RESUMO
Based on protein sequence, we have isolated a cDNA for intercellular adhesion molecule 3 (ICAM-3), the most recently defined counter-receptor for lymphocyte function-associated antigen 1 (LFA-1). Expression of the cDNA yields a product that reacts with monoclonal antibody to ICAM-3 and functions as a ligand for LFA-1. The deduced 518-amino acid sequence of the predicted mature protein defines a highly glycosylated type I integral membrane protein with five immunoglobulin (Ig)-like domains. The five Ig-like domains of ICAM-3 are highly homologous with those of human ICAM-1 (52% identity) and human ICAM-2 (37% identity).
Assuntos
Antígenos CD , Antígenos de Diferenciação , Moléculas de Adesão Celular/genética , Imunoglobulinas/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Receptores de Antígenos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , DNA , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Camundongos , Dados de Sequência Molecular , Receptores de Antígenos/química , Receptores de Antígenos/metabolismo , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.
Assuntos
Complemento C3b/metabolismo , Proteínas Inativadoras do Complemento/farmacologia , Fragmentos Fab das Imunoglobulinas/fisiologia , Receptores de Complemento/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação , Via Alternativa do Complemento , Humanos , Camundongos , Dados de Sequência Molecular , Receptores de Complemento 3b , Receptores de Complemento 3d , Sequências Repetitivas de Ácido NucleicoRESUMO
10 overlapping CR1 cDNA clones that span 5.5 kb were isolated from a tonsillar library and sequenced in whole or in part. A single long open reading frame beginning at the 5' end of the clones and extending 4.7 kb downstream to a stop codon was identified. This sequence represents approximately 80% of the estimated 6 kb of coding sequence for the F allotype of CR1. Three tandem, direct, long homologous repeats (LHRs) of 450 amino acids were identified. Analysis of the sequences of tryptic peptides provided evidence for a fourth LHR in the F allotype of CR1. Amino acid identity between the LHRs ranged from 70% between the first and third repeats to 99% between the NH2-terminal 250 amino acids of the first and second repeats. Each LHR comprises seven short consensus repeats (SCRs) of 60-70 amino acids that resemble the SCRs of other C3/C4 binding proteins, such as complement receptor type 2, factors B and H, C4 binding protein, and C2. Two additional SCRs join the LHRs to a single membrane-spanning domain of 25 amino acids; thus, the F allotype of CR1 probably contains at least 30 SCRs, 23 of which have been sequenced. Each SCR is predicted to form a triple loop structure in which the four conserved half-cystines form disulfide linkages. The linear alignment of 30 SCRs as a semi-rigid structure would extend 1,140A from the plasma membrane and might facilitate the interaction of CR1 with C3b and C4b located within the interstices of immune complexes and microbial cell walls. The COOH-terminal cytoplasmic domain of 43 residues contains a six-amino-acid sequence that is homologous to the sequence in the epidermal growth factor receptor that is phosphorylated by protein kinase C.
Assuntos
Receptores de Complemento/genética , Sequência de Aminoácidos , Sequência de Bases , Complemento C3b/metabolismo , Complemento C4/metabolismo , Complemento C4b , DNA/análise , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Receptores de Complemento/metabolismo , Receptores de Complemento 3b , Sequências Repetitivas de Ácido NucleicoRESUMO
Complement receptor type 2 (CR2;CD21), a member of the superfamily of proteins containing short consensus repeats (SCRs), is the B cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV), and for the C3dg protein of complement. By analysis of CR2 deletion mutants and chimeras formed with CR1 (CD35) we determined that of the 15 SCRs in CR2, the NH2-terminal two SCRs are necessary and sufficient to bind both gp350/220 and C3dg with affinities equivalent to those of the wild-type receptor. The epitope for OKB-7, a mAb that blocks binding of both EBV and C3dg and shares with these ligands B cell-activating capabilities, also requires both SCR-1 and SCR-2, whereas mAbs lacking these functions bind to other SCRs. Thus, EBV, a polyclonal activator of B cells, has selected a site that is proximate or identical to the natural ligand binding site in CR2, perhaps reflecting the relative immutability of that site as well as its signal transducing function.
Assuntos
Herpesvirus Humano 4/metabolismo , Receptores de Complemento/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sítios de Ligação , Quimera , Deleção Cromossômica , Epitopos/análise , Células L/imunologia , Camundongos , Receptores de Complemento 3d , TransfecçãoRESUMO
Complementary DNA clones encoding the NH2-terminal region of human CR1 have been isolated and sequenced. The deduced complete amino acid sequence of the F allotype of human CR1 contains 2,039 residues, including a 41-residue signal peptide, an extracellular domain of 1,930 residues, a 25-amino acid transmembrane domain, and a 43-amino acid cytoplasmic region. The extracellular domain is composed exclusively of 30 short consensus repeats (SCRs), characteristic of the family of C3/C4-binding proteins. The 28 NH2-terminal SCRs are organized as four long homologous repeats (LHRs) of seven SCRs each. The newly sequenced LHR, LHR-A, is 61% identical to LHR-B in the NH2-terminal two SCRs and greater than 99% identical in the COOH-terminal five SCRs. Eight cDNA clones were spliced to form a single construct, piABCD, that contained the entire CR1 coding sequence downstream of a cytomegalovirus promoter. COS cells transfected with piABCD transiently expressed recombinant CR1 that comigrated with the F allotype of erythrocyte CR1 on SDS-PAGE and that mediated rosette formation with sheep erythrocytes bearing C4b and C3b. Recombinant CR1 also had factor I-cofactor activity for cleavage of C3(ma). Analyses of six deletion mutants expressed in COS cells indicated that the NH2-terminal two SCRs of LHR-A contained a site determining C4 specificity and the NH2-terminal two SCRs of LHR-B and -C each had a site determining C3 specificity. The presence of these three distinct sites in CR1 may enable the receptor to interact multivalently with C4b/C3b and C3b/C3b complexes generated during activation of the classical and alternative pathways.
Assuntos
Complemento C3b/metabolismo , Complemento C4/metabolismo , Receptores de Complemento/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Complemento C4b , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Peso Molecular , Receptores de Complemento/genética , Receptores de Complemento/fisiologia , Receptores de Complemento 3b , Mapeamento por Restrição , TransfecçãoRESUMO
The organization and physical linkage of four members of a major complement locus, the RCA locus, have been determined using the technique of pulsed field gradient gel electrophoresis in conjunction with Southern blotting. The genes encoding CR1, CR2, DAF, and C4bp were aligned in that order within a region of 750 kb. In addition, the 5' to 3' orientation of the CR1 gene (5' proximal to CR2) was determined using 5'- and 3'-specific DNA probes. The proximity of these genes may be related to structural and functional homologies of the protein products. Overall, a restriction map including 1,500 kb of DNA was prepared, and this map will be important for positioning of additional coding sequences within this region on the long arm of chromosome 1.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 1 , Proteínas de Membrana/genética , Receptores de Complemento/genética , Antígenos CD55 , Mapeamento Cromossômico , Genes , Ligação Genética , Humanos , Integrina alfaXbeta2 , Receptores de Complemento 3b , Receptores de Complemento 3dRESUMO
Human CR1 exhibits an unusual form of polymorphism in which allotypic variants differ in the molecular weight of their respective polypeptide chains. To address mechanisms involved in the generation of the CR1 allotypes, DNA from individuals having the F allotype (250,000 Mr), the S allotype (290,000 Mr), and the F' allotype (210,000 Mr) was digested by restriction enzymes, and Southern blots were hybridized with CR1 cDNA and genomic probes. With the use of Bam HI and Sac I, an additional restriction fragment was observed in 20 of 21 individuals having the S allotype with no associated loss of other restriction fragments. Southern blot analysis with a noncoding genomic probe derived from the S allotype-specific Bam HI fragment showed hybridization to this fragment and to two other fragments that were also present in FF individuals. Thus, an intervening sequence may be repeated twice in the F allele and three times in the S allele. A restriction fragment length polymorphism (RFLP) unique to two individuals expressing the F' allotype was seen with Eco RV, but the absence of persons homozygous for this rare allotype prevented further comparisons with the F and S allotypes. Analysis of the CR1 transcripts associated with the three CR1 allotypes indicated that these differed by 1.3-1.5 kb and had the same rank order as the corresponding allotypes. Taken together, these findings suggest that the S allele was generated from the F allele by the acquisition of additional sequences, the coding portion of which may correspond to a long homologous repeat of approximately 1.4 kb that has been identified in CR1 cDNA. We saw two other RFLPs with Hind III and Pvu II that were in linkage dysequilibrium with the Bam HI-Sac I RFLPs associated with the S allotype, and a third polymorphism was seen with Eco RI that was not in linkage dysequilibrium with the other polymorphisms. Thus, 10 commonly occurring CR1 alleles can be defined, making this locus a useful marker for the long arm of chromosome 1 to which the CR1 gene maps.
Assuntos
Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Receptores de Complemento/genética , Sequências Repetitivas de Ácido Nucleico , Alelos , DNA/análise , Humanos , RNA Mensageiro/análise , Receptores de Complemento/análise , Transcrição GênicaRESUMO
A genetic basis for the regulation of the number of CR1 on E of different normal individuals was investigated by probing Southern blots of their genomic DNA with a 0.75-kb fragment of CR1 cDNA. Using Hind III, we observed a RFLP involving fragments of 7.4 kb and 6.9 kb that correlated with the number of CR1 on E. 32 individuals having only the 7.4-kb restriction fragment had a mean of 661 +/- 33 (SEM) CR1/E, 11 donors having both restriction fragments had a mean of 455 +/- 52 CR1/E, and 7 individuals having only the 6.9-kb fragment had a mean of 156 +/- 13 CR1/E, all means being significantly different (p less than 0.005). Cosegregation in a normal family of the Hind III restriction fragments with the S, F, and F' structural allotypes of CR1 confirmed that the regulatory element identified by these fragments is linked to the CR1 gene. Moreover, an analysis of the relative expression on E of these structural allotypes in association with either the 7.4-kb Hind III fragment or the 6.9-kb fragment showed that this regulatory element is cis-acting. In contrast, quantitation of CR1 of B lymphocytes and neutrophils revealed no differences in total CR1 expression between individuals homozygous for the 7.4-kb and 6.9-kb Hind III fragments. Thus, we have identified a genomic polymorphism that is linked to the CR1 gene and is associated with a cis-acting regulatory element for the expression of CR1 on E.
Assuntos
Enzimas de Restrição do DNA/genética , DNA/genética , Eritrócitos/metabolismo , Polimorfismo Genético , Receptores de Complemento/genética , Genes , Genes Dominantes , Humanos , Alótipos de Imunoglobulina/genética , Hibridização de Ácido Nucleico , Receptores de Complemento/metabolismo , Receptores de Complemento 3bRESUMO
Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1-MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL-immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.
Assuntos
Proteínas de Transporte/metabolismo , Receptores de Complemento 3b/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Proteínas de Transporte/isolamento & purificação , Adesão Celular/efeitos dos fármacos , Colectinas , Complemento C1q/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Fibronectinas/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Radioisótopos do Iodo , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Receptores de Complemento 3b/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Salmonella/imunologia , Salmonella/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The CD21/CD19/TAPA-1 complex of B lymphocytes amplifies signal transduction through membrane immunoglobulin (mIg), recruits phosphatidylinositol 3-kinase (PI3-kinase), and induces homotypic cellular aggregation. The complex is unique among known membrane protein complexes of the immune system because its components represent different protein families, and can be expressed individually. By constructing chimeric molecules replacing the extracellular, transmembrane, and cytoplasmic regions of CD19 and CD21 with those of HLA-A2 and CD4, we have determined that CD19 and TAPA-1 interact through their extracellular domains, CD19 and CD21 through their extracellular and transmembrane domains, and, in a separate complex, CD21 and CD35 through their extracellular domains. A chimeric form of CD19 that does not interact with CD21 or TAPA-1 was expressed in Daudi B lymphoblastoid cells and was shown to replicate two functions of wild-type CD19 contained within the complex: synergistic interaction with mIgM to increase intracellular free calcium and tyrosine phosphorylation and association with the p85 subunit of PI3-kinase after ligation of mIgM. The chimeric CD19 lacked the capacity of the wild-type CD19 to induce homotypic cellular aggregation, a function of the complex that can be ascribed to the TAPA-1 component. The CD21/CD19/TAPA-1 complex brings together independently functioning subunits to enable the B cell to respond to low concentrations of antigen.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Linfócitos B/metabolismo , Proteínas de Membrana/fisiologia , Receptores de Complemento 3d/fisiologia , Antígenos CD/química , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/química , Sequência de Bases , Linhagem Celular , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Receptores de Complemento 3b/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetraspanina 28 , Células Tumorais CultivadasRESUMO
Structural and quantitative polymorphisms have been described in human CR1. In the former, the S allotype is larger than the F allotype by 40-50 kD, the size of a long homologous repeat (LHR). In the latter, homozygotes for a 7.4-kb Hind III fragment express fourfold more CR1 per erythrocyte than do homozygotes for the allelic 6.9-kb restriction fragment. The basis for these genomic polymorphisms has been determined by restriction mapping the entire S allele and part of the F allele. The S allele is 158 kb and contains 5 LHRs of 20-30 kb, designated -A, -B/A, -B, -C, and -D, respectively, 5' to 3'. Extensive homology was found among the LHRs in their restriction maps, exon organization, and the coding and noncoding sequences. The presence of LHR-B/A in the S allele but not in the F allele accounts for the longer transcripts and polypeptide associated with the former allotype. At least 42 exons are present in the S allele, with distinct exons for the leader sequence, the transmembrane and cytoplasmic regions and most of the SCRs comprising the extracellular portion of CR1. Consistent with the mapping of the ligand binding site to the first two SCRs in each LHR, the second SCRs in LHR-A, -B/A, -B, and -C are encoded by two exons, reflecting a specialized function for this unit. The allelic 7.4/6.9-kb Hind III fragments extend from the 3' region of LHR-C to LHR-D. The 6.9-kb restriction fragment is the result of a new Hind III site generated by a single base change in the intron between the exons encoding the second SCR of LHR-D. A second cluster of genomic clones has been identified by hybridization to CR1 probes. Although they contain regions of hybridization to the cDNA and genomic probes derived from CR1, these cannot be overlapped with the structural gene owing to their distinct restriction maps. Three genomic polymorphisms previously identified by CR1 cDNA probes map to this region. These additional clones may represent part of a duplicated allele located nearby within the CR1 locus.
Assuntos
Alelos , Polimorfismo Genético , Receptores de Complemento/genética , Sequência de Aminoácidos , Sequência de Bases , Cosmídeos , Sondas de DNA , DNA Recombinante , Desoxirribonuclease BamHI , Desoxirribonuclease EcoRI , Desoxirribonuclease HindIII , Desoxirribonucleases de Sítio Específico do Tipo II , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Receptores de Complemento 3b , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
A human neutrophil lysosomal protease interacts at physiologic pH with a 62,000--67,000-mol wt plasma protein substrate to generate a vasoactive, smooth muscle-contracting "neutral" peptide. The peptide product of this system, previously designated the "neutral" peptide-generating pathway, was generated from purified components and purified by Bio-Gel P2 gel filtration and reverse-phase high performance liquid chromatography with a 50--60% yield of starting activity. The purified peptide had an amino acid composition of Asx, Pro, Val, Ile, Tyr, Phe, His, Arg, a composition identical to that of angiotensin II. The peptide and synthetic angiotensin II each filtered at 48--52% bed volume on Bio-Gel P2, had an isoelectric point of Ph 7.8--8.1 at 4 degrees C, migrated 3 cm toward the cathode during pH 6.4 low-voltage paper electrophoresis, and had a retention time of 44.8 min during reverse-phase high-performance liquid chromatography. In addition, the functional activity of the peptide at each purification step correlated with angiotensin II content determined by specific radioimmunoassay. The amino acid sequence of 25 nmol of the peptide was Asp-Arg-Val-Try-Ile-His-Pro-Phe, the same covalent structure as that of angiotensin II. Therefore, under physiologic conditions, in the absence of renin or angiotensin converting enzyme, a human neutrophil neutral protease cleaves a plasma protein to yield angiotensin II. This pathway provides a mechanism through which the neutrophil may alter local blood flow during inflammation by generation of a potent vasoactive peptide.
Assuntos
Angiotensina II/biossíntese , Neutrófilos/metabolismo , Aminoácidos/análise , Angiotensina I/análise , Angiotensina II/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Endopeptidases/sangue , Humanos , Ponto IsoelétricoRESUMO
The predominant lipoxygenase products of arachidonic acid were extracted and purified from synovial fluid and sonicates of synovial tissue of patients with rheumatoid arthritis (RA), spondyloarthritis (SA), or a noninflammatory arthropathy (NIA). The concentration of 5(S),12(R)-dihydroxy-6,8,10-(trans/trans/cis)-14-cis-eicosatetraenoic acid (leukotriene B4) in synovial fluid was elevated significantly in patients with RA and a positive latex test for rheumatoid factor (P < 0.05, n = 14) and in patients with SA (P < 0.05, n = 10), compared with that of subjects with NIA (n = 9). The content of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), but not of leukotriene B4, was elevated significantly in synovial tissue of seven patients with RA in comparison with that of four subjects with NIA (P < 0.05). A single intra-articular injection of corticosteroid significantly lowered the synovial fluid level of leukotriene B4 in six patients with RA. These data suggest an involvement of the potent chemotactic factors 5-HETE and leukotriene B4 in human inflammatory disease.
Assuntos
Ácidos Araquidônicos/análise , Ácidos Araquidônicos/metabolismo , Artrite Reumatoide/sangue , Fatores Quimiotáticos/sangue , Ácidos Hidroxieicosatetraenoicos , Neutrófilos/fisiopatologia , Espondilite Anquilosante/sangue , Leucotrieno B4 , Lipoxigenase/metabolismo , Líquido Sinovial/análiseRESUMO
Aggregated or immobilized complement C1q induces cellular responses in many different cell types. C1q-induced cellular responses may be involved in host defense and in protection against autoimmunity because C1q-deficient humans have infectious complications and a very high incidence of autoimmune disease. The search for the C1q receptor(s), which has been ongoing for 25 years, has led recently to the recognition that proteins identified as binding to C1q may be divided into two groups: C1q-binding molecules that are normally intracellular; and cell surface C1q receptors.
Assuntos
Proteínas de Transporte , Complemento C1q/imunologia , Receptores de Hialuronatos , Glicoproteínas de Membrana , Proteínas/imunologia , Receptores de Complemento/imunologia , Complemento C1q/metabolismo , Humanos , Imunidade Inata , Proteínas Mitocondriais , Proteínas/metabolismo , Receptores de Complemento/metabolismoRESUMO
We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (alphaMbeta2) but not lymphocyte function-associated antigen-1 (LFA-1; alphaLbeta2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1alpha in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1alpha were confirmed by expression of alphaM or alphaL in alphaL-deficient Jurkat cells. Moreover, expression of chimeras containing alphaL and alphaM cytoplasmic domain exchanges indicated that alpha cytoplasmic tails conferred the specific mode of regulation. Coexpressing alphaM or chimeras in mutant Jurkat cells with a "gain of function" phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the alphaL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of beta2 integrins. Our data suggest that a specific regulation of beta2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the alpha subunit cytoplasmic domains.
Assuntos
Antígenos CD18/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Antígeno de Macrófago 1/fisiologia , Receptores de Citocinas/fisiologia , Adesão Celular , Quimiocina CCL2/farmacologia , Quimiocina CCL4 , Quimiocina CCL5/farmacologia , Citoplasma/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/genética , Proteínas Inflamatórias de Macrófagos/farmacologia , Receptores CCR2 , Receptores de Quimiocinas/fisiologia , Receptores de Citocinas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.
Assuntos
Movimento Celular/fisiologia , Quimiocinas CXC/metabolismo , Genes ras/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas Serina-Treonina Quinases , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Endotélio/citologia , Endotélio/metabolismo , Genes ras/genética , Humanos , Integrina alfa4beta1 , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Células Jurkat , Leucócitos/fisiologia , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/farmacologia , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Isolated human polymorphonuclear neutrophils (PMN) responded to human C5a with an immediate, transient release of superoxide lasting from 0.5 to 5 min. This was followed by a second release of superoxide, which began at 10 min after addition of C5a, was sustained for more than 30 min, and required ICAM-1 immobilized in the wells. F(ab')2 monoclonal antibody (mAb) preparations were used to dissect the role of individual beta2 integrins and to avoid the confounding effects of ligating Fc receptors. Anti-CD18 mAb treatment of the PMN had no effect on the immediate first phase but completely inhibited the second, adhesion-dependent phase of superoxide production. Anti-CR3 mAb only inhibited the adhesion phase of superoxide production partially, implying that other beta2 integrins were involved. A mixture of anti-CD11a, anti-CD11b, and anti-CD11c was not able to block superoxide production completely, suggesting a role for alphad/beta2. Surprisingly, blocking anti-LFA-1 mAb had no effect on superoxide production. Consistent with this observation, immobilized, purified ICAM-2, a specific counter-receptor for LFA-1, did not support the adhesion-dependent phase of-superoxide production. Thus, PMN treated with C5a used signals via CR3, P150/95, and alphad/beta2, but not LFA-1, to support superoxide production. LFA-1 has been shown by others to mediate most of the adhesion necessary for transendothelial migration in vivo. The inability of LFA-1 ligation to stimulate superoxide production may be an important means of preventing blood-vessel damage when PMN migrate across the endothelium.
Assuntos
Antígenos CD18/fisiologia , Complemento C5a/farmacologia , Neutrófilos/metabolismo , Superóxidos/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos CD/metabolismo , Antígenos CD18/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/fisiologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estimulação QuímicaRESUMO
This report is devoted to methodologies used in analyzing the C3b/C4b receptor (CR1, CD35) on erythrocytes (E), its soluble form, the CRI structural or allotype polymorphism, and CR1 density polymorphism. In primates E CR1 serves as the main system for processing and clearance of complement opsonized immune complexes (IC). CR1 copy numbers decrease with aging of E in normal individuals. Erythrocyte CR1 is also decreased in pathological conditions such as systemic lupus erythematosus (SLE), HIV infection, certain hemolytic anemias, and many other conditions featuring immune complexes. Consequently, CRI on E has an important physiological role in immune complex handling and has interesting alterations in disease.
Assuntos
Eritrócitos/imunologia , Técnicas Genéticas , Técnicas Imunológicas , Polimorfismo Genético , Receptores de Complemento 3b/genética , Animais , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/imunologia , Southern Blotting , Mapeamento Cromossômico , Ativação do Complemento/genética , DNA/genética , Humanos , Processamento de Imagem Assistida por Computador , Família Multigênica , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Receptores de Complemento 3b/análise , Receptores de Complemento 3b/metabolismo , SolubilidadeRESUMO
Measurement of erythrocyte [red blood cells (RBC)] complement receptor type 1 (CR1, CD35) has the potential to serve as a sensitive assessment of complement activation and immune complex clearance. All previously reported monoclonal antibodies (MoAb) to the extracellular region of CR1 recognize epitopes within the long homologous repeats (LHR) of CR1 and the epitopes for the most frequently used MoAbs are repeated at least twice per CR1 molecule. Furthermore, CR1 exhibits structural polymorphism characterized by a variable number of LHR per molecule. Thus, accurate enumeration of cell surface CR1 using currently available MoAb would require that the results be corrected for the number of antibody epitopes per CR1 molecule encoded by each individual's alleles. To obtain a MoAb to a non-polymorphic epitope on human CR1, hybridomas were generated from mice immunized with recombinant soluble CR1 (sCR1) and MoAb were screened for those that recognized the full-length extracellular domain but failed to bind to all four recombinant LHR fragments. A single antibody, CR1-2B11, was identified and was found to recognize an epitope located wholly within SCR29-30 of CR1, NH2-terminal to an elastase cleavage site. Like other CR1 MoAb, the CR1-2B11 epitope expression decreased on old erythrocytes compared to younger cells and CR1-2B11 did not identify a CR1 'stump' on RBC. Importantly, CR1-2B11 immunofluorescence did not change with storage or handling of RBC, unlike the apparent decrease in immunofluorescence observed with other MoAb. CR1-2B11 should be useful for the accurate enumeration of RBC CR1.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/sangue , Receptores de Complemento 3b/imunologia , Animais , Western Blotting/métodos , Células CHO , Senescência Celular/imunologia , Cricetinae , Cricetulus , Eritrócitos/imunologia , Imunofluorescência/métodos , Humanos , Camundongos , Receptores de Complemento 3b/sangue , Proteínas Recombinantes/imunologia , TransfecçãoRESUMO
Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.