RESUMO
Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.
Assuntos
Síndromes de Imunodeficiência/genética , Mutação , Receptores de Adesão de Leucócito/genética , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/genética , Sequência de Bases , Antígenos CD18 , Adesão Celular , Linhagem Celular , Análise Mutacional de DNA , Citometria de Fluxo , Conversão Gênica , Humanos , Síndromes de Imunodeficiência/patologia , Síndrome da Aderência Leucocítica Deficitária , Leucócitos/patologia , Antígeno-1 Associado à Função Linfocitária , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fenótipo , RNA/genética , TransfecçãoRESUMO
Patients with the severe form of leukocyte adhesion deficiency syndrome do not express the CD11/CD18 adhesion complex on any of their leukocytes. Nevertheless, their lymphocytes, unlike their phagocytes, emigrate to extravascular sites of inflammation, demonstrating that surface proteins other than CD11/CD18 can mediate lymphocyte adherence to endothelium. Using a B-lymphoblastoid cell line (B-LCL) established from a CD11/CD18-deficient patient and cultured human umbilical vein endothelial cells (HEC), we investigated the CD11/CD18-independent mechanism(s) of lymphocyte adherence to endothelium. Monoclonal antibodies directed to the alpha 4 polypeptide (CD49d) and the beta 1 polypeptide (CD29) of the lymphocyte VLA-4 integrin receptor (CD49d/CD29), and to vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell significantly inhibited the adherence of the CD11/CD18-deficient B-LCL to untreated HEC and to HEC treated with recombinant human tumor necrosis factor-alpha. We suggest that the interaction of the lymphocyte receptor VLA-4 with the endothelial ligand VCAM-1 induced by cytokines at sites of inflammation or immune reaction represents a CD11/CD18-independent pathway of lymphocyte emigration.
Assuntos
Moléculas de Adesão Celular/fisiologia , Adesão Celular , Endotélio Vascular/fisiologia , Linfócitos/fisiologia , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/imunologia , Antígenos CD11 , Antígenos CD18 , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Integrina beta1 , Síndrome da Aderência Leucocítica Deficitária , Receptores de Antígeno muito Tardio/imunologiaRESUMO
Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.
Assuntos
Antígenos de Diferenciação/análise , Terapia Genética , Síndromes de Imunodeficiência/terapia , Integrinas/análise , Glicoproteínas de Membrana/análise , Receptores de Adesão de Leucócito/análise , Receptores de Adesão de Leucócito/genética , Transfecção , Animais , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/fisiologia , Northern Blotting , Antígenos CD18 , Moléculas de Adesão Celular/metabolismo , Agregação Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Síndromes de Imunodeficiência/genética , Síndrome da Aderência Leucocítica Deficitária , Antígeno-1 Associado à Função Linfocitária , Camundongos , RNA Mensageiro/análise , Receptores de Adesão de Leucócito/fisiologiaRESUMO
From the highly metastatic TAM2D2 T-cell hybridoma we have previously generated three independent mutants that were deficient in the surface expression of the adhesion molecule Leukocyte Function-associated Antigen 1 (LFA-1). Both the invasive capacity and the metastatic potential of these mutants were greatly reduced compared with TAM2D2 cells (F.F. Roossien et al., J. Cell Biol., 108: 1979-1985, 1989). We now show that, during in vivo transplantation, LFA-1 is reinduced in these mutants. From such revertant cell populations obtained after two to three i.p. passages, we isolated clones with different LFA-1 levels. Of each of the three mutant cell lines, the clone with the highest and the one with the lowest LFA-1 level were selected for further study. Invasiveness in fibroblast monolayers correlated strongly with LFA-1 level; i.e., the low-LFA-1 clones (mean LFA level, approximately 10% of TAM2D2) invaded as poorly as the original mutants, whereas the high-LFA-1 clones (greater than 25% of TAM2D2) were highly invasive. Metastatic potential was determined after tail vein injection of 10(6) cells in syngeneic AKR mice. A difference was observed between high- and low-LFA-1 clones, albeit less striking than previously found between LFA-1-negative mutants and parental TAM2D2 cells. The high-LFA-1 clones developed metastases in more mice (76 versus 43%) and earlier (mean survival, 30 versus 37 days). These results provide further evidence for an important role of LFA-1 in invasion and metastasis of mouse T-cell hybridomas.
Assuntos
Antígenos de Diferenciação/deficiência , Síndrome da Aderência Leucocítica Deficitária , Invasividade Neoplásica , Metástase Neoplásica , Antígenos de Diferenciação/biossíntese , Separação Celular , Citometria de Fluxo , Antígeno-1 Associado à Função Linfocitária , Mutação , Receptores de Adesão de Leucócito/biossíntese , Células Tumorais Cultivadas/patologiaRESUMO
FACS analysis together with PIPLC treatment was applied to PI-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylcholinesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.
Assuntos
Antígenos de Diferenciação/deficiência , Hemoglobinúria Paroxística/imunologia , Fosfatidilinositóis/imunologia , Adulto , Antígenos de Diferenciação/análise , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos CD55 , Antígenos CD59 , Proteínas Inativadoras do Complemento , Eritrócitos/imunologia , Citometria de Fluxo , Granulócitos/imunologia , Humanos , Receptores de Lipopolissacarídeos , Linfócitos/imunologia , Masculino , Proteínas de Membrana/análise , Monócitos/imunologia , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/farmacologia , Receptores Fc/análise , Receptores de IgGRESUMO
The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.
Assuntos
Antígenos de Diferenciação/deficiência , Síndrome da Aderência Leucocítica Deficitária , Neutrófilos/fisiologia , Circulação Pulmonar/fisiologia , Animais , Antígenos de Diferenciação/fisiologia , Tempo de Circulação Sanguínea , Antígenos CD11 , Antígenos CD18 , Adesão Celular/fisiologia , Cães , Fluoresceína-5-Isotiocianato , Fluoresceínas , Cinética , Microcirculação/fisiologia , Microscopia de Fluorescência , Neutrófilos/citologia , Receptores de Adesão de Leucócito/fisiologia , TiocianatosRESUMO
This report summarizes the clinical and laboratory findings from a group of our patients and literature reviews from several families who have a genetic deficiency in three related leukocyte membrane surface antigens known as CR3,LFA-1, and p150,95. Each surface antigen has an identical beta-chain noncovalenty linked to one of three distinct alpha-chain types. Patients affected by this autosomal recessive disease have now been identified by several investigators. The patients have an increased susceptibility to bacterial infections that is similar to patients who have other types of neutrophil functional deficiencies or neutropenia. Laboratory tests have indicated that isolated neutrophils from these patients have two major types of functional deficiencies that probably contribute to their reduced ability to overcome bacterial infections. This review focuses exclusively on the phagocytic and cytotoxic abnormalities of neutrophils from these patients.
Assuntos
Antígenos de Diferenciação/deficiência , Síndrome da Aderência Leucocítica Deficitária , Leucócitos/metabolismo , Receptores de Complemento/deficiência , Anticorpos Monoclonais , Adesão Celular , Granulócitos/metabolismo , Humanos , Integrina alfaXbeta2 , Antígeno-1 Associado à Função Linfocitária , Antígeno de Macrófago 1Assuntos
Adesão Celular , Leucócitos/fisiologia , Glicoproteínas de Membrana , Receptores de Superfície Celular , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Moléculas de Adesão Celular , Movimento Celular , Cães , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Inflamação , Integrina alfaXbeta2 , Antígeno-1 Associado à Função Linfocitária , Antígeno de Macrófago 1 , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Estrutura Molecular , Fagocitose , Coelhos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores de Complemento/metabolismo , Receptores de Complemento 3bAssuntos
Antígenos de Diferenciação/deficiência , Infecções Bacterianas , Úlcera da Perna/complicações , Glicoproteínas de Membrana/deficiência , Adolescente , Feminino , Humanos , Úlcera da Perna/sangue , Antígeno-1 Associado à Função Linfocitária , Antígeno de Macrófago 1 , Cicatrização/efeitos dos fármacosAssuntos
Antígenos de Diferenciação/deficiência , Síndrome da Aderência Leucocítica Deficitária , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/fisiologia , Humanos , Integrina alfaXbeta2 , Antígeno-1 Associado à Função Linfocitária , Antígeno de Macrófago 1 , Peso Molecular , Receptores de Adesão de Leucócito/genética , Receptores de Adesão de Leucócito/fisiologiaAssuntos
Antígenos de Diferenciação/imunologia , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Receptores Fc/imunologia , Animais , Antígenos de Diferenciação/deficiência , Humanos , Antígeno-1 Associado à Função Linfocitária , Receptores de Superfície Celular/imunologia , Receptores de Complemento/imunologia , Receptores de LipoproteínasRESUMO
Leukocyte adhesion deficiency (LAD) is a recessive autosomal disease characterized by life-threatening recurrent bacterial infections, by defective functions of leukocytes and by deficient membrane expression of leukocyte adhesion glycoproteins. These proteins, LFA-1, Mac-1 and p150,95, are alpha 1/beta 1 heterodimers composed of different alpha chains and of a common beta chain. Patients with the severe phenotype of the disease completely lack the three glycoproteins on cell surface. Previous studies showed a conserved synthesis of the LFA-1 alpha chain precursor in cytosol of patients' cells and an inconstant presence of the beta chain precursor. When present, precursors are in free form and not associated to alpha/beta complexes in the cells of patients with the severe phenotype. The availability of the beta chain cDNA probe allowed us to examine the beta chain gene expression in the lymphoblastoid cell lines of 4 patients. On the basis of the results obtained both at protein and RNA levels we can distinguish 3 types of mutations characterized by (a) barely detectable beta subunit messenger RNA and undetectable beta precursor, (b) decreased level of beta subunit mRNA and undetectable beta precursor and (c) normal beta subunit mRNA level and detectable beta precursor of normal size.
Assuntos
Antígenos de Diferenciação/fisiologia , Adesão Celular , Síndromes de Imunodeficiência/genética , Leucócitos/citologia , Antígenos de Diferenciação/análise , Antígenos de Diferenciação/deficiência , Northern Blotting , Eletroforese em Gel Bidimensional , Humanos , Ponto Isoelétrico , Antígeno-1 Associado à Função Linfocitária , Peso Molecular , Testes de PrecipitinaRESUMO
Three consecutive patients with the severe phenotype of leukocyte adhesion deficiency characterized by a defective expression of LFA-1, Mac-1 (CR3), and p150.95 on leukocytes have received HLA partially incompatible bone marrow transplantation (BMT). The degree of HLA incompatibility between related donors and recipients was 2 HLA antigens in one and one full haplotype in the two others. Graft-v-host disease (GVHD) prophylaxis consisted in T-cell depletion of the bone marrow inoculum and a 60-day course of cyclosporin A. A first attempt led to autologous recovery in one patient. The second transplant in this patient and the first transplant in the two others led to stable partial engraftment of lymphocytes and phagocytic cells, as shown by expression of adhesion molecules (LFA-1, Mac-1) on leukocytes and by HLA typing and restriction fragment-length polymorphism studies using minisatellite probes. Although the level of mixed chimerism was lower in one patient (7% to 30% donor cells) and greater than 50% in the two others, recovery of lymphocyte and phagocytic cell functions was sufficient enough to allow the patient to lead a normal life, infection free in the three cases. These patients, now 57, 32, and 19 months post-transplant, are in good condition without any therapy. These results lead us to propose that the LFA-1 molecule plays a role in HLA-incompatible graft rejection, probably by mediating adhesion of cytotoxic T and non-T lymphocytes to their targets.
Assuntos
Transplante de Medula Óssea , Adesão Celular , Doenças Hematológicas/terapia , Leucócitos/fisiologia , Formação de Anticorpos , Antígenos de Diferenciação/deficiência , Medula Óssea/imunologia , Quimera , Antígenos HLA/análise , Humanos , Integrina alfaXbeta2 , Células Matadoras Naturais/fisiologia , Antígeno-1 Associado à Função Linfocitária , Antígeno de Macrófago 1RESUMO
Understanding the molecular basis of a rare inherited disease, Leu-CAM deficiency in humans, has underscored the importance of the cellular component of inflammation and unravelled the complex series of homotypic and heterotypic cell interactions necessary for mobilization of leukocytes to infected sites. Furthermore, this disease has shown that several apparently distinct cellular inflammatory responses (e.g. aggregation, adhesion to endothelium, directed migration and phagocytosis) are mechanistically related and mediated by a set of molecules which belong to a larger group of adhesion molecules (Integrins) mediating similar phenomena critical for immune surveillance, lymphocyte homing, morphogenesis and thrombogenesis. This disease also showed the relative biologic importance of CD11/CD18 in leukocytes. CD11/CD18 are more critical for the functions of phagocytic cells as compared to lymphocytes although similar inhibitory effects of anti-CD11/CD18 mAbs can be demonstrated in vitro. Expression and function of CD11/CD18 is regulated at several levels which include formation of stable heterodimers, qualitative changes in the receptor and quantitative changes in the levels of expression of the receptors and their ligands. We have identified inherited single amino acid substitutions on CD18 which impair heterodimer formation and cell surface expression, thus accounting for the pathogenesis of Leu-CAM deficiency. We also found a stimulus-induced phosphorylation of CD18, which is transient in nature when elicited through other surface receptors. This may be important in regulation of CD11/CD18 receptor avidity, recycling, endocytosis and cross-talk with other receptors. Finally, realization of the profound impairment in the acute cellular inflammatory response present in Leu-CAM deficiency has permitted novel ways of controlling the inflammatory response in several situations were inflammation serves an injurious rather than a beneficial role to the host.
Assuntos
Inflamação/imunologia , Síndrome da Aderência Leucocítica Deficitária , Sequência de Aminoácidos , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/metabolismo , Antígenos de Diferenciação/fisiologia , Antígenos CD11 , Antígenos CD18 , Humanos , Inflamação/fisiopatologia , Inflamação/terapia , Leucócitos/imunologia , Leucócitos/patologia , Ligantes , Dados de Sequência Molecular , Prognóstico , Conformação Proteica , Receptores de Adesão de Leucócito/metabolismo , Receptores de Adesão de Leucócito/fisiologiaRESUMO
The healthy mother of a child with transient immune neutropenia was found to be "NA-null." The mother's neutrophils did not react with anti-NA1 and anti-NA2 antibodies (polyclonal human alloantibodies and mouse monoclonal antibodies). A healthy donor was discovered during routine neutrophil antigen typing whose neutrophils were also "NA-null." This NA-phenotype was due to the absence of FcRIII (CD16 antigen) on neutrophils as demonstrated with anti-FcRIII monoclonal antibodies. The neutrophils of these two individuals were not able to bind dimeric immunoglobulin G. However, their cells had a normal expression of other phosphatidylinositol (PI)-linked membrane glycoprotein (CD24, CD67, and CLB gran/5 antigens), ruling out the existence of a PI-linkage defect, such as paroxysmal nocturnal hemoglobinuria. The mother (propsitus) had isoantibodies in her blood against neutrophil-FcRIII without allospecificity, apparently produced during pregnancy and responsible for the neutropenia of her child. The expression of FcRIII on natural killer lymphocytes of both individuals was normal. FcRIII is encoded by two separate genes, one (FcRIII-1) for the neutrophil-PI-linked receptor, another (FcRIII-2) for the natural killer cell and macrophage-transmembrane receptor. By messenger RNA and DNA analysis (with an FcRIII-cDNA probe and restriction endonucleases) the neutrophil-FcRIII deficiency appeared to be due to deletion of the FcRIII-1 gene in both individuals, while the FcRIII-2 gene was normally present. The parents of the propositus were found to be heterozygous for this defect. Thus, FcRIII-1 gene deficiency of the mother may be a cause of (iso)immune neutropenia of the newborn. Whether this deficiency may have other clinical consequences has to be studied.
Assuntos
Antígenos de Diferenciação/genética , Imunidade Materno-Adquirida , Doenças do Recém-Nascido/genética , Neutropenia/genética , Neutrófilos/imunologia , Receptores Fc/genética , Anticorpos Monoclonais , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Deleção Cromossômica , Mapeamento Cromossômico , Feminino , Humanos , Recém-Nascido , Doenças do Recém-Nascido/imunologia , Doenças do Recém-Nascido/patologia , Neutropenia/imunologia , Neutropenia/patologia , Polimorfismo de Fragmento de Restrição , Gravidez , Receptores Fc/deficiência , Receptores Fc/imunologia , Receptores de IgGRESUMO
The CD11/CD18 complex of leukocyte adhesion molecules has been shown to bind LPS on the surface of gram negative bacteria and LPS-coated erythrocytes (J. Exp. Med. 164:1876, 1986). LPS elicits several responses in leukocytes including secretion of TNF-alpha and IL-1 beta, and priming for enhanced release of oxygen radicals such as superoxide anion. To determine if expression of CD18 molecules is necessary for these effects of LPS, we have examined the responses of leukocytes from CD18-deficient patients. Three of the patients in this study are characterized for the first time here, and three were described elsewhere. Monocytes and macrophages from CD18-deficient patients synthesized normal amounts of IL-1 beta and TNF-alpha in response to LPS. Further, PMN and monocytes from CD18-deficient patients showed normal priming for enhanced release of superoxide anion in response to LPS. Although a small contribution of CD18 molecules to some responses cannot be ruled out by our data, we may conclude that CD18 molecules are not essential for cellular responses to LPS.
Assuntos
Antígenos CD/deficiência , Interleucina-1/biossíntese , Síndrome da Aderência Leucocítica Deficitária , Leucócitos Mononucleares/fisiologia , Lipopolissacarídeos/farmacologia , Neutrófilos/fisiologia , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Actinas/metabolismo , Antígenos CD/análise , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/fisiologia , Antígenos CD11 , Antígenos CD18 , Citometria de Fluxo , Humanos , Receptores de Adesão de Leucócito/fisiologiaRESUMO
When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC.
Assuntos
Antígenos de Diferenciação/fisiologia , Proteínas Sanguíneas/fisiologia , Proteínas Inativadoras do Complemento/fisiologia , Via Alternativa do Complemento , Eritrócitos/fisiologia , Hemoglobinúria Paroxística/sangue , Hemólise , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Animais , Antígenos de Diferenciação/deficiência , Antígenos de Diferenciação/farmacologia , Antígenos CD55 , Antígenos CD59 , Cobaias , Hemólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/farmacologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/farmacologia , Valores de ReferênciaRESUMO
We studied the expression and function of 2 cell surface markers that induce T cell activation, CD3 and CD5, in 19 patients with primary Sjögren's syndrome (SS). The expression of CD3+ lymphocytes was normal, but CD3 function was moderately reduced, as measured by anti-CD3-induced T cell proliferation. Anti-CD3-induced stimulation of T cell help for Ig production and non-major histocompatibility complex-restricted (natural) killing were normal. In contrast, there was reduced expression and function of the CD5 molecule on peripheral blood lymphocytes of the SS patients. The ratio of CD5+ to CD3+ lymphocytes was 0.45 in SS patients compared with 0.85 in normal subjects, indicating that the CD3+ cells are relatively CD5-deficient in SS patients. Anti-CD5 monoclonal antibody did not augment suboptimal anti-CD3 stimulation of whole peripheral blood lymphocytes or of purified T lymphocytes from SS patients, which indicated impaired functioning of the CD5 molecule. A significant impairment in proliferation was found in response to phorbol myristate acetate and to ionomycin in combination, suggesting defective intracellular signaling. Findings from serial studies of individual patients suggested that normal or low expression of CD5 on T cells can be stable over periods as long as 2 years. However, in 2 patients who required systemic therapy, a correction of the CD5 lymphocyte abnormality occurred in association with clinical remission, which suggests the potential reversibility of this condition. Our findings suggest a possible defect in intracellular signaling in primary SS, related in part to the CD5 molecule, which may provide a molecular explanation for the T cell hyporesponsiveness that is characteristic of this disease.