RESUMO
The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein. Additional studies with human neuroblastoma cells conducted to address the functional role of C5a receptors revealed that C5a triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-kappa B transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protected terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid A beta peptide. Thus, normal rodent hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results have implications for understanding the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer's disease.
Assuntos
Antígenos CD/biossíntese , Complemento C5a/metabolismo , Neurônios/imunologia , Neurônios/metabolismo , Fragmentos de Peptídeos/biossíntese , Receptores de Complemento/biossíntese , Idoso , Peptídeos beta-Amiloides/toxicidade , Animais , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Sítios de Ligação/imunologia , Química Encefálica/imunologia , Cálcio/metabolismo , Diferenciação Celular/imunologia , Sobrevivência Celular/imunologia , Humanos , Imuno-Histoquímica , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Microesferas , Fatores de Crescimento Neural/fisiologia , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Receptor da Anafilatoxina C5a , Receptores de Complemento/metabolismo , Receptores de Complemento/fisiologia , Transdução de Sinais/imunologia , Células Tumorais CultivadasRESUMO
The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.
Assuntos
Proteínas de Membrana , Receptores de Complemento/química , Receptores de Complemento/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Citometria de Fluxo , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
1 The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. 2 The rabbit C5a receptor stably expressed in RBL cells binds human 125I-C5a (2 nM). Unlabelled C5a and the C-terminal analogue N-acetyl-Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg (Ac-YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. 3 The order of potency human C5a>Ac-YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. 4 The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).
Assuntos
Antígenos CD/efeitos dos fármacos , Antígenos CD/genética , Receptores de Complemento/efeitos dos fármacos , Receptores de Complemento/genética , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Sequência de Bases , Ligação Competitiva/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Clonagem Molecular , Complemento C5a/metabolismo , Complemento C5a/farmacologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Coelhos , Ensaio Radioligante , Receptor da Anafilatoxina C5a , Receptores de Complemento/biossíntese , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Human C3a receptor (huC3aR) belongs to the G-protein coupled receptor family chacterized by having seven transmembrane domains. The huC3aR is a unique member of this family having a large extracellular (EC) loop of 175 amino acids between the 4th and 5th transmembrane domains. Based on a comparison of C3aR sequences from several species, a number of charged and conserved amino acids (Asp182, Asp309, Asp310, and Arg331) in and near the large EC loop of guinea pig C3aR were replaced using site-directed mutagenesis. Competitive binding assays showed that changing Arg331 in guinea pig C3aR to Ala (or Gln), but not changing Asp182, Asp309, or Asp310 to Ala, resulted in complete loss of ligand binding activity. These results and major EC loop deletions demonstrated that an essential C3a binding site is present in the transmembrane portion of C3aR, but not in the large EC loop. Replacement of Arg331 by a noncharged residue was sufficient to eliminate ligand-receptor interactions.
Assuntos
Complemento C3a/metabolismo , Proteínas de Membrana , Receptores de Complemento/metabolismo , Animais , Arginina/genética , Sítios de Ligação , Ligação Competitiva , Cobaias , Humanos , Ligantes , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Receptores de Complemento/genética , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.
Assuntos
Proteínas de Membrana , Receptores de Complemento/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Cobaias , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Ratos , Receptores de Complemento/agonistas , Receptores de Complemento/química , Relação Estrutura-AtividadeRESUMO
The anaphylatoxin C3a is released from C3 during complement activation. C3a is a potent spasmogen and has recently been described as an eosinophil and mast cell chemotactic factor that mediates a number of inflammatory reactions. Previously, we demonstrated the presence of a specific C3a receptor (C3aR) on guinea pig platelets. We report here the isolation of cDNA clones encoding for two isoforms of guinea pig C3aR (gpC3aR). Hydropathy analysis of the deduced amino acid sequence of both gpC3aR clones indicated seven transmembrane domains with a large extracellular (EC) loop between the fourth and fifth transmembrane domains, which is a known characteristic of the human C3aR. Northern blot analysis revealed that the gpC3aR was abundantly expressed on macrophages and in the spleen. A comparison of the deduced amino acid sequence of the larger gpC3aR (gpC3aR-L) with the recently cloned human C3aR indicated a 59.5% identity. The deduced amino acid sequence of the second, smaller cDNA clone was identical with gpC3aR-L, except that it lacked 35 amino acids in the large EC loop. Our evidence indicates that alternative splicing occurred in the large EC loop that accounts for these two isoforms. L cells separately expressing one of these two isoforms of the gpC3aR showed similar high-affinity C3a binding. An RT-PCR analysis documented that both forms of the C3aR were expressed in a variety of guinea pig tissues. The cloning and expression of these two natural forms of gpC3aR cDNA indicated that the deletion of the 35-residue portion of the large EC loop of gpC3aR-L did not alter C3a binding.
Assuntos
Processamento Alternativo/imunologia , Complemento C3a/metabolismo , Proteínas de Membrana , Estrutura Terciária de Proteína , Receptores de Complemento/química , Receptores de Complemento/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Plaquetas/metabolismo , Northern Blotting , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Citometria de Fluxo , Cobaias , Humanos , Isomerismo , Células L , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Reação em Cadeia da Polimerase , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de Complemento/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção/imunologiaRESUMO
A 47-year-old woman was admitted to hospital complaining of swelling and pain of the extremities, accompanied by high fever and generalized erythema. Laboratory examination showed marked blood eosinophilia with elevation of IgM, IgE, and C-reactive protein. All autoantibodies examined were negative. The heart and lungs showed no untoward findings. Biopsies of the skin and muscle revealed cellular infiltration of eosinophils around small blood vessels. Quantitation of C5a receptor (C5aR) expression by flow cytometry using anti-C5aR antibody showed upregulated expression of C5aR on blood eosinophils but downregulated expression on neutrophils. The abnormal C5aR expression on eosinophils and neutrophils became normal after spontaneous resolution of symptoms and blood eosinophilia. The possibility that C5aR expression on granulocytes is related to the pathogenesis of this syndrome may be considered.
Assuntos
Angioedema/complicações , Antígenos CD/metabolismo , Eosinofilia/complicações , Eosinófilos/metabolismo , Receptores de Complemento/metabolismo , Regulação para Cima , Angioedema/sangue , Angioedema/imunologia , Proteína C-Reativa/análise , Complemento C5a/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina E/sangue , Imunoglobulina M/sangue , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Receptor da Anafilatoxina C5aRESUMO
In these studies, we determined whether there are receptors for the anaphylatoxin C5a (C5aR, CD88) on human mesangial cells (HMC). To prepare Abs to C5aR, we first synthesized an immunogenic peptide spanning residues 8-32 of the molecule, and this peptide was used to immunize rabbits. Anti-C5aR antiserum, but not preimmune serum, stained fixed and unfixed HMC in culture. By Western blotting anti-C5aR, Abs identified a 49.6-kDa protein in HMC. By reverse-transcription PCR, a cDNA product of 558 bp was amplified corresponding to the expected size of C5aR cDNA. A cDNA of the same size was amplified simultaneously from human PBL. Restriction mapping of the products amplified from HMC and from PBL gave restriction fragments of the same size. Incubation of HMC with increasing doses of C5a caused a progressive increase in the levels of the transcription factors activator protein-1 (AP-1) and cAMP response element binding protein (CREB), but C5a had no effect on the level of nuclear factor-kappaB (NF-kappaB). The effects of C5a on AP-1 were concentration and time dependent and peaked after 60 min. In contrast, the C5a metabolite C5adesArg had no significant effect on AP-1 levels. Preincubation of HMC with rabbit anti-C5aR antiserum inhibited partially the effect of C5a on AP-1. However, anti-C5aR Abs alone had no appreciable effects on AP-1. C5a caused a significant up-regulation of mRNA for the early response genes c-jun and c-fos on HMC. These results provide evidence for the presence of C5aR in adult HMC in culture and indicate that, after binding to C5aR, the anaphylatoxin C5a causes significant up-regulation of certain transcription factors and early response genes.
Assuntos
Antígenos CD/análise , Complemento C5a/metabolismo , Mesângio Glomerular/metabolismo , Receptores de Complemento/análise , Adulto , Antígenos CD/genética , Células Cultivadas , Complemento C5a/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genes fos/efeitos dos fármacos , Genes fos/imunologia , Genes jun/efeitos dos fármacos , Genes jun/imunologia , Mesângio Glomerular/imunologia , Humanos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/imunologiaRESUMO
The anaphylatoxin C5a receptor (C5aR, CD88 in man) plays a prominent role in mediating inflammatory and host defense processes. Direct evidence of C5aR involvement in host defense mechanisms was demonstrated recently using C5aR knockout mice. Mice deficient in C5aR were unable to clear intrapulmonary-instilled bacteria. The guinea pig system is perhaps unique for exhibiting cross-reactivity with human complement components and its high sensitivity to anaphylatoxins. Therefore, we cloned the guinea pig C5aR from a megakaryocyte cDNA library. The deduced amino acid sequence of guinea pig C5aR is 67% identical to human, 61.6% to dog, 60.2% to mouse and 63.6% to rat C5aR. Transient expression of guinea pig C5aR in COS-7 cells and stable expression on L cell fibroblasts were confirmed by FACS analysis. Competitive binding studies using [125I]C5a and stimulation of calcium mobilization by C5a proved that functional C5aR was expressed on these stably transfected L cells. The N-terminal extracellular region of guinea pig C5aR was five to seven residues shorter than the same region in C5aR from other species and sequence homology was limited to 11%. Other outer membrane loops were also poorly conserved (8-33%) when compared across five species. Transmembrane segments were highly conserved between these various species (46-86%). Guinea pig C5aR binds human C5a, therefore residues critical for C5a binding have been conserved between these species. Sequence comparison of C5aR from multiple species permits conserved elements of the ligand binding sites to be elucidated.
Assuntos
Antígenos CD/genética , Receptores de Complemento/genética , Sequência de Aminoácidos , Animais , Antígenos CD/análise , Antígenos CD/química , Sequência de Bases , Clonagem Molecular , Cães , Cobaias , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Receptor da Anafilatoxina C5a , Receptores de Complemento/análise , Receptores de Complemento/química , Especificidade da EspécieRESUMO
The anaphylatoxin C3a, generated during complement activation, is a factor known to mediate various inflammatory reactions. The human C3a receptor (C3aR) was recently cloned and identified to be a member of the G-protein-coupled receptor family. C3aR is characterized by seven transmembrane domains including a large second extracellular loop that appears to be a unique feature of this receptor. Here we report the isolation of the rat C3aR clone and confirm that the isolated cDNA coded for rat C3aR based on C3a binding analysis to stably transfected cells. Northern blot analysis of rat C3aR revealed expression in various tissues, similar to that of human C3aR but dissimilar to rat C5aR. We found that expression of rat C3aR in various tissues did not increase significantly after LPS injection, whereas rat C5aR expression is greatly increased. These results suggest that expression of C3aR and C5aR is independently regulated in rat cells and tissues.
Assuntos
Antígenos CD/química , Regulação da Expressão Gênica/genética , Lipopolissacarídeos/farmacologia , Proteínas de Membrana , Receptores de Complemento/química , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/fisiologia , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Complemento C3a/metabolismo , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica/efeitos dos fármacos , Células L , Camundongos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/análise , Ratos , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Complemento/fisiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transfecção/genéticaRESUMO
We have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N-formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.
Assuntos
Antígenos CD/análise , Neutrófilos/química , Receptores de Complemento/análise , Receptores de Interleucina/análise , Animais , Antígenos CD/genética , Feminino , Fibroblastos/química , Humanos , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Imunoeletrônica , Ativação de Neutrófilo/imunologia , Neutrófilos/ultraestrutura , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Interleucina-8A , TransfecçãoRESUMO
Activation of the complement cascade plays an essential role in the early stages of inflammation. C5a and its receptor are particularly active in anaphylaxis. To determine the pathological roles played by C5a and C5a receptor (C5aR) in rats, we cloned C5aR cDNA and analyzed distribution of its mRNA in various organs including lung from an LPS-stimulated rat. Furthermore, we generated a polyclonal antiserum which specifically recognizes rat C5aR, as confirmed by its specific interaction with cells transfected with rat C5aR cDNA.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Expressão Gênica , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Animais , Antígenos CD/imunologia , Sequência de Bases , Clonagem Molecular , Citometria de Fluxo , Pulmão/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Receptor da Anafilatoxina C5a , Receptores de Complemento/imunologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
The mouse anaphylatoxin C3a receptor (mC3aR) gene was isolated using a human C3aR cDNA probe. The genomic fragment contains an open reading frame of 1431 base pairs that encodes a peptide of 477 amino acids. A cDNA with identical sequence was subsequently obtained from the mouse pre-B cell line 70Z/3 by reverse transcriptase polymerase chain reaction based on sequence of the mC3aR gene. Northern blot analysis suggested expression of the mC3aR in lung and heart, and to a lesser extent, in brain, liver, muscle, kidney, and testis. The deduced amino acid sequence of the mouse C3aR is 65% identical to that of the human C3aR. Like the human receptor, mouse C3aR contains a predicted large extracellular loop of approximately 165 amino acids (residues 161-325) between the fourth and fifth transmembrane domains. This loop, however, is the least conserved structure (45% identical sequence) of all the extracellular and intracellular domains between the mouse and human C3aRs. The mouse gene product bound 125I-labeled human C3a with a Kd of 2.54 nM when expressed in the stably transfected rat basophilic leukemic cell line RBL-2H3. Bound C3a could be effectively displaced by excess quantities of unlabeled C3a, but not by C4a or C5a. C3a induced dose-dependent calcium mobilization in the transfected cells, which could be blocked by pertussis toxin treatment. These results confirm that the cloned gene encodes a functional C3aR capable of coupling to a pertussis toxin-sensitive G protein. The sequence divergence of the large extracellular loop does not appear to affect C3a binding and transmembrane signaling.
Assuntos
Proteínas de Membrana , Receptores de Complemento/genética , Sequência de Aminoácidos , Anafilatoxinas/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Receptores de Complemento/biossíntese , Receptores de Complemento/metabolismo , Análise de Sequência , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , TransfecçãoRESUMO
In summary, recent advances in molecular cloning of anaphylatoxins and the anaphylatoxin receptors add new dimensions to our investigations and understanding of the molecular mechanisms involved in anaphylatoxin action. Combining knowledge accumulated from peptide modeling of the ligands with mutagenesis studies of these ligands and their receptors makes it possible to more accurately model interactive sites and understand the sequence of molecular interactions required for cellular activation. In addition, these new developments provide valuable tools for investigating, yet unknown, activities and cellular targets of the anaphylatoxin molecules.
Assuntos
Anafilatoxinas/imunologia , Anafilaxia/imunologia , Ativação do Complemento/imunologia , Receptores de Complemento/genética , Sequência de Aminoácidos , Anafilatoxinas/química , Anafilatoxinas/genética , Anafilaxia/genética , Complexo Antígeno-Anticorpo , Clonagem Molecular , Ativação do Complemento/genética , Complemento C3a/análogos & derivados , Complemento C3a/genética , Complemento C3a/imunologia , Complemento C4a/química , Complemento C4a/genética , Complemento C4a/imunologia , Complemento C5a/química , Complemento C5a/genética , Complemento C5a/imunologia , Humanos , Dados de Sequência Molecular , Mutagênese/genética , Mutagênese/imunologia , Receptores de Complemento/química , Receptores de Complemento/imunologiaRESUMO
The anaerobic bacteria P. gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacteria and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Collagenases and cysteine proteinases (i.e., the gingipains) have been characterized as the predominant vesicular enzymes produced by this bacterium. It has been shown that an arginine-specific cysteine proteinase from P. gingivalis, called gingipain-1 or Arg-gingipain, can selectively cleave complement components C3 and C5. In the case of C5, cleavage by Arg-gingipain results in the generation of C5a, a potent chemotactic factor for PMNs. Since these bacterial proteinases are capable of generating pro-inflammatory factors at sites of infection, we examined the possibility that gingipains or other proteinases from this bacterium might attack or destroy cell surface proteins, such as receptor molecules. Using an affinity-purified rabbit antibody raised against residues 9-29 of the C5a receptor (i.e., C5aR; CD88), the signal transmitting element for the pro-inflammatory mediator C5a, we demonstrated that the mixture of proteinases in P. gingivalis vesicles cleaves the C5a receptor on human neutrophils. This vesicular proteinase activity did not require cysteine activation which indicates that proteinases other than the gingipains may be responsible for cleavage of the C5aR molecule. in addition, the purified Lys-gingipain, but not Arg-gingipain, also cleaved C5aR on the human neutrophils. The N-terminal region of CaR (residues 9-29, PDYGHYDDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin, but not by trypsin, despite the presence of potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of C5aR 9-29 peptide cleavage were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain. These studies suggest that the proteolytic activity in the bacterial vesicles that is responsible for cleaving C5aR is primarily a non-tryptic proteinase, distance from either Arg- or Lys-gingipain. Consequently, there appear to be additional proteinase(s) in the vesicles that attacks the cell surface molecule C5aR which are not the same (i.e., Arg- and Lys-gingipain) as were shown to generate pro-inflammatory activity from complement components C3 and C5. Evidence that the proteinases which attack the inflammatory precursor molecules (i.e., C3 and C5) exhibit different specificities than those that attack receptors to these bioactive complement products makes a particularly interesting story of how this bacteria avoids major host defense mechanisms. It is well known that generation of pro-inflammatory factors such as C3a and C5a at extra-vascular sites can promote edema, leukocyte recruitment and cellular activation responses that could lead to the release of toxic oxygen products and to phagocytosis of the bacteria. Destruction of receptors to these cellular activating factors generated by bacterial proteinases may eliminate the ability of these (i.e., complement-derived) and other mediators to carry out their anti-bacterial actions and thereby limit the host's defense mechanisms in responses to the infecting bacteria. The concept of anti-bacterial responses (i.e., oxygen radical generation and phagocytosis) being effectively eliminated at the injury site, by bacterial proteinases acting at the cellular receptor level, has not been studied in detail. In this case, the situation is particularly unusual because, once the bacterial gingipains generate potent plasma-derived inflammatory factors that can enhance edema and deliver essential nutrients to the bactgeria, other bacterial proteinases may destsroy their cellular receptors. These receptors transmit the signal activation mechanisms in the infiltrating cells that elicit bacterial killing.(ABSTRACT TRUNCATED)
Assuntos
Antígenos CD/sangue , Complemento C5a , Cisteína Endopeptidases/sangue , Endopeptidases/sangue , Hemaglutininas/sangue , Leucócitos/metabolismo , Porphyromonas gingivalis/enzimologia , Receptores de Complemento/sangue , Adesinas Bacterianas , Sequência de Aminoácidos , Cisteína Endopeptidases/isolamento & purificação , Endopeptidases/isolamento & purificação , Cisteína Endopeptidases Gingipaínas , Hemaglutininas/isolamento & purificação , Humanos , Dados de Sequência Molecular , Receptor da Anafilatoxina C5a , Transdução de Sinais/fisiologiaRESUMO
A series of decapeptide analogues corresponding to the C-terminal region of the human C5a anaphylatoxin (C5a65-74) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminal region (residues 71-74). These analogues behaved as full agonists of natural C5a in their ability to induce shape change (polarization) and the release of enzyme (beta-glucuronidase) from human neutrophils (PMNs). There was a significant pharmacological correlation between the polarization and enzyme-release assays, suggesting similarities in PMN responsiveness toward these constrained peptides. Good correlations were also observed between these two PMN responses and spasmogenic activity (smooth muscle contraction of human fetal artery). A structure-function analysis for PMN polarization and enzyme release led to the identification of the following preferred backbone conformations: a twisted, helix-like conformation for residues 65-69, an extended conformation for residues 70-71, and a beta-turn of type V for residues (71)72-74. The existence of a C-terminal, type V beta-turn is supported by the NOE (nuclear Overhauser effect) results of two peptides from this series. These conformational features are reminiscent of those that were shown to correlate with the expression of spasmogenic and platelet aggregatory activities in an earlier investigation (Sanderson, S.D.; et al. J. Med. Chem. 1994, 37, 3171). These results suggest that PMNs and the cells responsible for smooth muscle contraction possess C5a receptors that respond to similar topochemical features presented by the agonist peptide ligand.
Assuntos
Anafilatoxinas/química , Complemento C5a/agonistas , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Células Cultivadas , Glucuronidase/metabolismo , Humanos , Dados de Sequência Molecular , Oligopeptídeos/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Acute inflammation is characterized by increased production of acute phase proteins in the liver. The induction of the hepatocytic response is primarily mediated through soluble cytokines such as IL-1, IL-6, TNF-alpha, and transforming growth factor beta, which bind to specific cell surface receptors and regulate gene expression of acute-phase proteins. Hepatoma cell lines, such as HepG2, represent a model system for studying acute-phase protein synthesis. HepG2 is induced to produce a variety of acute-phase proteins, including alpha 1-antitrypsin, alpha 1-antichymotrypsin, fibrinogen, alpha 1-acid glycoprotein, and haptoglobin, upon stimulation with cytokines. Analysis of HepG2 by reverse transcriptase PCR indicated that this cell line synthesized mRNA specific for the human C5a receptor (CD88). Flow cytometric analysis of HepG2 cells indicated that these cells bound anti-CD88 Ab, thus confirming our RT-PCR data by demonstrating that these cells also express the C5a receptor. Because C5a has been shown to be a potent mediator of inflammation and HepG2 cells express CD88, we assessed the possibility that C5a was capable of stimulating acute-phase protein synthesis by HepG2 cells. The results indicate that binding of human C5a to CD88 on HepG2 cells resulted in an increased production of alpha 1-antitrypsin- and alpha 1-antichymotrypsin-specific mRNA as assayed by RT-PCR. Analysis of culture supernatants derived from C5a-stimulated HepG2 cells showed an increased production of alpha 1-antitrypsin as measured by solid-phase ELISA. alpha 1-antitrypsin production by HepG2 cells was a direct result of C5a stimulation as evidenced by the fact that anti-C5a receptor Ab inhibited the response. These results suggest that C5a may be an important mediator of APP production in the regulation of the inflammatory response.
Assuntos
Proteínas de Fase Aguda/biossíntese , Antígenos CD/fisiologia , Complemento C5a/fisiologia , RNA Mensageiro/análise , Receptores de Complemento/fisiologia , Proteínas de Fase Aguda/genética , Animais , Antígenos CD/genética , Complemento C5a/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Células Tumorais Cultivadas , alfa 1-Antiquimotripsina/genéticaRESUMO
We have shown previously that the extracellular cysteine proteinase of Entamoeba histolytica trophozoites activates the alternative pathway of complement by specifically cleaving C3. This unique mechanism of complement activation leads to passive lysis of nonpathogenic, but not of pathogenic strains. In an attempt to investigate the relationship between the cleavage of complement components C3 and C5 and the pathogenesis of amebiasis, we investigated the production of the anaphylatoxins C3a and C5a, which have diverse effects on the host immune response. The concentration of proteinase required to cleave purified C5 was at least 5 to 10 times that needed for C3 cleavage, but these levels are easily obtainable as demonstrated by cleavage of 125I-labeled C5 during incubation with purified trophozoites. When the C3a-like cleavage fragments were purified by gel filtration, they were found to be extensively degraded during a 1-h incubation of C3 with the proteinase. Subsequent evaluations of the C3a- and C5a-like cleavage products generated earlier in the reaction using immunoblots and cellulose acetate electrophoresis revealed rapid degradation, even during incubation periods as short as 5 min. Because C-terminal fragments as small as 20 amino acid residues can mimic the biologic functions of C3a or C5a, we tested cleavage products for activity. In sensitive bioassays, including guinea pig platelet aggregation for C3a activity and chemotaxis for C5a activity, we demonstrated that proteolysis renders these molecules inactive. These studies suggest that the extracellular cysteine proteinase of E. histolytica, which is capable of activating the complement system, may also provide a mechanism to circumvent normal host immunity by inactivating the proinflammatory factors C3a and C5a.
Assuntos
Anafilatoxinas/antagonistas & inibidores , Anafilatoxinas/metabolismo , Cisteína Endopeptidases/farmacologia , Entamoeba histolytica/enzimologia , Animais , Quimiotaxia de Leucócito/fisiologia , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Cisteína Endopeptidases/imunologia , Imunidade Inata/efeitos dos fármacos , Immunoblotting , Agregação Plaquetária/fisiologia , Fatores de TempoRESUMO
Inflammatory action of the potent chemotaxin C5a has been well characterized on a variety of human cell types, including neutrophils, monocytes, basophils, and eosinophils. The cellular effects of C3a are less well defined. Contradictory reports have been published for C3a activation of neutrophils. Recent reports that C3a activates both basophils and eosinophils prompted us to reinvestigate the effects of C3a stimulation on eosinophils. We hypothesized that C3a activation of eosinophils, cells that are present in most neutrophil preparations, might lead to neutrophil activation. Using neutrophils of 98% purity, we observed no evidence of cellular activation after stimulation with either C3a, recombinant human C3a (rhC3a), or the synthetic C3a analogue C3a 57-77, Y57. Eosinophils purified to > 98% purity displayed concentration-dependent polarization, chemotaxis, and enzyme release by stimulation with C3a, rhC3a, and the synthetic C3a analogue. An inactive form of C3a, C3adesArg, failed to stimulate either eosinophils or neutrophils. Using neutrophil preparations containing 5-9% eosinophils, up to 20% of neutrophils became polarized after exposure to C3a. Likewise, we demonstrated that supernatant from C3a-stimulated eosinophils promotes neutrophil chemotaxis. Eosinophil polarization experiments were repeated in the presence of antibody to the C5a receptor (C5aR) to show that C3a and C5a interact with different receptors. C3a activates eosinophils in the presence of anti-C5aR antibody at concentrations that fully block C5a activation. We conclude that eosinophils are directly activated by either C3a or C5a, whereas C3a failed to activate neutrophils. C3a acts on eosinophils via a receptor that is distinct from C5aR. Since neutrophils are indirectly stimulated by C3a, eosinophils contaminating neutrophil preparations may explain earlier reports that C3a activates human neutrophils.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Complemento C3a/farmacologia , Eosinófilos/fisiologia , Ativação de Neutrófilo , Neutrófilos/fisiologia , Sequência de Aminoácidos , Quimiotaxia de Leucócito/efeitos dos fármacos , Complemento C5a/farmacologia , Relação Dose-Resposta a Droga , Eosinófilos/efeitos dos fármacos , Glucuronidase/análise , Glucuronidase/sangue , Humanos , Cinética , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
There is increasing interest in the role of polymorphonuclear (PMN) leukocytes in the evolution of focal cerebral infarction. Surgical preparation of focal cerebral ischemia models may alter leukocyte reactivity and thereby make interpretation of leukocyte function following ischemia/reperfusion difficult. The effects of surgical preparation and of experimental ischemia/reperfusion on granulocyte function have been examined prospectively in a baboon model. Twenty-six adolescent male baboons underwent surgical preparation, of which 21 underwent middle cerebral artery occlusion/reperfusion. Four additional animals served as nonsurgical controls. Peripheral venous blood specimens were taken for performing assays of leukocyte function at defined intervals before and after both the surgical preparation (i.e., the overall procedure for implantation of the middle cerebral artery occlusion device) and occlusion/reperfusion. A stress-related elevation in total leukocyte number was attributed mainly to an increase in the number of circulating PMN leukocytes. Values rose from 13.9 +/- 4.9 x 10(3) to 27.8 +/- 5.8 x 10(3)/microliters, (+/- SD; n = 21) for total leukocyte number, with p < 0.001, and from 4.3 +/- 2.1 x 10(3) to 15.9 +/- 4.7 x 10(3)/microliters (n = 21) for PMN leukocytes, with p < 0.001. Surgical preparation had no effect (p > or = 0.4) on the ability of PMN leukocytes, isolated 24 h after the implantation procedure, to display polarization, O2.- production, or beta-glucuronidase release when stimulated with human C5a. A moderate decrease in the chemotactic response to C5a resolved within the 7-day postsurgery (preocclusion) period. Three-hour middle cerebral artery occlusion and 1-h reperfusion resulted in a significant reduction in C5a-induced polarization.(ABSTRACT TRUNCATED AT 250 WORDS)