RESUMEN
This unit describes procedures for measuring the adhesion of lymphoid cells to immobilized ligands or adherent cell monolayers. The basic protocol, which is optimized for human T cells, can be easily modified to study almost any cell type in suspension if the corresponding adhesive ligand is available. Alternate protocols describe modifications to the basic protocol that allow analysis of cell adhesion in the presence of stimulators of lymphocyte activation or monoclonal antibodies (MAbs) that react with adhesion molecules or their ligands, or analysis of cell adhesion to adherent cell monolayers. A support protocol is provided for determining the optimal concentration of adhesion ligands and choosing the appropriate type of microtiter plate for static adhesion assays.
Asunto(s)
Adhesión Celular/fisiología , Técnicas Inmunológicas , Linfocitos/citología , Animales , Adhesión Celular/inmunología , Humanos , Linfocitos/inmunología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Animal models of allergic lung inflammation have provided important insight into the cellular and biochemical factors involved in the pathogenesis of human asthma. Herein, we describe an adoptive transfer model of OVA-specific eosinophilic lung inflammation in the mouse that is used to characterize the cells involved in mediating the pulmonary inflammatory response. We report that freshly isolated spleen cells from OVA-sensitized mice are unable to prime naive recipient mice to respond to a subsequent OVA aerosol challenge. Subjecting the spleen cells to short term restimulation with Ag in vitro, however, renders the cells competent to transfer activity. The magnitude and the kinetics of the eosinophilic pulmonary inflammation in the adoptive transfer recipients are nearly identical with those generated by a more conventional active sensitization/challenge protocol, with the notable exception of differential production of plasma IgE in the two models. Extensive negative and positive selection of splenocyte subtypes indicates that the transfer of Ag-primed CD4+ T cells is both necessary and sufficient to establish full responsiveness in the recipient mice. Additional phenotypic characterization of the transfer-reactive CD4+ T cells indicates that they are found within the CD62LlowCD25+ subset and secrete high levels of IL-5 in response to Ag stimulation. Limiting dilution analysis-derived minimal frequency estimates indicate that approximately 1 in 8500 of the sensitized, cultured spleen cells produces IL-5 in response to OVA stimulation in vitro, suggesting that eosinophilic lung inflammation can be induced in naive mice by the transfer of <1200 Ag-specific CD4+ T cells.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD4-Positivos/inmunología , Selectina L/biosíntesis , Pulmón/patología , Receptores de Interleucina-2/inmunología , Hipersensibilidad Respiratoria/inmunología , Subgrupos de Linfocitos T/inmunología , Administración por Inhalación , Traslado Adoptivo/métodos , Aerosoles , Animales , Linfocitos B/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Eosinofilia/inmunología , Eosinofilia/patología , Femenino , Inmunoglobulina E/sangre , Interleucina-5/biosíntesis , Interleucina-5/metabolismo , Cinética , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Hipersensibilidad Respiratoria/patología , Bazo/citología , Bazo/trasplante , Subgrupos de Linfocitos T/patología , Factores de TiempoRESUMEN
The very late Ag-4 (VLA-4) integrin supports both rolling and firm adhesion of leukocytes on VCAM-1 under shear flow. The molecular basis for the unique ability of a single adhesion molecule to mediate these versatile adhesive processes was investigated. VLA-4 occurs in multiple activation states, with different affinities to ligand. In this study we tested how these states regulate VLA-4 adhesiveness under shear flow in Jurkat T cells and PBL. VLA-4 on nonstimulated Jurkat cells supported rolling and spontaneous arrest on VCAM-1, whereas a Jurkat activation mutant with reduced VLA-4 affinity failed to spontaneously arrest after tethering to or during rolling on VCAM-1. The contribution of VLA-4 affinity for ligand to rolling and spontaneous arrests on immobilized VCAM-1 was dissected using soluble VLA-4 ligands, which selectively block high affinity states. VLA-4 saturation with ligand completely blocked spontaneous adhesion strengthening post-tethering to VCAM-1, but did not impair rolling on the endothelial ligand. High affinity VLA-4 was found to comprise a small subset of VLA-4 on resting Jurkat cells and PBL. This subset is essential for firm adhesion but not for tethering or rolling adhesions on VCAM-1. Interestingly, low and high affinity VLA-4 states were found to mediate similar initial tethering to ligand. High affinity VLA-4, constitutively expressed on circulating T cells, may control their early adhesion strengthening on VCAM-1-expressing endothelium before exposure to vascular chemokines and activation of additional integrins.
Asunto(s)
Movimiento Celular/inmunología , Integrinas/biosíntesis , Receptores Mensajeros de Linfocitos/biosíntesis , Receptores de Antígeno muy Tardío/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Molécula 1 de Adhesión Celular Vascular/fisiología , Adhesión Celular/genética , Adhesión Celular/inmunología , Movimiento Celular/genética , Fibronectinas/fisiología , Humanos , Integrina alfa4beta1 , Integrinas/genética , Integrinas/metabolismo , Integrinas/fisiología , Interfase/inmunología , Células Jurkat , Activación de Linfocitos/genética , Microscopía Inmunoelectrónica , Mutación , Receptores Mensajeros de Linfocitos/genética , Receptores Mensajeros de Linfocitos/metabolismo , Receptores Mensajeros de Linfocitos/fisiología , Receptores de Antígeno muy Tardío/genética , Receptores de Antígeno muy Tardío/metabolismo , Receptores de Antígeno muy Tardío/fisiología , Reología , Subgrupos de Linfocitos T/fisiología , Subgrupos de Linfocitos T/ultraestructura , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.
Asunto(s)
Antígenos CD2/química , Antígenos CD2/fisiología , Integrina beta1/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Prolina , Linfocitos T/fisiología , Secuencia de Aminoácidos , Antígenos CD/biosíntesis , Antígenos CD/química , Antígenos CD/fisiología , Antígenos CD2/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citoplasma , Activación Enzimática , Fibronectinas/fisiología , Glutatión Transferasa/metabolismo , Células HL-60 , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Linfocitos T/inmunología , TransfecciónRESUMEN
The role of intercellular adhesion molecule-1 (ICAM-1) in murine lung inflammation was examined in vivo. Ovalbumin (Ova)-sensitized and -challenged ICAM-1-deficient (KO) mice had decreased accumulation of leukocytes in the bronchoalveolar lavage fluid compared with wild-type (WT) mice. Lung tissue inflammation was also attenuated. Ova immunization and challenge produced equivalent plasma levels of Ova-specific immunoglobulin (Ig) G1 and higher concentrations of IgE in KO versus WT mice. Ova-dependent induction of cytokines in vitro, as measured by enzyme-linked immunosorbent assay, was impaired in splenocytes from KO mice compared with the comparable release of interleukin (IL)-5 and IL-10 from anti-CD3-stimulated WT and KO splenocytes. Methacholine-induced increases in trapped gas in lungs of Ova-sensitized and -challenged WT mice were greater than those of KO mice. The activation of lung tissue nuclear factor-kappa B was diminished in KO mice after Ova provocation. This suggests that ICAM-1 was important for activation of the inflammatory cascade leading to the recruitment of leukocytes but was not critical for the generation of antibody responses in vivo.
Asunto(s)
Citocinas/biosíntesis , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Molécula 1 de Adhesión Intercelular/fisiología , Leucocitos/fisiología , Pulmón/fisiología , Linfocitos T/inmunología , Animales , Formación de Anticuerpos , Antígenos de Diferenciación/análisis , Líquido del Lavado Bronquioalveolar/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inflamación , Molécula 1 de Adhesión Intercelular/genética , Leucocitos/inmunología , Pulmón/inmunología , Pulmón/patología , Cloruro de Metacolina/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Ovalbúmina , Bazo/inmunologíaRESUMEN
The functional activity of integrin receptors on T cells is dynamically regulated so that T cells can alternate rapidly between adhesive and nonadhesive states. The CD7 Ag is one of several molecules on T cells that can transduce intracellular signals that rapidly up-regulate integrin-mediated adhesion. We demonstrate in this report that the signaling pathway that CD7 utilizes to regulate integrin activity involves the lipid kinase phosphatidylinositol 3-kinase (PI 3-K). CD7 stimulation of both Jurkat T cells and resting human peripheral blood CD4+ T cells results in rapid association and activation of PI 3-K with CD7. Phosphopeptide competition assays demonstrate that the association of CD7 with PI 3-K is dependent on tyrosine phosphorylation of the SH2 binding motif Tyr-Glu-Asp-Met (YEDM) in the CD7 cytoplasmic domain. A role for PI 3-K in the regulation of integrin function by CD7 is demonstrated by: 1) the ability of two structurally distinct PI 3-K inhibitors, wortmannin and LY294002, to inhibit CD7-mediated increases in beta1 integrin function of human T cells; and 2) inhibition of CD7-mediated activation of beta3 integrin function in human T cells by expression of a dominant negative form of the p85 subunit of PI 3-K. These results demonstrate that the CD7 Ag on human T cells is coupled to PI 3-K and that this association is relevant to CD7-mediated signaling events, specifically CD7-induced increases in integrin adhesiveness. Furthermore, these studies provide important new evidence implicating PI 3-K in the regulation of integrin adhesiveness by multiple cell surface signaling receptors.
Asunto(s)
Antígenos CD7/inmunología , Integrinas/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Adhesión Celular/inmunología , Humanos , Células Jurkat , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Linfocitos T/patología , Tirosina/metabolismoRESUMEN
Eosinophils and mast cells have long been considered as the major effector cells ultimately responsible for bronchial obstruction and airway hyper-responsiveness in asthmatics. However, there is now accumulating evidence that products of Th2 lymphocytes may orchestrate the generation, accumulation, and activation of these cells within the airway wall. Since the first report by Mosmannet al. in 1986 that murine helper T-cell clones could be divided into two subsets, Th1 and Th2, depending on their pattern of cytokine secretion, and observations that polarisation of Th1- or Th2-dependent cytokine production could be correlated with distinct autoimmune and allergic disorders, there has been an increasing interest in the possibility that pharmacological manipulation of the Th1/Th2 paradigm could provide novel treatments for human disease. This review summarises the evidence to date, attempts to explain some apparent discrepancies, and indicates opportunities for therapeutic intervention.
RESUMEN
Cross-linking of the CD28 Ag on T cells results in increased beta 1-integrin-mediated adhesion to fibronectin. Chimeric contructs containing the CD28 cytoplasmic domain fused to the extracellular and transmembrane regions of CD2 were expressed in HL60 cells to investigate CD28-mediated regulation of adhesion. Ab cross-linking of the CD2/28 chimera resulted in increased beta 1-dependent adhesion of HL60 transfectants to fibronectin. Induced binding was completely inhibited by the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Cross-linking of the CD2/28 chimera also induced association of the p85 subunit of PI 3-K with the CD2/28 cytoplasmic domain. In contrast, cross-linking of a CD2/28 chimera containing a tyrosine to phenylalanine substitution in the YMNM motif did not result in increased adhesion to fibronectin and did not lead to association of the chimera with PI 3-K. These results directly implicate the YMNM motif and PI 3-K in the regulation of beta 1-integrin activity by the CD28 Ag.
Asunto(s)
Antígenos CD28/metabolismo , Antígenos CD28/farmacología , Integrina beta1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Regulación hacia Arriba/inmunología , Secuencia de Aminoácidos , Androstadienos/farmacología , Secuencia de Bases , Antígenos CD2/genética , Línea Celular , Fibronectinas/metabolismo , Vectores Genéticos , Humanos , Integrina beta1/efectos de los fármacos , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Proteínas Recombinantes/análisis , Tirosina/genética , Tirosina/fisiología , Regulación hacia Arriba/efectos de los fármacos , WortmaninaRESUMEN
beta 1-integrins expressed on resting T cells support only minimal adhesion to integrin ligands. T cell activation through multiple stimuli, including phorbol ester treatment and Ab cross-linking of the CD3/TCR complex, results in a rapid and transient switch in integrin function from low to high avidity binding. The exact nature of the intracellular signals involved in this avidity switch remain poorly defined, but the ability of phorbol esters to induce such up-regulation implicates a role for protein kinase C (PKC). We have used a genetic approach to identify factors other than PKC that regulate activation-dependent beta 1-integrin function on T cells. We isolated mutants of the Jurkat T cell line that express beta 1- and beta 2-integrins but do not exhibit increased integrin activity in response to PMA stimulation or CD3 cross-linking. PKC activity appears to be normal in the mutants. One mutation is associated with an altered form of the mitogen-activated protein kinase ERK1 and an inability to produce IL-2. Another mutant with defective integrin function has IL-2 production intact. Complementation analysis verified that these two types of mutants are genetically distinct. Thus, two mutations downstream of PKC have been identified that alter the process of integrin regulation without affecting T cell viability or proliferative capacity. These mutants represent novel reagents for the identification of integrin regulatory factors and indicate possible sites of pharmacologic intervention that could prevent integrin-dependent migration and localization in the process of inflammation, while leaving other T cell functions intact.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Integrinas/genética , Activación de Linfocitos/genética , Linfoma de Células T/enzimología , Mutación , Proteína Quinasa C/genética , Antígenos CD18/genética , Adhesión Celular/genética , Separación Celular , Regulación Neoplásica de la Expresión Génica/inmunología , Prueba de Complementación Genética , Humanos , Integrina beta1/genética , Integrinas/biosíntesis , Integrinas/fisiología , Interleucina-2/biosíntesis , Interleucina-2/genética , Linfoma de Células T/genética , Proteína Quinasa C/deficiencia , Proteína Quinasa C/fisiología , Proteínas Quinasas/genética , Células Tumorales CultivadasRESUMEN
The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen-specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of beta 1 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses beta 1 integrins that mediate adhesion to fibronectin and VCAM-1 in an activation-dependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased beta 1-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in beta 1 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in beta 1 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylation-dependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2-induced increases in beta 1 integrin function, and CD2 stimulation increases F-actin content in a wortmannin-sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of beta 1 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of beta 1 integrin functional activity by the CD2 antigen.
Asunto(s)
Antígenos CD2/metabolismo , Integrina beta1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Actinas/metabolismo , Androstadienos/farmacología , Adhesión Celular/fisiología , Inhibidores Enzimáticos/farmacología , Expresión Génica/fisiología , Células HL-60/enzimología , Humanos , Integrina beta1/genética , Leucemia Promielocítica Aguda , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Linfocitos T/enzimología , Transfección , Regulación hacia Arriba/fisiología , WortmaninaRESUMEN
Activation of murine T cells by Ag or mitogens results in changes in the expression of several cell-surface adhesion molecules that alter the way in which these cells migrate and localize in tissues in vivo. As naive CD8 precursor cells in lymph nodes (LN) differentiate into effector CTL in response to a skin allograft, they up-regulate their expression of Pgp-1 (CD44), VLA-4, and LFA-1 (CD11a/18), while down-regulating L-selectin (CD62L). All cytolytic activity is therefore present in this minor population of L-selectin- Pgp-1high LN T cells. We now report that, late after rejection of minor histocompatibility Ag-disparate skin grafts, the majority of memory CD8 T cells express both L-selectin and Pgp-1 and thus would be expected to migrate via the classical route of recirculation through LN. Furthermore, restimulation of these memory cells by Ag causes them to down-regulate L-selectin, so that memory-effector cells have the same L-selectin- Pgp-1high phenotype as do primary effector cells. These results are in contrast to recent reports that murine and ovine CD4 memory cells do not express L-selectin or recirculate through LN high endothelial venules. In addition, although virgin and naive CD4 cells may be divided based upon their expression of CD45RA or CD45RB, memory CD8 cells cannot be differentiated by their expression of CD45 isoforms.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Antígenos de Histocompatibilidad Menor/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Pruebas Inmunológicas de Citotoxicidad , Femenino , Citometría de Flujo , Rechazo de Injerto/inmunología , Técnicas de Dilución del Indicador , Selectina L , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Trasplante de Piel/inmunologíaRESUMEN
T lymphocytes isolated from human peripheral blood express beta 1 (VLA) and LFA-1 integrins, but strong binding to integrin ligands occurs only after the delivery of an activation stimulus to the T cell. To gain further insight into activation-dependent regulation of integrin function, we have analyzed integrin activity on three different T-leukemic cell lines: Jurkat, CEM, and H9. This analysis shows important mechanistic differences in integrin regulation. First, phorbol ester treatment results in increased beta 1 integrin-dependent adhesion of both Jurkat and CEM cells to fibronectin, but decreased adhesion of H9 cells. Second, certain activation stimuli that upregulate beta 1 integrin activity in peripheral T cells are nonfunctional in these T-cell lines. Third, analysis of a panel of Jurkat mutants lacking surface expression of CD2 and/or CD3 shows that CD2-mediated upregulation of beta 1 integrin activity is dependent on expression of CD3, whereas CD28-mediated upregulation is not dependent on either CD2 or CD3 expression. Fourth, all T-cell lines tested show an inability to adhere to purified ICAM-1 via LFA-1. The selective alterations in integrin regulation in these cell lines relative to peripheral blood T cells provide important insights into the intracellular processes involved in integrin activation.
Asunto(s)
Adhesión Celular , Integrinas/fisiología , Linfocitos T/inmunología , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos CD2 , Línea Celular , Células Clonales , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Integrinas/biosíntesis , Integrinas/efectos de los fármacos , Cinética , Leucemia de Células T , Receptores Inmunológicos/biosíntesis , Células Tumorales CultivadasRESUMEN
Integrins are a large family of cell surface receptors that mediate the adhesion of cells to other cells and to components of the extracellular matrix. Various divalent cations, particularly Ca2+ and Mn2+, have been shown to modulate the functional activity of many different integrins expressed on a wide variety of cell types. In this study, we have characterized the divalent cation requirements for the adhesion of human peripheral CD4+ T cells to four distinct integrin ligands: the alpha 4 beta 1 and alpha 5 beta 1 ligand fibronectin, the alpha 4 beta 1 ligand VCAM-1, the LFA-1 ligand ICAM-1, and the alpha 4 beta 1 bacterial ligand invasin. We find that there are distinct divalent cation requirements for T cell adhesion to each of these ligands: 1) Mg2+/EGTA treatment selectively up-regulates T cell adhesion to ICAM-1; 2) Mn2+ coordinately up-regulates adhesion to ICAM-1, fibronectin, and VCAM-1, with a peak response at 100 microM Mn2+; 3) Ca2+ can selectively support adhesion to VCAM-1 induced by activation and inhibit Mn(2+)-dependent adhesion to ICAM-1; and 4) binding to invasin is maximal in the presence of Ca2+, Mg2+, or Mn2+. Furthermore, divalent cation modifications do not fully up-regulate T cell adhesion to fibronectin, VCAM-1, and ICAM-1, because additional cell activation with phorbol ester treatment can further enhance adhesion in the presence of Mn2+. These results suggest that modification of divalent cations may provide a mechanism by which an individual integrin receptor/ligand interaction can be specifically and selectively regulated.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/fisiología , Linfocitos T CD4-Positivos/fisiología , Moléculas de Adhesión Celular/fisiología , Fibronectinas/fisiología , Integrinas/fisiología , Calcio/farmacología , Adhesión Celular , Ácido Egtácico/farmacología , Humanos , Molécula 1 de Adhesión Intercelular , Manganeso/farmacología , Acetato de Tetradecanoilforbol/farmacología , Regulación hacia Arriba , Molécula 1 de Adhesión Celular VascularRESUMEN
Adhesion molecules allow lymphocytes to interact with and respond to the extracellular environment. Since these interactions must be essentially transient in nature, the function of lymphocyte adhesion molecules must be precisely regulated. Studies of integrin receptors vividly illustrate the various mechanisms by which the function of these adhesion molecules can be regulated. These include: (1) activation-dependent changes in functional activity; (2) changes in levels of expression due to differentiation events; (3) cell-specific differences in integrin binding; and (4) differential binding to distinct ligands by the same integrin. These mechanisms provide highly precise and specific modes of regulating lymphocyte interactions with a wide variety of potential counter-receptors and ligands.
Asunto(s)
Integrinas/fisiología , Linfocitos T/fisiología , Animales , Diferenciación Celular , Humanos , Integrinas/análisis , Activación de LinfocitosRESUMEN
A variety of adhesion molecules regulate the traffic and tissue localization of lymphocytes in vivo by mediating their binding to vascular endothelial cells. The homing receptor gp90MEL-14 (gp90), also known as LECAM-1 or L-selectin, mediates the adhesion of lymphocytes to specialized high endothelial venules in lymph nodes (LN) and is the primary molecule regulating lymphocyte recirculation and homing to LN, whereas other adhesion molecules have a major role in the localization of lymphocytes in inflammatory sites. We used four-color flow cytometric analysis to examine the regulation of adhesion receptor expression on LN CD8 T cells responding to skin allografts in vivo. In normal mice, greater than 95% of LN CD8 T cells are gp90+, being either gp90+Pgp1- (Population (Pop.) 1 or gp90+Pgp-1+ (Pop.2). Allografting induces the down-regulation of gp90 and up-regulation of Pgp-1 on a subset of cells, resulting in the appearance of CD8+gp90-Pgp-1hi (Pop. 3) cells. Pop. 3 cells also express high levels of LFA-1, ICAM-1, and ICAM-2, and a subset of them are VLA-4 alpha-positive. Purified Pop. 3 cells have potent cytolytic activity directed against donor alloantigen, whereas no such activity is present in Pop. 1 or 2 cells. Correlating with this is the high granzyme activity in Pop. 3 cells. In addition, Pop. 3 lymphocytes, but not Pop. 1 or 2, secrete a large amount of IFN-gamma in response to Ag. Finally, the CD8 T cells that infiltrate sponge matrix allografts are markedly enriched for the Pop. 3 subset. These results show that, during the immune response to alloantigen in vivo, a small subset of CD8 T cells down-regulates the LN homing receptor while increasing the expression of other adhesion molecules, as they differentiate into highly active cytolytic T lymphocytes. Thus, the differential regulation of LN homing receptors and receptors for peripheral vascular endothelium provides a mechanism that would redirect the traffic of activated effector cells away from lymphoid tissue and to sites of Ag deposition, where they would participate in the inflammatory response.