RESUMO
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.
Assuntos
Anticorpos Biespecíficos/imunologia , Patógenos Transmitidos pelo Sangue , Modelos Animais de Doenças , Receptores de Complemento/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Bacteriófago phi X 174/imunologia , Relação Dose-Resposta Imunológica , Eritrócitos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , PapioRESUMO
LFA-1 exists in a low avidity state on resting leukocytes and is believed to adopt a high avidity state when the cells are exposed to a stimulus. Current evidence supports both aggregation of LFA-1 on the cell surface and conformational changes in the reversible acquisition of a high avidity state. We studied this regulation by selecting a Jurkat T cell clone, J-lo1.3, that expresses LFA-1 yet fails to bind to purified ICAM-1 despite treatment of the cells with PMA or Mn2+. Several lines of evidence demonstrated the absence of any changes within LFA-1 itself. LFA-1 protein purified from the J-lo1.3 clone and the wild-type Jurkat clone, Jn.9, were found to be functionally equivalent. The cDNA sequences encoding the LFA-1 alpha- and beta-chains from J-lo1.3 were identical with the published sequences except for nine base pairs. However, these differences were also found in a Jurkat mutant with a constitutively avid phenotype, J+hi1.19 or the wild-type Jn.9 genomic or cDNA. Fusion of J-lo1.3 with Jn.9 yielded hybrids that exhibited the J-lo1.3 adhesion phenotype, which indicated a dominant mutation in J-lo1.3. This phenotype was relatively specific for LFA-1 among all integrins expressed by Jurkat. Interestingly, the J-lo1.3 cells had a 1.2-fold faster doubling time than did the Jn.9 cells. Reversion of J-lo1.3 to the wild-type adhesion phenotype by mutagenesis and selection also decreased the growth rate. These data support a connection between cellular growth and cellular adhesion in lymphocytes.
Assuntos
Substâncias de Crescimento/genética , Substâncias de Crescimento/metabolismo , Células Jurkat/citologia , Células Jurkat/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Mutagênese Sítio-Dirigida , Animais , Células CHO , Adesão Celular/genética , Adesão Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Células Clonais , Cricetinae , DNA Complementar/análise , Substâncias de Crescimento/fisiologia , Humanos , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Fenótipo , Ligação Proteica/genética , Ligação Proteica/imunologia , Análise de Sequência de DNA , Transfecção , Células Tumorais CultivadasRESUMO
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
To investigate the role of LFA-1 in the immune defects in DS patients, we analysed lymphocytes from DS patients in LFA-1 expression and LFA-1 mediated cell adhesion. DS patients less than 2 years of age expressed a higher level of LFA-1 when compared with age-matched controls. The difference in LFA-1 expression was much less significant in older DS patients when compared with age-matched children. Although older children (2-15-year-old groups) without DS tend to increase their expression of lymphocyte LFA-1 when compared with younger normal children (0-2 years old), DS patients showed no age-associated increase in lymphocyte LFA-1 expression. Two-colour analysis with CD4/CD8 and LFA-1 in patients and controls showed that proportions of CD4 + lymphocytes were comparable in DS patients and controls, while the proportion of CD8 + lymphocytes was higher in older DS patients. Expression levels of LFA-1 on both CD4 + and CD8 + lymphocytes in younger DS patients were higher when compared with age-matched controls and close to the expression levels in the older DS group. Proportions of memory lymphocytes expressing the CD45RO isoform were higher in both younger and older DS patients when compared with age-matched control groups. Noticeably, the LFA-1 expression levels on CD45RO lymphocytes from younger DS patients were higher than the levels of the controls and declined in the older DS group. We tested lymphocytes (EBV transformed B cells, resting and anti-CD3 stimulated T cells) for cellular adhesion to recombinant ICAM-1 and found that lymphocytes from DS patients were less adhesive, even though their beta2 integrin expression was comparable with that of normal controls. These results suggest that more generalized pathological processes, such as early senescence of the immune system or ineffective lymphocyte activation, and subsequent integrin dysfunction may underlie the immune defects in DS patients.
Assuntos
Adesão Celular , Síndrome de Down/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/biossíntese , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/imunologia , Adolescente , Fatores Etários , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Linhagem Celular Transformada , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Antígenos Comuns de Leucócito/metabolismo , Regulação para CimaRESUMO
We have identified the integrin alpha(v)beta3 as a ligand for mouse gp49B1, thus identifying a new class of ligand for a member of the family of inhibitory immunoreceptors that bear C2-type immunoglobulin-like domains. The specific interaction was shown by both cell-protein and cell-cell binding assays. In addition, we found that the interaction of alpha(v)beta3 with gp49B1 on bone marrow-derived mouse mast cells inhibited antigen-induced immunoglobulin E-mediated cell activation. Because neither gp49B1 nor alpha(v)beta3 exhibit substantive allelic variation, their newly appreciated interaction may reflect an innate pathway for down-regulating the activity of mast cells.
Assuntos
Antígenos de Superfície/metabolismo , Integrinas/metabolismo , Mastócitos/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Vitronectina/metabolismo , Animais , Antígenos de Superfície/genética , Ligação Competitiva , Células da Medula Óssea/imunologia , Imunoglobulina E/imunologia , Integrinas/genética , Ligantes , Glicoproteínas de Membrana/genética , Camundongos , Ligação Proteica , Receptores Imunológicos/genética , Proteínas Recombinantes/metabolismo , Vitronectina/metabolismoRESUMO
A 74-year-old woman presented to her rheumatologist with classic symptoms of giant cell arteritis. The temporal arteries were strikingly swollen, warm, and erythematous. On biopsy of the right temporal artery, a focal dissection was found associated with a pan-arteritis and giant cells. Isolated temporal artery dissection in giant cell arteritis has not been reported previously. We propose that the unusually intense vascular inflammation may have weakened the vessel wall, so that the dissection occurred during the routine physical exam or biopsy. We believe this case illustrates that physicians should take special care in the examination of floridly inflamed vessels, because vigorous palpation might lead to dissection. In the case of patients with giant cell arteritis, dissection may result in an increased risk of ischemic complications, such as scalp necrosis.
RESUMO
For some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful. A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable. Since relatively few recombinants are obtained after subtraction, this protocol is for a cDNA library constructed in the lambda>10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.
Assuntos
Clonagem Molecular/métodos , DNA Complementar/genética , Biblioteca Gênica , Técnica de Subtração , Bacteriófago lambda/genética , Desoxirribonuclease EcoRI , Escherichia coli , Vetores Genéticos/genética , Indicadores e Reagentes , RNA Mensageiro/genética , Manejo de EspécimesRESUMO
This protocol may be used for genomic DNA or cDNA libraries. A freshly packaged and titered library is adsorbed to log phase plating bacteria. The mixture is then plated at high density and allowed to grow until the plaques are just subconfluent. The phage are eluted from the plate by overnight incubation with phage buffer and the library is titered and stored.
Assuntos
Bacteriófagos/genética , DNA/genética , Amplificação de Genes , DNA Complementar , Escherichia coli/genéticaRESUMO
Enzymatic conversion of mRNA into double-stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide-primed synthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. The following protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt-ended cDNA that can then be ligated to linkers for subsequent cloning into a unique restriction site such as EcoRI. The Alternate Protocol is a variation that requires fewer enzymatic manipulations and allows construction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The Alternate Protocol takes advantage of a linker-primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt-ended cDNA. The internal XhoI sites on the individual cDNA molecules are protected by incorporation of 5-methyl-dCTP in the first-strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI/XhoI-digested vectors after ligation of EcoRI adaptors to the 5' end and digestion by XhoI to release the 3' XhoI sites that were incorporated into the cDNA by the linker-primer. These changes result in a considerably streamlined procedure that is substantially faster and easier than the basic protocol.
Assuntos
DNA Complementar/química , RNA Mensageiro/genética , Clonagem Molecular , Indicadores e Reagentes , SoluçõesRESUMO
The Basic Protocol describes the strategy of producing a genomic DNA library. A number of small-scale ligations are performed using a set amount of vector and varying amounts of insert. Test ligations are transformed into bacteria (plasmid vectors) or packaged and plated on host bacteria (lambda and cosmid vectors). The number of clones in the different ligations is compared, and the optimum ratio of vector to insert is indicated by the ligation with the most recombinant clones. A large-scale ligation is then set up using this optimum ratio. This protocol employs a bacteriophage vector; however, cosmid or plasmid vectors can be used with minor modifications.
Assuntos
DNA Complementar/química , DNA/química , Cromatografia em Gel , Eletroforese em Gel de Ágar , Vetores Genéticos , PlasmídeosRESUMO
A complete cDNA library is one that contains at least one cDNA clone representing each mRNA in the cell. A method is provided in this unit for calculating the required base of a cDNA library such that the library should contain at least one copy of every mRNA. Either a phage or plasmid vector can be used in constructing a complete cDNA library, and protocols are described for both. Procedures for evaluating these cDNA libraries are also provided along with a procedure for evaluating the quality of the lambda arms used in constructing complete phage cDNA libraries.
Assuntos
DNA Complementar/genética , Bacteriófago lambda/genética , Plasmídeos , RNA Mensageiro/genética , TransfecçãoRESUMO
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
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
OBJECTIVE: To investigate the role of intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins in the production of superoxide (O2-) by C1q-stimulated human polymorphonuclear leukocytes (PMN). METHODS: PMN were pretreated with F(ab')2 fragments of monoclonal antibodies (mAb) that blocked or did not block beta2 integrin-mediated adhesion. The cells were added to wells coated with C1q, and the production of O2- was monitored kinetically as a color change due to reduction of cytochrome c. In some experiments, C1q was co-immobilized with purified ICAM-1. RESULTS: Blocking mAb to the shared beta2 integrin subunit, CD18, completely inhibited the O2- response triggered by immobilized C1q, while blocking mAb to the alpha subunits of the beta2 integrins each partially blocked the O2- response. PMN treated with C1q were found to activate the beta2 integrins lymphocyte function-associated antigen 1 and CR3 for binding to ICAM-1. Co-immobilization of ICAM-1 with C1q cooperatively triggered O2- production by PMN. CONCLUSION: beta2 integrin binding to an ICAM provided an essential costimulatory signal for O2-production triggered by C1q in PMN. Our findings suggest a model for PMN activation in which 2 stimuli are required for O2- production: a first signal that also activates PMN beta2 integrins, followed by a second, beta2 integrin-mediated signal, which occurs physiologically upon PMN binding to ICAM-1. The requirement for this dual signal for PMN generation of O2- would serve as a regulatory mechanism to limit the production of O2- to a tissue environment where C1q, or some other stimulus, is colocalized with stromal cells bearing up-regulated ICAM-1. This mechanism may explain why all tissues can express ICAM-1 and may explain in part why inhibitors of tumor necrosis factor alpha, a major physiologic stimulus of ICAM-1 up-regulation, are potent antiinflammatory agents.
Assuntos
Antígenos CD18/fisiologia , Complemento C1q/farmacologia , Molécula 1 de Adesão Intercelular/fisiologia , Neutrófilos/metabolismo , Superóxidos/metabolismo , Anticorpos Bloqueadores/fisiologia , Antígenos CD18/imunologia , Complemento C1q/antagonistas & inibidores , Humanos , Inflamação/fisiopatologia , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno-1 Associado à Função Linfocitária/fisiologia , Antígeno de Macrófago 1/metabolismo , Antígeno de Macrófago 1/fisiologia , Receptores de Complemento 3b/imunologiaRESUMO
Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.
Assuntos
Complemento C1q/metabolismo , Complemento C4b/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Receptores de Complemento 3b/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Colágeno/metabolismo , Complemento C1/metabolismo , Proteínas Inativadoras do Complemento 1/farmacologia , Proteínas do Sistema Complemento/metabolismo , Ácido Edético/farmacologia , Temperatura Alta , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Cinética , Ligantes , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Receptores de Complemento 3b/imunologia , Toxoide Tetânico/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
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
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
The ras-related small GTPases of the rho family coordinate the assembly of complex cytoskeletal structures crucial for cell motility and polarization. Cdc42 controls filopodia formation in some cell types; however, other physiological functions, agonists and subsequent signaling cascades remain to be elucidated. Expression of cdc42 mutants in monocytic cells showed that CC chemokines regulate the rearrangement of the actin cytoskeleton via cdc42, i.e. formation of filopodia-like projections was induced by CC chemokines or dominant active cdc42, while dominant inactive cdc42 prevented its stimulation with CC chemokines. Both cdc42 mutants inhibited CC chemokine-induced monocyte migration across bare filters or across filters coated with endothelium, implicating cdc42 activity and its effector functions in chemotaxis. In contrast, cdc42 mutants did not affect the rho-dependent activation integrin avidity by CC chemokines. The chemokine-induced signaling pathways involved phosphoinositide 3-kinase upstream of cdc42, as shown by inhibition of cytoskeletal reorganization with wortmannin. These data identify CC chemokines as physiological agonists of cdc42 and reveal its functional importance in chemotaxis and extravasation of monocytes.