RESUMEN
Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14(high) CD16(neg/low)), intermediate (CD14(high) CD16(high)) and non-classical (CD14(low) CD16(high)) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0.01). Patients with PR3-ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0.01); however, levels in patients with MPO-ANCA disease in remission were lower than active MPO-ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0.79, P < 0.0001 and r = 0.42, P < 0.005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3-ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Monocitos/inmunología , Receptores de IgG/biosíntesis , Adulto , Anciano , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/biosíntesis , Humanos , Masculino , Persona de Mediana Edad , Mieloblastina/biosíntesis , Peroxidasa/biosíntesisRESUMEN
Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Interleucina-2/farmacología , Interleucina-7/farmacología , Activación de Linfocitos/efectos de los fármacos , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Línea Celular , Activación Enzimática , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Piridinas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
IL-7 acts on both resting and activated T cells. The functional IL-7R is reported to consist of two subunits, the ligand binding IL-7R and the common gamma-chain (gamma c chain), where IL-7R:gamma c chain association is driven solely by ligand binding. However, we now demonstrate that in primary T cells this event is also controlled by cellular activation. We show that IL-7R:gamma c chain complexes are detected in activated, but not in resting, T cells despite similar levels of gamma c chain expression, implying that the gamma c chain is not associated with the IL-7R in unstimulated T cells. Furthermore, IL-7R:gamma c chain association correlates with the expression of JAK-3 in T cells, but not in transfected COS-7 cells. The finding that IL-7R:gamma c chain assembly is controlled at a level beyond that of receptor expression has important implications for the control of cytokine function in T cells.
Asunto(s)
Interleucina-7 , Activación de Linfocitos/fisiología , Receptores de Interleucina/análisis , Linfocitos T/inmunología , Anticuerpos Monoclonales , Humanos , Pruebas de PrecipitinaRESUMEN
We have investigated signaling events associated with the cloned 90-kDa (p90) interleukin-7 receptor (IL-7R) to determine whether changes in the signaling pathways initiated by this molecule can explain the ability of T cells to proliferate to IL-7 following activation. Using in vitro kinase assays we find that the p90 IL-7R in both unstimulated and activated human T cells is physically associated with two molecules with intrinsic kinase activity. Western blotting analysis reveals these proteins to be the src kinase enzymes, p59fyn and p56lck. Binding of human recombinant IL-7 to the p90 IL-7R results in increased activity of both receptor-associated kinases in both resting and activated mature T cells. Thus, the signaling pathways initiated via the p90 IL-7R-associated src kinases are unlikely to be solely responsible for the proliferation of only activated T cells in response to IL-7. Additional signals, which may derive from other IL-7R-associated molecules such as the gamma c, are clearly required for IL-7-driven proliferation of activated primary T cells.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-7/farmacología , Activación de Linfocitos , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal/fisiología , Linfocitos T/efectos de los fármacos , Familia-src Quinasas/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Sustancias Macromoleculares , Proteínas Proto-Oncogénicas c-fyn , Receptores de Interleucina-7 , Proteínas Recombinantes/farmacología , Linfocitos T/enzimologíaRESUMEN
Although both unstimulated and activated human T cells express high affinity IL-7R, only activated T cells can proliferate to IL-7. This responsiveness may occur as a direct result of changes in the structure of the IL-7R during T-cell activation. We have previously demonstrated such changes by affinity cross-linking studies, and have shown that unstimulated human T cells express a single IL-7R of 90 kDa, whereas activated T cells express an additional 76-kDa IL-7 binding protein. In this study the origin and function of the p90 and p76 molecules have been investigated. To determine the role of each of these receptors in IL-7 driven proliferation, IL-7R expression and proliferative capacity were monitored during mitogenic stimulation. These analyses showed that the ability of PBMC to proliferate to IL-7 correlated with expression of the p76 IL-7R, and not with expression of the p90 IL-7R. IL-7-driven proliferation is mediated via high affinity IL-7R, and accordingly, Scatchard analysis revealed that, like the p90 IL-7R, the p76 IL-7R bound IL-7 with dual (high; Kd 38 pM and low; Kd 360 pM) affinity. Deglycosylation studies showed that the p90 and p76 IL-7R are not simply differently glycosylated isoforms of a single receptor. In agreement, mAb to the previously cloned IL-7R were found to stain unstimulated T cells that express only the p90 IL-7R but not T-cell clones that express predominantly the p76 IL-7R. These antibodies also immunoprecipitated the cloned IL-7R as a 90-kDa species from both 125-I-surface-labeled resting and activated T cells, but were unable to precipitate the 76-kDa IL-7R. In addition, PCR analysis of p76-expressing cells could not detect splicing of the extracellular domain of the cloned IL-7R, thereby excluding the possibility that the p76 IL-7R is a previously undescribed splice variant of the cloned IL-7R. These data demonstrate that the p90 IL-7R is the T-cell homologue of the cloned IL-7R, and imply that the p90 and p76 IL-7R have different extracellular domains. Taken together these data suggest that the 76-kDa receptor is a novel high affinity IL-7R that may be necessary for IL-7 driven proliferation in human T cells.
Asunto(s)
Interleucina-7/farmacología , Activación de Linfocitos , Receptores de Interleucina/análisis , Linfocitos T/química , Secuencia de Bases , Células Cultivadas , Células Clonales , Glicosilación , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , ARN Mensajero/análisis , Receptores de Interleucina/genética , Receptores de Interleucina/fisiología , Receptores de Interleucina-7 , Linfocitos T/inmunologíaRESUMEN
Like interleukin (IL)-2 and IL-4, IL-7 can act as a growth factor for activated T lymphocytes. Upon screening a panel of growth factor-dependent T cell lines, we found that only the cell line CT6 responded to IL-7, indeed as vigorously as to IL-2. Obviously, these findings challenge the validity of previous results on IL-2 production obtained using the CT6 cell line. However, they also demonstrate a novel and sensitive system for the bioassay of IL-7. The ability of the surveyed T cell lines to proliferate to IL-7 corresponded with the expression of IL-7 receptors (IL-7R) on the cell surface. The murine IL-7R on CT6 was shown to bind IL-7 with dual affinity and was visualized as an affinity cross-linked complex of 93 kDa. This IL-7R appears similar to that seen on murine splenic T cells and on 70Z/3, the pre-B cell line from which the murine IL-7R was cloned.
Asunto(s)
Interleucina-7/análisis , Receptores Inmunológicos/metabolismo , Linfocitos T Citotóxicos/fisiología , Animales , Bioensayo , Línea Celular , Técnicas In Vitro , Interleucina-2/farmacología , Interleucina-7/fisiología , Activación de Linfocitos/efectos de los fármacos , Ratones , Receptores de Interleucina-7 , Linfocitos T Citotóxicos/citologíaRESUMEN
Activation of human peripheral blood T cells renders them capable of proliferating to IL-2, -4, and -7, and upregulates the receptors for IL-2 and -4. In this study the effect of activation on the receptor for IL-7 has been investigated. Scatchard analysis showed dual affinity binding of IL-7 to peripheral blood mononuclear cells (PBMC). Furthermore, activation of PBMC with anti-CD3 antibodies resulted in a 4-fold downregulation of both the high and low affinity IL-7 receptors. SDS-PAGE analysis of [125I]IL-7 cross-linked resting PBMC revealed a major complex of 104/107 kDa (reduced/non-reduced) and a minor complex of 184/178 kDa (reduced/non-reduced). In contrast, cross-linking of activated PBMC revealed a third prominent complex of 93 kDa (non-reduced) not seen on unstimulated cells. This 93 kDa complex was observed on purified activated peripheral blood T cells and T cell blasts. Moreover, on a panel of IL-7 responsive T cell clones the 93 kDa complex was the only major cross-linked product observed. These results demonstrate that T cell activation causes changes in both the level of expression of the IL-7 receptor and the nature of the proteins associated with the receptor. It is postulated that these changes in receptor structure may be related to the acquisition of responsiveness to the IL-7 growth signal.
Asunto(s)
Regulación de la Expresión Génica , Activación de Linfocitos/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3 , Relación Dosis-Respuesta Inmunológica , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Humanos , Interleucina-7/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Inmunológicos/química , Receptores de Interleucina-7RESUMEN
We have isolated, by transient expression and screening using an oligonucleotide probe, cDNA clones encoding the rat interleukin 2 receptor (IL 2R) alpha and beta chains, respectively. Both chains are approximately 60% identical at the amino acid level to their human counterparts. In common with the human and mouse IL 2R beta chains, the rat beta chain is a member of the cytokine receptor family. The rat IL 2R beta chain contains no conventional signal transduction machinery and, like the mouse IL 2R beta chain, has a significant number of deletions in its cytoplasmic tail in comparison to the human protein. Northern analysis indicates that the rat beta chain message is constitutively transcribed in normal resting lymphocytes, whereas the alpha chain message is absent. Transcription of both chains of the receptor is up-regulated by cellular activation. However, kinetic studies have shown that whereas alpha chain message is rapidly induced, and can be detected 5 h after the addition of phytohemagglutinin, levels of beta chain mRNA increase at a much later time point and are not seen to rise until approximately 24 h after the addition of mitogen. These data suggest that the expression of high-affinity IL 2R is controlled by factors which influence transcription of both the alpha and the beta chain genes.
Asunto(s)
Receptores de Interleucina-2/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Sustancias Macromoleculares , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Fitohemaglutininas/farmacología , Ratas , Receptores de Interleucina-2/biosíntesis , Homología de Secuencia de Ácido Nucleico , Regulación hacia ArribaRESUMEN
Tolerance to alloantigen may be induced in rats by administration of blood followed by transplantation of a renal allograft. The mechanism of this tolerance was investigated by directly analyzing the functional activity of graft-infiltrating cells. We have previously shown cytotoxic T lymphocyte infiltration of, and major histocompatibility complex induction on, grafts of tolerant animals. We now report that cells isolated from the grafts of tolerant rats show a reduced expression of the p55 interleukin 2 receptor (IL-2R) chain on the cell surface compared with that seen on the cells of untreated animals. Scatchard analysis further reveals low expression of high affinity IL-2R. This is due to reduced transcription of both IL-2R alpha and beta chain mRNAs and results in a reduced ability of cells to proliferate in response to IL-2. Cells isolated from tolerant animals are unable to make biologically active IL-2 in culture, whereas cells from untreated animals make high levels. This is not reflected at the mRNA level as the IL-2 gene is induced in both tolerant and untreated animals to similar levels. The induction of tolerance is abrogated by administration of recombinant IL-2 to animals at the time of transplantation. Thus, we conclude that an altered regulation of the IL-2 pathway results in tolerance in these alloantigen-treated and transplanted animals.