, C5L2 transfected into RBL-2H3 cells does not support degranulation or increases in intracellular [Ca(2+)] and is not rapidly internalized in response to ligand binding. However, ligation of C5L2 by anaphylatoxin did potentiate the degranulation response to cross-linkage of the high affinity IgE receptor by a pertussis toxin-sensitive mechanism. These results suggest that C5L2 is an anaphylatoxin-binding protein with unique ligand binding and signaling properties.     

Hyperglycemia-Induced Protein Kinase C Activation Inhibits Phagocytosis of C3b- and Immunoglobulin G–Opsonized Yeast Particles in Normal Human Neutrophils

The aim of this study was to investigate the effects of elevated glucose concentrations on complement receptor– and Fcγ receptor–mediated phagocytosis in normal human neutrophils. D-Glucose at 15 or 25 mM dose-dependently inhibited both complement receptor– and Fcγ receptor– mediated phagocytosis, as compared to that at a normal physiological glucose concentration. The protein kinase C (PKC) inhibitors GF109203X and Go6976 both dose dependently and completely reversed the inhibitory effect of 25 mM D-glucose on phagocytosis. Complement receptor– mediated phagocytosis was dose-dependently inhibited by the cell permeable diacylglycerol analogue 1,2-dioctanoylsn- glycerol (DAG), an effect that was abolished by PKC inhibitors. Furthermore, suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect on complement receptor–mediated phagocytosis. The authors conclude that elevated glucose concentrations can inhibit complement receptor and Fcγ receptor–mediated phagocytosis in normal human neutrophils by activating PKCα and/or PKCβ, an effect possibly mediated by DAG.     

氟氏佐剂诱导大鼠慢性前列腺炎疼痛模型中L5-S2脊髓中枢小胶质细胞的活化

目的:观察大鼠慢性前列腺炎疼痛模型中L5-S2脊段背角小胶质细胞活化的变化.方法:通过前列腺完全弗氏佐剂注射制作大鼠慢性前列腺炎疼痛模型,对照组注射生理盐水,观察时间为0、4、12、24d,用热辐射痛阈测定法和前列腺病理进行疼痛模型鉴定,采用实时荧光定量RT-PCR检测L5-S2脊段后角中小胶质细胞标志物IBA-1的表达.结果:成功建立慢性前列腺炎疼痛大鼠模型,并观察到L5-S2脊髓背角中存在小胶质细胞的活化.结论:慢性前列腺炎疼痛可以引起L5-S2脊髓中枢小胶质细胞活化,小胶质细胞异常活化可引起神经炎性疼痛,有可能与慢性前列腺炎疼痛的持续和泛化有密切关系.     

Calcium Regulation of EGF-Induced ERK5 Activation: Role of Lad1-MEKK2 Interaction

The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.