RESUMEN
Impaired fibrinolysis and complement activation in Systemic Lupus Erythematosus contributes to disease amplification including increased risk of thrombosis and tissue Ischemia/Reperfusion (IR) injury. Previous work has demonstrated complement is a key regulator of tissue injury. In these studies inhibitors had varying efficacies in attenuating injury at primary versus systemic sites, such as lung. In this study the role of coagulation factors in tissue injury and complement function was evaluated. Tissue Factor Pathway Inhibitor (TFPI), an extrinsic pathway inhibitor, and Anti-Thrombin III, the downstream common pathway inhibitor, were utilized in this study. TFPI was more effective in attenuated primary intestinal tissue injury. However both attenuated systemic lung injury. However, ATIII treatment resulting in enhanced degradation of C3 split products in lung tissue compared to TFPI. This work delineates the influence of specific early and late coagulation pathway components during initial tissue injury versus later distal systemic tissue injury mechanism.
Asunto(s)
Anticoagulantes/farmacología , Antitrombina III/farmacología , Antitrombinas/farmacología , Intestino Delgado/efectos de los fármacos , Lipoproteínas/farmacología , Pulmón/efectos de los fármacos , Lupus Eritematoso Sistémico , Animales , Intestino Delgado/metabolismo , Intestino Delgado/patología , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Isquemia Mesentérica/metabolismo , Isquemia Mesentérica/patología , Ratones , Ratones Endogámicos MRL lpr , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
B6.MRL/lpr mice, an autoimmune strain, have an accelerated injury time course, increased intensity of tissue damage, and increased CD4+ T cell infiltration in the mesenteric ischemia/reperfusion injury model. In this study, the mechanism by which CD4+ T cells were recruited into injured tissue was addressed. Fingolimod (FTY720) was utilized to assess the role of infiltrating CD4+ T cells. FTY720 treatment was more effective in attenuating injury in B6.MRL/lpr mice then in control mice. Reduced CD4+ cell infiltration and tissue injury correlated with decreased neutrophil infiltration and pro-inflammatory cytokine generation. Inhibiting downstream Sphingosine-1-phosphate (S1P) receptor signaling, specifically GαI mediated signaling, did not inhibit injury, suggesting differential utilization of the S1P receptors between control and MRL/lpr strains. Analysis of S1P receptor expression exposed a predominance of S1P2 in the B6.MRL/lpr strain. Reliance on alternate S1P receptors in the autoimmune strain will alter the progress of inflammation and tissue injury.
Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Clorhidrato de Fingolimod/uso terapéutico , Inmunosupresores/uso terapéutico , Receptores de Lisoesfingolípidos/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Animales , Autoinmunidad , Vasos Sanguíneos/patología , Movimiento Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Neutrófilos/inmunología , Receptores de Lisoesfingolípidos/genética , Daño por Reperfusión/inmunología , Receptores de Esfingosina-1-FosfatoRESUMEN
Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.
Asunto(s)
Linfocitos B/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas/fisiología , Proteínas RGS , Transducción de Señal/inmunología , Animales , Linfocitos B/inmunología , Células COS , Regulación hacia Abajo/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/fisiología , Humanos , Células Jurkat , Células K562 , Unión Proteica/inmunología , Biosíntesis de Proteínas , Ratas , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/fisiología , Células Tumorales CultivadasRESUMEN
The development and establishment of the B Cell Repertoire is the net result of both genetic and environmental forces. The primary event at the genetic level is Ig gene rearrangement resulting in numerous possible combination of genes which can be further modified by somatic events such as N segment addition and somatic mutation. Environmental forces in the form of self and exogenous Ags also shape the repertoire by positively or negatively selecting B cells according to the specificity of their Ig receptors. These are dynamic processes beginning with the earliest expression of immunoglobulins in fetal life and continuing throughout life. In this review we discuss the genetic and selective mechanisms responsible for differences in the early immune system compared to that of the adult.
Asunto(s)
Linfocitos B/citología , Hematopoyesis , Ratones/inmunología , Animales , Diversidad de Anticuerpos , Antígenos de Diferenciación de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Médula Ósea/embriología , Médula Ósea/crecimiento & desarrollo , Células de la Médula Ósea , Antígenos CD5/fisiología , Linaje de la Célula , Movimiento Celular , Reordenamiento Génico de Linfocito B , Genes de Inmunoglobulinas , Sistema Inmunológico/embriología , Sistema Inmunológico/crecimiento & desarrollo , Sistema Inmunológico/inmunología , Ratones/embriología , Ratones/genética , Ratones/crecimiento & desarrollo , Ratones Transgénicos , Transducción de Señal , Especificidad de la EspecieRESUMEN
Antibodies bearing the T15 idiotype dominate the murine primary immune response to phosphocholine (PC). Analysis of antigen binding of antibodies derived from V1:DFL16.1:JH1 (VH1) germline and N region-derived variant heavy (H) chains and kappa 22, kappa 24, and kappa 8 light (L) chains demonstrates that the T15H:kappa 22L (T15) antibody binds PC at least 20-40 times better than other antibodies derived from alternate germline forms of the VH1 H chain and kappa 22, kappa 24, or kappa 8 L chains. To achieve affinities in the same range as the T15 antibody, kappa 24 and kappa 8 L chain-containing antibodies must have H chains derived from variant N region or somatically mutated VH1 genes. Single amino acid differences at the VD junction of the various germline and N region variant VH1 H chains dictate the L chain that can associate with the H chain to produce a PC-specific antibody. Several H:L combinations give rise to T15 or M167 idiotype-positive antibodies that lack specificity for PC, and single amino acid substitutions or insertions at the VH1:D junction result in the loss of T15 or M167 idiotopes. Based on these observations, our data support a molecular model involving both preferential gene rearrangement and antigen-driven B cell selection to explain T15 idiotype dominance in the immune response to PC. In the absence of N region diversification, large numbers of neonatal B cells bearing the T15H:kappa 22L surface immunoglobulin M (sIgM) receptors would be selected and expanded by autologous or environmental PC antigen into the long-lived peripheral B cell pool.
Asunto(s)
Genes Dominantes , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Idiotipos de Inmunoglobulinas/genética , Idiotipos de Inmunoglobulinas/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Fosforilcolina/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo , Secuencia de Bases , Línea Celular , Estimulación Eléctrica , Ensayo de Inmunoadsorción Enzimática , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Idiotipos de Inmunoglobulinas/biosíntesis , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , RatasRESUMEN
Seven human ocular melanoma cell lines were established in vitro and 3 of these, GU-4, LLN-40 and its subline C17-11, were characterized. Mice were immunized with these ocular melanoma cell lines, and 2 hybridomas producing monoclonal IgG1 antibodies (MAb) were produced. MAb 2/10SN recognizes a 44-kDa monomeric protein, whereas MAb 10/12SN reacts with an 83/65-kDa heterodimeric protein. These melanoma-associated antigens (MAA) are detected at high concentrations in the cytoplasm of ocular melanoma cells. However, cell-surface labelling techniques suggest that these MAA are also associated with the cell-surface membrane. These 2 ocular MAA are also expressed by several skin melanoma cell lines. Immunohistochemical studies have localized these antigens to ocular and skin melanomas, to sweat ducts and basal squamous cells in normal skin, with limited expression in several other normal tissues and some carcinomas. Biodistribution studies in nude mice with human ocular melanomas have demonstrated good localization of 125I-labeled MAb 2/10SN at the tumor sites. Therefore, these 2 MAbs, 2/10SN and 10/12SN, recognize MAA which appear to be unique and may prove useful for imaging purposes.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Neoplasias del Ojo/inmunología , Melanoma/inmunología , Animales , Especificidad de Anticuerpos , Antígenos de Neoplasias/química , Sitios de Unión de Anticuerpos , Unión Competitiva , Citometría de Flujo , Humanos , Hibridomas/inmunología , Técnicas para Inmunoenzimas , Sustancias Macromoleculares , Melanoma Experimental/inmunología , Ratones , Ratones Desnudos , Radioinmunoensayo , Neoplasias Cutáneas/inmunología , Células Tumorales Cultivadas , Neoplasias de la Úvea/inmunologíaRESUMEN
The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Inmunoglobulina M/inmunología , Linfoma de Células B/patología , Fosfatidilinositoles/metabolismo , Proteína Quinasa C/fisiología , Transducción de Señal , Tirosina/metabolismo , Activación Enzimática , Humanos , Hidrólisis , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The activation, growth, and differentiation of three B-cell- and one non-B-cell-derived human lymphoma cell lines were examined after treatment with protein kinase C-activating phorbol esters. Treatment with these agents resulted in early activation events similar to those observed in normal B cells. However, in contrast to their growth-promoting effect on normal human B lymphocytes, exposure to these phorbol esters induced profound growth inhibition of the three B-cell-derived lymphoma lines. Maximal inhibition was achieved within 24 hours of culture initiation and could be reversed if the phorbol ester was removed after 12, but not 20, hours in culture. Cell-cycle analysis of phorbol ester-treated lymphoma cells revealed a G1/S block in one line, whereas cells from the other two lines accumulated in G2/M. These data demonstrate that protein kinase C-binding phorbol esters can interrupt the cell cycle in two places in actively dividing human B-lymphoma cells. These findings may prove valuable with regard to potential therapy of human malignant lymphomas.
Asunto(s)
Linfocitos B/citología , Linfoma no Hodgkin/patología , Ésteres del Forbol/farmacología , Antígenos de Diferenciación de Linfocitos B/análisis , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Citometría de Flujo , Reordenamiento Génico de Linfocito B , Reordenamiento Génico de Linfocito T , Inhibidores de Crecimiento , Humanos , Técnicas In Vitro , Ionomicina/farmacología , Activación de Linfocitos/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Qa-5 is a Class I MHC Ag expressed on mature T cells and natural killer (NK) cells of mice, and is defined by specific reactivity with an IgM secreting hybridoma (B16-167). The availability of IgG antibodies with Qa-5 specificity would greatly facilitate in vivo studies of Qa-5 positive NK cells. Using a sequential subline technique in conjunction with ELISA screening, several isotype variants of the B16-167 parent line were selected and cloned. An IgG2a, an IgG2b, and two IgG1 switch variants were selected, and it was demonstrated that one of these cloned lines produced both IgM and IgG1 simultaneously. Binding of the variant antibodies to Qa-5+ cells was not detected by flow cytometry analysis, nor by the ability to deplete NK activity from Qa-5+ large granular lymphocytes (LGL). However, all of the variant lines appeared to use the same VH region genes by Southern blot analysis. Furthermore, IgG2a from the switch variants inhibited binding of the parental IgM anti-Qa-5 when used in excess, and reduction of the parental IgM anti-Qa-5 to monomeric form abolished its binding capacity. These data together indicate that the weak binding by the IgG variants may be due to loss of an innate low affinity/high avidity binding of the parent IgM. Therefore, the isotype switch detection procedure is a useful technique for selection of antibodies with various effector functions, provided the initial antibody is an antibody with sufficient binding affinity.