RESUMEN
The beta2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of beta2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the alpha and beta subunit. As an approach to identify and characterize regulatory domains of the beta2 subunit, we have generated two different subdomains of the beta2 subunit for expression on the surface of mammalian cells through a phosphatidyl-inositol glycan anchor. The first subdomain contains the putative beta2 MIDAS motif implicated in ligand binding [beta2(LB)], whereas the second beta2 subdomain contains the cysteine-rich region [beta2(CR)]. Cells expressing alphaM and beta2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant alphaMbeta2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete alphaMbeta2 heterodimer. In contrast, cells expressing alphaMbeta2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected alpha or beta chains alone failed to adhere to immobilized iC3b. These results indicate that along with alphaM, the beta2(LB) subdomain contains the sufficient components within the beta2 subunit essential for ligand recognition. These findings support the hypothesis that the beta2 subunit cooperates with site(s) within the alphaM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction.
Asunto(s)
Antígenos CD18/biosíntesis , Antígenos CD18/química , Antígeno de Macrófago-1/metabolismo , Animales , Antígenos CD18/genética , Células COS , Células Cultivadas , Complemento C3b/metabolismo , Ligandos , Antígeno de Macrófago-1/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
We have previously demonstrated that Asp134 and Ser136 of the beta2 subunit are essential for alphaLbeta2 and alphaMbeta2 ligand recognition. It has been proposed that these residues may be part of a metal ion-dependent adhesion site (MIDAS) within the beta subunit homologous to the alphaM I domain MIDAS structure (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638). In the present study, we evaluated the role of additional candidate metal ion-coordinating residues in the beta2 subunit in ligand interactions. Cells bearing the recombinant alphaLbeta2 or alphaMbeta2 mutant(s) were tested for the ability to bind to immobilized ligands. Alanine substitution at Asp232 in beta2 produced a complete loss in the capacity of both alphaLbeta2 and alphaMbeta2 to support cell adhesion and suppressed the expression of a divalent cation-dependent conformation recognized by mAb 24. Alanine substitution at Glu235 differentially affected receptor function dependent upon the co-transfected alpha subunit. Cells expressing alphaLbeta2 with a substitution at Glu235 failed to adhere to intercellular adhesion molecule 1 (ICAM-1) but did retain the capacity to bind mAb 24. Moreover, cells expressing alphaMbeta2 with a substitution at Glu235 failed to adhere to fibrinogen or ICAM-1 and did not bind mAb 24. However, these cells did retain the capacity to adhere to iC3b following antibody-induced activation. These results implicate Asp232 and Glu235, along with Asp134 and Ser136, in ligand binding function of alphaLbeta2 and alphaMbeta2. These findings provide evidence in support of the existence of a MIDAS structure in beta2 analogous to that seen in the alphaM I domain.
Asunto(s)
Antígenos CD18/química , Cationes Bivalentes , Moléculas de Adhesión Celular/química , Complemento C3b/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Metales , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Células COS , Adhesión Celular , Cricetinae , Fibrinógeno/metabolismo , Ligandos , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Oil-in-water emulsions are being used increasingly for the delivery of lipophilic drugs, but the fundamental physicochemical principles governing such delivery have not been explored. We determined the kinetics and thermodynamics of delivery from emulsions to cells in culture for two lipophilic compounds, U74006 and U74500. Two fundamental properties dominate the delivery, (a) the concentration of the compound in the lipid phase of the emulsion is directly proportional to the concentration of the compound in cells at equilibrium, and (b) the rate of transfer is directly proportional to the concentration of particles in contact with the cells. Thus, the transfer is consistent with direct partitioning from the lipid phase of the emulsion to cells and occurs by the direct collision of emulsion particles with cells. The details of the mechanism of delivery differ between the two compounds. Specifically, delivery of U74006 is first-order with respect to the drug accumulating in the cells. The transfer of U74500 is best described as a sum of two simultaneous pseudo first-order processes consistent with delivery from a single donor compartment to two receiver compartments. Furthermore, two molecules of U74500 appear to be involved in each transfer event. Our results show that relatively simple principles govern the delivery of compounds from oil-in-water emulsions to cells.
Asunto(s)
Antioxidantes/metabolismo , Sistemas de Liberación de Medicamentos , Pregnatrienos/metabolismo , Animales , Antioxidantes/farmacología , Emulsiones , Cinética , Ratones , Neuroblastoma/metabolismo , Neuronas/metabolismo , Fosfatidilcolinas/metabolismo , Ratas , Termodinámica , Trioleína/metabolismo , Células Tumorales CultivadasRESUMEN
An extensive comparison of TCR alpha beta V-region usage by CD8 beta-CD4+CD8+ intraepithelial lymphocytes (IEL), CD4-CD8+ IEL, and lymph node (LN) T cell subsets in three minor lymphocyte stimulating (Mls)-disparate, MHC-identical mouse strains revealed novel TCR selection patterns. In cases where forbidden V regions were expressed by CD8 beta- CD4-CD8+ IEL, the same TCRs were deleted from CD8 beta- CD4+CD8+ IEL, indicating that lack of CD8 beta expression was not solely responsible for forbidden V-region expression. These results also suggested that CD4 may be involved in negative selection of CD4+CD8+ IEL TCRs. In C57BR/cdJ (Mls-1b2b) mice, a major increase in V beta 3+CD4+CD8+ IEL but not in other IEL or LN subsets was noted suggesting a subset-specific expansion of V beta 3+ cells. Negative selection of V beta 14+ cells in only the CD4+CD8+ IEL subset further supported the existence of intestine-specific TCR selection processes. Analysis of V-region expression of CD8 beta + and CD8 beta-CD4-CD8+ IEL subsets revealed that forbidden V-region expression was not strictly confined to the CD8 beta- subset in all cases. Overall, the data point to a dynamic, gut-specific TCR selection process that may be antigen driven.
Asunto(s)
Intestinos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/inmunología , Animales , Células Epiteliales , Epitelio/inmunología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunofenotipificación , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos AKR/inmunología , Ratones Endogámicos C3H/inmunología , Ratones Endogámicos C57BL/inmunología , Antígenos Estimulantes de Linfocito Menor/genética , Selección GenéticaRESUMEN
Intraepithelial lymphocytes (IEL) of the mouse small intestine were examined for their potential to respond to TCR signalling in vitro. Purified IEL subsets were activated using mAbs specific for CD3, TCR alpha beta or TCR gamma delta. Thy-1+ IEL, regardless of TCR type, proliferated equally well in response to anti-TCR mAb with or without exogenous IL-2. In contrast, Thy-1- TCR alpha beta, CD8 beta- IEL required exogenous IL-2 for proliferation. No such requirement was observed for Thy-1- TCR gamma delta IEL proliferation. IEL proliferation in the absence of added IL-2 was due to an IL-2 secretion/IL-2 receptor (IL-2R) autocrine pathway, since mAbs specific for IL-2 and IL-2R inhibited IEL proliferation. Thy-1+ CD8 beta- CD4+CD8+ IEL were unresponsive to TCR-induced proliferation but exhibited high levels of cytolytic activity upon TCR-triggering. Thy-1- non-cytolytic IEL were induced to express Thy-1 and cytolytic activity following activation in vitro. In addition, the involvement of the co-stimulatory molecule CD28 in IEL activation was tested. CD28 was weakly expressed by fresh IEL and anti-CD28 mAb had no effect on TCR-triggered proliferation. However, anti-TCR stimulation increased CD28 expression on a subset of TCR alpha beta IEL and the addition of anti-CD28 mAb resulted in increased IL-2 production, but not in increased proliferation. Our results indicate that IEL, including the purported extrathymic CD8 beta- subset, can respond to TCR-driven signals via proliferation and/or cytolytic activity.
Asunto(s)
Complejo CD3/inmunología , Intestino Delgado/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos CD28 , Células Epiteliales , Epitelio/inmunología , Femenino , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL/inmunologíaRESUMEN
The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.
Asunto(s)
Antígenos de Diferenciación/inmunología , Antígenos de Histocompatibilidad/inmunología , Mucosa Intestinal/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Antígenos de Superficie/análisis , Secuencia de Bases , Exones , Citometría de Flujo , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , Mucosa Intestinal/citología , Antígenos Comunes de Leucocito , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Procesamiento Proteico-Postraduccional , Antígenos Thy-1RESUMEN
Ocular exposure of mice to live elementary bodies of Chlamydia trachomatis serovar A results in immunological sensitization of the mice. This reactivity is manifested by the development of early (5 h) and delayed-type (24 h) dermal reactivity and serovar-specific antibody formation against either live or irradiated (100 kilorads) elementary bodies. Parallel ocular exposure of mice to irradiated elementary bodies does not result in this sensitization. The early and late dermal immune responses induced by ocular exposure to live organisms can be transferred to unexposed mice by serum and lymphoid cell transfers, respectively. It appears that successful murine ocular sensitization by human C. trachomatis serovar A elementary bodies is an ability manifested by live organisms and not by inactivated but antigenic organisms.
Asunto(s)
Chlamydia trachomatis/inmunología , Conjuntiva/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/patología , Chlamydia trachomatis/efectos de la radiación , Ensayo de Inmunoadsorción Enzimática , Inmunización Pasiva , Linfocitos/inmunología , Ratones , Ratones Endogámicos , Piel/inmunologíaRESUMEN
A rapid hydrogenase assay has been developed which may be useful in separating the species Campylobacter jejuni and C. coli from the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. This assay employs the impermeant redox dye benzyl viologen, and positive determinations can be made within 20 min. All strains of C. jejuni and C. coli were found to be strongly hydrogenase positive. All strains of C. fetus subsp. fetus and C. fetus subsp. venerealis were negative for hydrogenase when the assay was performed at a benzyl viologen concentration of 2 mM and an incubation temperature of 30 degrees C. Some strains of C. fetus had low levels of hydrogenase as determined with cell extracts but were hydrogenase negative by the benzyl viologen assay. Since there are few rapid diagnostic tests available for screening Campylobacter isolates, we hope that the rapid hydrogenase assay will prove useful.
Asunto(s)
Campylobacter/enzimología , Catalasa/análisis , Oxidorreductasas/análisis , Campylobacter/clasificación , Hidrogenasas , Serotipificación , Especificidad de la EspecieRESUMEN
A study of the electron transport chain of the human intestinal pathogen Campylobacter jejuni revealed a rich complement of b- and c-type cytochromes. Two c-type cytochromes were partially purified: one, possibly an oxidase, bound carbon monoxide whereas the other, of high potential was unreactive with carbon monoxide. Respiratory activities determined with membrane vesicles were 50- to 100-fold higher with formate and hydrogen than with succinate, lactate, malate, or NADH as substrates. Evidence for three terminal respiratory components was obtained from respiratory kinetic studies employing cyanide, and the following Ki values for cyanide were determined from Dixon plots: ascorbate + reduced N,N,N', N'-tetramethyl-p-phenylenediamine, K1 + 3.5 muM; malate, K1 = 55 muM; and hydrogen, K1 = 4.5 muM. Two oxidases (K1 = 90 muM, 4.5 mM) participated in the oxidation of succinate, lactate, and formate. Except with formate, 37 muM HQNO inhibited respiration by approximately 50%. Carbon monoxide had little inhibitory effect on respiration except under low oxygen tension (less than 10% air saturation). The stoichiometry of respiratory-driven proton translocation (H+/O) determined with whole cells was approximately 2 for all substrates examined except hydrogen (H+/) = 3.7) and formate (H+/O = 2.5). The higher stoichiometries observed with hydrogen and formate are consistent with their respective dehydrogenase being located on the periplasmic face of the cytoplasmic membrane. The results of this study suggest that the oxidation of hydrogen and formate probably serves as the major sources of energy for growth.