RESUMEN
The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99YPPPY104 motif are adjacent to a patch of charged surface residues on the A'GFCC' face of the protein. Mutation of these residues, which are conserved in the CTLA-4/CD28 family, significantly reduces binding to CD80 and/or CD86, implicating this patch as a ligand binding site.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación/química , Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Inmunoconjugados , Glicoproteínas de Membrana/metabolismo , Abatacept , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/genética , Antígeno B7-2 , Sitios de Unión , Antígeno CTLA-4 , Secuencia Conservada , Dimerización , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Soluciones , SulfurosRESUMEN
CD80 and CD86 (B7-1 and B7-2) are the ligands on antigen-presenting cells (APCs) which bind CD28 and deliver the costimulatory signals necessary for T cell activation. The reasons for the existence of two CD28 binding molecules are not well understood. We created a mutant version of CTLA4-Ig that could selectively bind CD80 and block CD28-CD80 interaction but leave CD28-CD86 binding intact. CD80 blockade prevented antigen-induced accumulation of eosinophils and lymphocytes in the lung of immunized mice, but did not block antigen induced systemic blood eosinophilia or IgE antibody production. No preferential expression of CD80 could be demonstrated on a population of lung APC consisting mainly of macrophages. These results indicate that CD80 costimulation is not necessary for the induction of Th2 immune responses but rather for the maintenance or amplification of lung inflammatory responses.
Asunto(s)
Antígenos de Diferenciación/farmacología , Antígeno B7-1/fisiología , Eosinofilia/fisiopatología , Eosinófilos/fisiología , Inmunoconjugados , Inflamación , Enfermedades Pulmonares/fisiopatología , Linfocitos/fisiología , Abatacept , Secuencia de Aminoácidos , Animales , Antígenos CD , Antígeno B7-1/efectos de los fármacos , Antígenos CD28/efectos de los fármacos , Antígenos CD28/fisiología , Células CHO , Antígeno CTLA-4 , Secuencia Conservada , Cricetinae , Eosinofilia/prevención & control , Eosinófilos/efectos de los fármacos , Citometría de Flujo , Humanos , Cinética , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/prevención & control , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC'C" beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC'C" face of the IgV domain and the ABED face of the IgC domain.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Inmunoconjugados , Abatacept , Secuencia de Aminoácidos , Antígenos CD , Antígeno B7-1/genética , Secuencia de Bases , Antígeno CTLA-4 , Secuencia Conservada , Cartilla de ADN , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
T cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (approximately 20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of the CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-analogous regions.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/química , Antígenos CD28/biosíntesis , Antígenos CD28/química , Secuencia Conservada , Inmunoconjugados , Linfocitos T/inmunología , Abatacept , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD , Sitios de Unión , Células CHO , Antígeno CTLA-4 , Línea Celular , Chlorocebus aethiops , Cricetinae , Humanos , Activación de Linfocitos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Serum levels of circulating oncostatin-M (OM) were compared among cases of Kaposi's sarcoma associated with acquired immune deficiency syndrome (AIDS-KS) and multiple controls, including a homosexual man infected with human immunodeficiency virus type 1 (HIV-1), an HIV-1-uninfected homosexual man, and a heterosexual man; and among classic KS cases and heterosexual controls. Cases were selected from abstracts collected by a population-based cancer registry and from local AIDS clinics. Controls for the AIDS-KS cases were matched to the cases by age, sex, and race and were either friends of the cases or residents from the cases' neighborhoods; controls for the classic KS cases were similarly matched, but were obtained solely from neighborhood residents. Blood samples were obtained from participants, serum levels of OM were determined by enzyme-linked immunosorbent assay (ELISA), and CD4 cell counts were obtained by flow cytometry. Geometric mean levels of OM were compared among the risk groups adjusted for age and CD4 cell count. No differences in adjusted OM levels were found between AIDS-KS cases and HIV-1-infected homosexual controls (8.4 pg/ml vs. 10.2) or between classic KS cases and controls (13.3 pg/ml vs. 9.6); however the HIV-1-infected controls (both homosexual and heterosexual) matched to the AIDS-KS cases had higher levels than did the HIV-1-infected cases and controls. Among the HIV-1-infected groups, an inverse correlation between OM and CD4 cell count was observed and was statistically significant for the cases. Among all heterosexual controls (matched to either case group), serum OM was inversely related to age.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Sustancias de Crecimiento/sangre , VIH-1 , Péptidos/sangre , Sarcoma de Kaposi/sangre , Adulto , Factores de Edad , Anciano , Linfocitos T CD4-Positivos , Estudios de Casos y Controles , Citocinas/sangre , Homosexualidad , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Oncostatina M , Sarcoma de Kaposi/etiología , Sarcoma de Kaposi/patología , Conducta SexualRESUMEN
A sensitive and specific enzyme immunoassay was developed for detecting oncostatin M (OM) in human plasma and serum. The assay utilizes three anti-OM monoclonal antibodies that recognize mutually exclusive epitopes, including a neutralizing epitope. A sensitivity of 24 pg/ml was routinely obtainable. The assay showed no cross-reactivity with leukemia inhibitory factor (LIF) or interleukin-6 (IL-6), other members of the cytokine family that includes OM. The utility of the enzyme immunoassay (EIA) was demonstrated by detecting the time-dependent accumulation of OM in plasma from lipopolysaccharide (LPS)-treated human whole blood. The concentration of OM in human sera from normal donors was generally below the detection limits of the assay. However, concentrations of OM greater than 25 pg/ml were found in 17 of 212 serum samples from apparently normal donors. The detection of OM in human plasma and serum demonstrates that the EIA could be a useful tool in examining the role of OM in physiologic and pathologic states.
Asunto(s)
Análisis Químico de la Sangre/métodos , Citocinas/sangre , Técnicas para Inmunoenzimas , Péptidos/sangre , Análisis Químico de la Sangre/normas , Análisis Químico de la Sangre/estadística & datos numéricos , Citocinas/normas , Estudios de Evaluación como Asunto , Humanos , Técnicas para Inmunoenzimas/normas , Técnicas para Inmunoenzimas/estadística & datos numéricos , Lipopolisacáridos , Oncostatina M , Péptidos/normas , Plasma/química , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
Oncostatin M (OM) is a novel cytokine which exhibits pleiotropic effects on a wide variety of normal and transformed cell lines. To determine some of the physiological functions of OM we have characterized several monoclonal antibodies to the recombinant molecule. Antibodies OM1 and OM2 bound native, but not denatured OM, suggesting they recognize non-contiguous epitopes. A third antibody, OM6, bound predominantly denatured OM. Of the two antibodies which detect discontinuous epitopes, OM2, but not OM1, was identified as a neutralizing antibody based on its ability to abrogate OM activity in the growth inhibition assay (GIA) and to inhibit OM binding in the radioreceptor assay (RRA). OM2 was equally effective in abrogating the functional effects of either natural or recombinant OM, thereby demonstrating that the active sites of these molecules are structurally similar, if not identical.
Asunto(s)
Anticuerpos Monoclonales , Péptidos/antagonistas & inhibidores , Receptores de Citocinas , Animales , Células CHO , División Celular/efectos de los fármacos , Cricetinae , Epítopos/inmunología , Femenino , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C/inmunología , Oncostatina M , Péptidos/metabolismo , Péptidos/farmacología , Receptores de Superficie Celular/metabolismo , Receptores de Oncostatina M , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/farmacologíaRESUMEN
When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.
Asunto(s)
Granulocitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Receptores de Superficie Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Electroforesis en Gel de Poliacrilamida , Granulocitos/ultraestructura , Humanos , Cinética , Ligandos , Microscopía Electrónica , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Formil PéptidoRESUMEN
Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.
Asunto(s)
Factores Quimiotácticos/metabolismo , Granulocitos/fisiología , Metionina/análogos & derivados , N-Formilmetionina/análogos & derivados , Oligopéptidos/metabolismo , Transporte Biológico , Granulocitos/efectos de los fármacos , Granulocitos/ultraestructura , Humanos , Cinética , Microscopía Electrónica , N-Formilmetionina/metabolismo , N-Formilmetionina/farmacología , N-Formilmetionina Leucil-Fenilalanina , Oligopéptidos/farmacología , Fracciones Subcelulares/metabolismo , TritioRESUMEN
Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83000 X g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40-60% were achieved with a 20-70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g X cm-3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimented with specific granules (p = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg2+-ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg2+-ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.
Asunto(s)
Adenosina Trifosfatasas/sangre , Granulocitos/metabolismo , Oligopéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , ATPasa de Ca(2+) y Mg(2+) , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Factores Quimiotácticos/metabolismo , Granulocitos/ultraestructura , Humanos , N-Formilmetionina/análogos & derivados , N-Formilmetionina/metabolismo , N-Formilmetionina Leucil-Fenilalanina , Receptores de Formil PéptidoRESUMEN
Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.