RESUMEN
The expression and catalytic activity of the protein tyrosine kinase (PTK) ZAP-70 are needed for normal intracellular signaling through the T-cell receptor (TCR)/CD3 complex. However, the possible effect of aging on the catalytic activity of ZAP-70 in human peripheral blood T cells stimulated via the TCR/CD3 complex is unknown. The current studies show that T cells from a substantial proportion of elderly humans (12) exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. In addition, the reduced catalytic activity of ZAP-70 in T cells from elderly subjects was not restored to the normal levels in response to ligation of CD4 receptors, suggesting defects in PTKs linked to both CD3 and CD4 receptors. Other experiments demonstrated that the age-related impairments of ZAP-70 activation in anti-CD3-stimulated T cells were accompanied by decreased tyrosine phosphorylations of zeta-chains and autophosphorylations of the PTKs p561ck/p59fyn. Moreover, the age-related defects in these early TCR/CD3-mediated phosphorylation events were readily detectable in both CD45RO+ memory and CD45RA+ naive T cells. Thus, these results suggest that defects in early TCR/CD3-mediated phosphorylation events among CD45RO+ memory and CD45RA+ naive T cells from certain elderly humans may con tribute to impaired induction of ZAP-70 catalytic activity.
Asunto(s)
Envejecimiento/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Tirosina/metabolismo , Anciano , Anciano de 80 o más Años , Envejecimiento/sangre , Envejecimiento/inmunología , Complejo CD3/inmunología , Células Cultivadas , Activación Enzimática , Humanos , Memoria Inmunológica , Antígenos Comunes de Leucocito/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fyn , Linfocitos T/citología , Proteína Tirosina Quinasa ZAP-70RESUMEN
Aging is often accompanied by altered T-cell signaling and functions. Signals mediated through the T-cell receptor (TCR)/CD3 complex are associated with tyrosine phosphorylations of zeta-chains by the regulated activities of protein tyrosine kinases p56(lck) and p59(fyn) as well as protein tyrosine phosphatases. In the present investigation, the coupling and phosphorylation of zeta-chains to TCR/CD3 immunocomplexes were examined in peripheral blood T-cells from 13 elderly and young humans stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. Western blots analyzing the non-covalent coupling of zeta-chains to TCR/CD3 immunocomplexes from Brij-96 detergent lysates of anti-CD3 ligated T-cells showed that the levels of zeta-chains within TCR/CD3 immunocomplexes from T-cells of elderly and young subjects did not significantly differ. By contrast, the levels of phosphorylated zeta-chains generated during in vitro phosphorylations of TCR/CD3 immunocomplexes from elderly subjects were significantly reduced and averaged 44% of those observed for anti-CD3epsilon ligated T-cells from young subjects. Analyses of the levels of zeta-chain coupling and phosphorylations in T-cells from each of the 13 elderly individuals also showed that the reductions in zeta-chain phosphorylations were heterogeneous and unrelated to modest reductions in coupling. Furthermore, the age-related decreases in zeta-chain phosphorylations were not due to diminished frequencies of CD3epsilon+ cells or densities of CD3epsilon surface receptors and could be observed without reductions in epsilon-chain phosphorylations. These results suggest that aberrancies of zeta-chain phosphorylations can occur in T-cells of elderly humans independent from any uncoupling of zeta-chains to activated TCR/CD3 complexes.
Asunto(s)
Envejecimiento/inmunología , Proteínas de la Membrana/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Anciano , Anciano de 80 o más Años , Complejo CD3/metabolismo , Antígenos CD4/metabolismo , Femenino , Humanos , Masculino , FosforilaciónRESUMEN
Optimal signal transduction through the T cell receptor (TCR)/CD3 complex requires the coordinated activities of protein tyrosine kinases (PTKs) Fyn and Lck in addition to protein tyrosine phosphatases (PTPases) such as CD45. Although T cells stimulated with anti-CD3 monoclonal antibodies (mAb) exhibit age-related reductions in tyrosine phosphorylations of cellular proteins, it is unknown if the reduction represent abnormalities in PTKs or PTPases. In the current studies, immune complex kinase assays showed that the stimulation of peripheral blood T (PBT) cells from young humans with cross-linked anti-CD3 epsilon mAb OKT3 induced increased Fyn catalytic activity while anti-CD3 stimulation failed to induce significant increases in Lck activation. By contrast, Fyn activation in anti-CD3 stimulated PBT cells from a substantial proportion of elderly humans was reduced compared to anti-CD3 stimulated PBT cells from young humans. Also, we failed to find any increase in anti-CD3 stimulation of Lck activity in PBT cells from elderly subjects that could compensate for the decline in Fyn activity. However, no age-related alterations were detected in PBT cell expression of Fyn or Lck that might contribute to the changes in enzymatic activity. The results of other experiments demonstrated that the functional activities of PTPases in PBT cells from elderly subjects were equivalent to PBT cells from young subjects. These observations suggest that aberrant regulation of TCR/CD3 coupled PTKs may contribute to the age-related defects in signaling cascades and immune responsiveness of human T cells.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Linfocitos T/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Catálisis , Células Cultivadas , Activación Enzimática , Femenino , Humanos , Antígenos Comunes de Leucocito/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Masculino , Datos de Secuencia Molecular , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-fyn , Linfocitos T/citologíaRESUMEN
Age-related changes in the functional properties of human T cells are well described, but less is known about possible changes in T cell signaling pathways. The signaling pathways mediated by mitogen-activated protein kinases (MAPK) are considered essential for normal cellular growth and function. Several stimuli trigger MAPK activation in human T cells and MEK (MAPK or ERK kinases) are immediate upstream inducers of MAPK activation. The current study investigated if aging might influence the activation and expression of MAPK and MEK in human T cells. Exposure of peripheral blood T cells from young subjects to PHA or cross-linked anti-CD3 monoclonal antibodies stimulated rapid increases in MAPK and MEK enzymatic activity. By contrast, significant reductions of MAPK and MEK activation were observed in stimulated T cells from 7 of 13 elderly subjects. Kinetic studies showed that the age-related impairments represented reduction in both the levels and duration of MAPK activation. In addition, Western immunoblot analysis did not reveal significant age-related differences in T cell expression of p42mapk/ERK2, p44mapk/ERK1, or MEK, suggesting impairments in upstream inducers of MEK/MAPK activation. Other experiments determined if agents that directly stimulate upstream Ras or Raf kinase components of the early MAPK cascade might reverse the age-related impairments of MAPK activation. Treatment of elderly T cells with fluoroaluminate (AlF(-)4), phorbol esters/Ca2+ ionophores, or okadaic acid stimulated increased MAPK activation compared to anti-CD3. However, these agents failed to restore MAPK activation in elderly T cells to the levels seen in young T cells. These results suggest that aberrancies in the MAPK activation cascade may underlie the age-related reductions of MAPK activation in human T cells stimulated via the TCR/CD3 complex.
Asunto(s)
Envejecimiento/inmunología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Isoenzimas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Muromonab-CD3/farmacología , Fitohemaglutininas/farmacología , Procesamiento Proteico-Postraduccional/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Transducción de Señal/fisiología , Linfocitos T/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Activación Enzimática/efectos de los fármacos , Éteres Cíclicos/farmacología , Femenino , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Lectinas/farmacología , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Masculino , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Ácido Ocadaico , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Sublethal concentrations of reactive oxygen intermediates including H2O2 can alter human T cell function and inhibit proliferative responses but relatively little is known about the effects of low levels of oxidant stress on signaling pathways. In the present study, we investigated whether the exposure of Jurkat T cells to micromolar concentrations of H2O2 might influence the activity of certain serine/threonine kinases and protein phosphatases important for T cell signaling as well as initiation of nuclear events. Jurkat cells treated with 100-200 microM H2O2 exhibited rapid increases in cytosolic protein kinase C (PKC) activity without detectable translocation of PKC to the membrane/particulate compartment. The stimulation of PKC activity by H2O2 was associated with an increase in the activation of kinases phosphorylating myelin basic protein (MBP), a substrate for mitogen-activated protein (MAP) kinase and RRLSSLRA (S6 peptide; a substrate for the approximately 90-kDa ribosomal S6 kinases). Optimal activation of MAP kinase in cells treated with H2O2 was preceded by increases in protein tyrosine phosphorylations and occurred at sublethal concentrations of H2O2 which did not markedly deplete intracellular ATP. Pretreatment of cells with the PKC inhibitors sangivamycin and H7 suppressed but did not block the stimulation of MAP kinase activity in response to H2O2 or phytohemagglutinin. The activities of both protein tyrosine phosphatase (PTP) and protein phosphatase 2A (PP2A) were reduced after H2O2 treatment of intact cells. Furthermore, kinetic studies showed that H2O2 was capable of suppressing the activities of PTP and PP2A before inducing optimal increases in MAP kinase activity. These results demonstrate that the exposure of T cells to sublethal levels of oxidant stress acutely stimulates the MAP kinase cascade and suggest that this activation may involve PKC-dependent and -independent pathways as well as inhibition of certain protein phosphatases.
Asunto(s)
Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/enzimología , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/toxicidad , Cinética , Datos de Secuencia Molecular , Estrés Oxidativo , Péptidos/química , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Fosfatasa 2 , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal , Especificidad por Sustrato , Linfocitos T/efectos de los fármacos , Tirosina/metabolismoRESUMEN
The expression of alpha- and beta-isoforms of protein kinase C (PKC) was analyzed in the peripheral blood T and B cells from 11 elderly and young humans. Immunoblot analysis with isoenzyme specific antibodies showed that T cells from five of 11 elderly subjects exhibited selective reductions in PKC alpha which was < 60% of those in young subjects whereas the levels of PKC beta were comparable to T cells of young subjects. No age-related reductions of PKC alpha or beta were observed in B cells. Among individual elderly subjects, the reductions in T cell PKC alpha were not associated with lower levels of PKC beta thereby resulting in only approximately 60-70% reductions of combined PKC alpha plus PKC beta. In addition, the functional properties of PKC in stimulated T cells of elderly subjects with respect to activation/translocation were comparable to T cells of young subjects. These results suggest that selective alterations in PKC isoenzymes can occur in human T cells during aging which may not be readily apparent in standard enzymatic assays and may contribute to aberrancies in intracellular signal transduction.
Asunto(s)
Envejecimiento/metabolismo , Linfocitos B/enzimología , Células Sanguíneas/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Linfocitos T/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Transporte Biológico , Calcio/fisiología , Femenino , Humanos , Immunoblotting , Masculino , Fosfolípidos/fisiologíaRESUMEN
Prior studies have suggested that intracellular phosphorylation events and cellular redox mechanisms may interact in regulating a variety of cellular functions, including the transcriptional activation of gene expression. Increased activity of transcriptional factors NF kappa B and AP1 has been described in cells exposed to oxidative stress and following the direct stimulation of protein kinase C (PKC) by phorbol diesters. However, the mechanisms that may contribute to redox regulation of PKC are unknown. We studied the expression of PKC activity and several second messengers in human Jurkat T cells exposed to oxidative stress in the form of H2O2. Micromolar concentrations of H2O2 rapidly induced increased cytosolic PKC enzymatic activity in Jurkat T cells that was associated with a marked arrest of cellular proliferation. The increase in cytosolic PKC activity in cells treated with H2O2 was accompanied by elevations in intracellular free calcium ([Ca2+]i), generation of inositol phosphates, and release of arachidonic acid. Functional studies showed that H2O2 enhancement of cytosolic PKC activity required phospholipase C activity but was not primarily mediated by arachidonic acid. The response of PKC to oxidative stress displayed a lack of Ca2+ dependence and was uncoupled from the activity of protein tyrosine kinases (PTK). Furthermore, the reduced activation requirements of PKC from cells treated with H2O2 were associated with shifts in elution profiles of PKC enzymatic activity after Mono-Q chromatography. These shifts appeared to represent intrinsic changes in the conformation of PKC induced by oxidative stress because western blotting failed to reveal any PKC cleavage products or reductions in native PKC alpha or beta. These findings indicate that oxidative regulation of intracellular events can intersect phosphorylation events mediated by PKC through the release of second messengers as well as direct changes in PKC activation requirements. Moreover, redox regulation of PKC is distinct from T cell receptor signaling in that the activity of PKC is uncoupled from the regulatory influences of PTK.
Asunto(s)
Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/enzimología , Secuencia de Aminoácidos , Ácido Araquidónico/farmacología , Calcio/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Oxidación-Reducción , Fosforilación Oxidativa , Estrés Oxidativo , Péptidos/química , Péptidos/farmacología , Fosfolipasas A/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.
Asunto(s)
Linfocitos B/efectos de los fármacos , Bucladesina/farmacología , División Celular/efectos de los fármacos , Proteínas Quinasas/efectos de los fármacos , Sulfonamidas , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos , Bucladesina/antagonistas & inhibidores , Sinergismo Farmacológico , Humanos , Ionomicina/farmacología , Isoquinolinas/farmacología , Datos de Secuencia Molecular , Ésteres del Forbol/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas QuinasasRESUMEN
Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.
Asunto(s)
Envejecimiento/fisiología , Aminoácidos/metabolismo , Linfocitos B/fisiología , Calcio/metabolismo , Citosol/metabolismo , Transducción de Señal/fisiología , División Celular/fisiología , Células Cultivadas , Humanos , Fosforilación , Proteína Quinasa C/sangre , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiología , Ribonucleasas/antagonistas & inhibidores , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismoRESUMEN
Age-related reductions in the DNA replication of human peripheral blood B cells have been reported after stimulation by cross-linking surface immunoglobulins (sIg) with the polyclonal activator Staphylococcus aureus Cowan I (SAC). However, little is known about the mechanisms of these age-related impairments. To examine whether these impairments represented defects unique to sIg mediated signalling, B cells from elderly humans were stimulated with SAC, immobilized anti-IgM and with monoclonal antibodies (mAbs) specific for B cell CD20 and CD40 determinants. Regardless of the stimuli or combinations of stimuli, the proliferative responses of B cells from elderly subjects remained 50% or less of the values observed for B cells from young subjects. Also, the failure to fully restore the age-related impairments of B cells could not be attributed to an absolute lack of potentially reactive cells. Supplementation of anti-IgM stimulated B cells from elderly subjects with IL-2, IL-4 or B cell growth factor (BCGF) revealed that BCGF was able to improve the reduced responses to levels approximating B cells of young subjects. The age-related defects were not restricted to B cell DNA replication because reductions in G1 progression of stimulated B cells from elderly subjects were directly demonstrated by decreased [3H]uridine incorporation into de novo RNA synthesis. However, the age-related impairments in RNA synthesis were less severe than those in DNA replication consistent with progressively greater reductions in the abilities of B cells to traverse the entire cell cycle. Other results showed that the reduced DNA replication of B cells from elderly subjects to immobilized anti-IgM with and without IL-2 did not represent a premature exit of B cells from DNA replication and accelerated maturation into antibody producing cells. Thus, these studies demonstrate that age-related impairments exist in activation signals mediated by several types of human B cell determinants and that abnormalities can be detected during pre-S phase events.
Asunto(s)
Envejecimiento/inmunología , Linfocitos B/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD , Antígenos CD20 , Antígenos de Diferenciación de Linfocitos B , Linfocitos B/citología , Linfocitos B/metabolismo , Antígenos CD40 , Ciclo Celular , Replicación del ADN , Humanos , Técnicas In Vitro , Activación de Linfocitos , Persona de Mediana Edad , ARN/biosíntesis , Receptores de Antígenos de Linfocitos B , Transducción de Señal/inmunologíaRESUMEN
Age-related changes are known to occur in the function of human T cells but less information is available about human B cells during aging. In this study, B cells obtained by negative selection from the peripheral blood of young and elderly subjects were stimulated in vitro with anti-IgM, Staphylococcus aureus Cowan strain I (SAC), or Staph protein A (SpA) from SAC. Their proliferative capabilities with and without lymphokines were quantitated by [3H]thymidine uptake. Stimulated B cells from elderly subjects were reduced in their overall ability to sustain normal levels of proliferation observed for B cells from young subjects. However, time course studies analyzing early proliferative responses revealed that B cells from one subset of elderly displayed persistent hyporesponsiveness whereas another subset demonstrated early hyperresponsiveness compared to B cells from young adults. Experiments to determine the frequencies of B cells with transferrin receptors (TfR) and low-affinity receptors for IgE (Fc epsilon RII/CD23) showed reductions in the expression of these two glycoproteins among stimulated B cells from elderly with the persistent decreases in proliferation. By contrast, unstimulated B cells of elderly subjects with early hyperresponsiveness displayed increased frequencies of TfR-positive cells, which became reduced after stimulation. Further, stimulated B cells from this group demonstrated greater frequencies of CD23 positive cells than young adults (13 vs. 8%). Thus two distinct profiles of proliferative abnormalities can be observed in early cohorts of activated B cells from elderly humans. The association of these abnormalities with differences in TfR and CD23 suggests that certain age-related defects occur relatively early during the B cell activation scheme.
Asunto(s)
Envejecimiento/inmunología , Linfocitos B/citología , Activación de Linfocitos/fisiología , Anciano , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos B/metabolismo , División Celular/fisiología , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina E/metabolismo , Activación de Linfocitos/efectos de los fármacos , Receptores Fc/metabolismo , Receptores de IgE , Receptores de Transferrina/metabolismo , Factores de TiempoRESUMEN
Utilizing a dual liquid/semisolid culture system with human B cells suspended in a semisolid matrix, T4 helper cells activated in the autologous mixed-lymphocyte reaction (AMLR) were much more effective than T8 suppressor cells in promoting B-cell colony formation in the presence of Staph protein A. C-reactive protein (CRP) inhibited the promotion of B-cell colony formation by autoactivated T cells and inhibition was observed only when CRP was present during the early AMLR. Coculture experiments revealed that CRP directly reduced T4 promotion of colony formation rather than inducing T8-mediated suppression of T4 cells. B cells preincubated with CRP responded normally to facilitative signals from autoactivated T cells; however, monocytes or T cells preincubated with CRP were reduced in AMLR promotion of colony formation. Other results indicated that CRP inhibition of colony formation during the AMLR was associated with considerable reductions in the proliferation of autoreactive T cells. These data demonstrate that CRP can directly reduce the ability of autoreactive T4 cells to expand the B-cell compartment and that CRP blocks T-cell activation events proximal to proliferation.
Asunto(s)
Linfocitos B/inmunología , Proteína C-Reactiva/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T , Antígenos de Superficie/análisis , Autoantígenos , Humanos , Cooperación Linfocítica , Prueba de Cultivo Mixto de Linfocitos , Macrófagos/inmunología , Proteína Estafilocócica A/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
The abilities of T cells from young and elderly humans to cap membrane T3 determinants, proliferate in response to anti-OKT3 antibody, and elaborate soluble factors required for the growth of B-cell colonies were investigated. The results showed that T cells from approximately 50 to 80% of elderly subjects had reductions in ligand-induced T3-mediated membrane events and proliferative responses in addition to decreased elaboration of soluble B-cell growth factors. Thus, these previously unrecognized abnormalities in T cells might contribute to the decline of immunocompetence observed among some elderly humans.
Asunto(s)
Envejecimiento , Antígenos de Superficie/inmunología , Sustancias de Crecimiento/análisis , Recubrimiento Inmunológico , Activación de Linfocitos , Linfocinas/análisis , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación de Linfocitos T , Colchicina/farmacología , AMP Cíclico/análisis , Citocalasina B/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Interleucina-2/biosíntesis , Interleucina-4 , CinéticaRESUMEN
The abilities of human B cells from young and aged subjects to form colonies in semisolid cultures stimulated with Staphylococcus protein A were investigated. Approximately three-fourths of aged adults had significantly diminished colony responses compared to young adults. In 55% of these aged adults, the in vitro blocking of monocyte prostaglandin synthesis lead to a 1.5-fold or greater augmentation of the depressed colony responses. Other experiments showed that the improvement with indomethacin could not be explained by the greater sensitivity of aged versus young B-cell colony precursors to prostaglandin suppression. However, indomethacin failed to improve the depressed colony responses of the remaining aged adults. This failure could not be attributed to deficient interleukin 1 production, detectable alterations in accessory cell subsets of monocytes, or the lack of potential colony precursors bearing sIgD/M. Instead, the B cells from these aged subjects demonstrated a substantial decrease in the capping of sIgD/M compared to the B cells of aged subjects which displayed improved colony responses with indomethacin and compared to the B cells from young adults. Thus, these data indicate that the diminished B-cell colony responses of aged humans represent aberrancies within both the B-cell and monocyte lineages which might coexist.
Asunto(s)
Envejecimiento , Linfocitos B/inmunología , Monocitos/inmunología , Adulto , Anciano , Alprostadil , Células Clonales , Humanos , Recubrimiento Inmunológico , Indometacina/farmacología , Interleucina-1/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Prostaglandinas E/farmacología , Receptores de Antígenos de Linfocitos B/análisis , Receptores Fc/análisisRESUMEN
The present investigation was performed to determine whether the activation of human B cells by Staphylococcal protein A (SpA) in liquid and semi-solid cultures might be dependent on distinct subsets of peripheral blood mononuclear-phagocytes (M phi) defined by the expression of HLA-DR and HLA-DS determinants. Highly pure HLA-DR- M phi functioned as effectively as HLA-DR+ MO in supporting B cell liquid proliferative responses when SpA was continuously present in cultures. However, HLA-DR+ M phi were two to three times more effective than HLA-DR- M phi in promoting B cell proliferative responses when either M phi or B cells were pulsed with SpA and were then cultured without supplemental SpA. Similarly, B cell activation in semisolid cultures was crucially dependent on HLA-DR+ M phi because colony responses were reduced fivefold in the presence of M phi expressing low/intermediate HLA-DR levels compared to M phi-containing cells with high HLA-DR levels. HLA-DS- M phi isolated by two different techniques were more effective than HLA-DS+ M phi in supporting both liquid proliferative and colony responses of B cells. Flow microcytofluorometry analysis of the dual expression of HLA-DR and HLA-DS on highly pure HLA-DR- M phi and HLA-DR+ M phi revealed that both HLA-DR- and HLA-DR+ M phi expressed low levels of HLA-DS. Importantly, the expression of HLA-DS on HLA-DR- M phi was bimodal, with an HLA-DR-, DS+ subset and an HLA-DR-, DS-subset being present. Other experiments supported the conclusions that the differential abilities of the HLA-DR-, -DS-defined subsets of M phi to support B cell activation did not represent M phi suppressive effects or differences in IL 1 production. Collectively, these results indicate that B cell activation can be directly supported by M phi whose predominant phenotype is HLA-DR+, -DS-. Thus, the accessory cell pathway of B cell activation described here is distinct from the pathway known to be required for T cell responsiveness, and could serve to provide early alternative or ancillary signals for triggering B cells.
Asunto(s)
Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Activación de Linfocitos , Monocitos/clasificación , Adulto , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Antígenos HLA-DQ , Antígenos HLA-DR , Humanos , Interleucina-1/biosíntesis , Monocitos/inmunología , Fenotipo , Proteína Estafilocócica A/farmacologíaRESUMEN
Human monocytes (M phi) preexposed to cyclosporine (CsA) concentrations ranging between 1.0 and 10.0 micrograms/ml were impaired in their ability to stimulate autologous and allogeneic mixed lymphocyte reactions (MLR) when they were compared with control M phi unexposed to CsA. M phi preexposed to CsA and M phi preexposed to PGE2 displayed reduced expression of HLA-DR. Indomethacin protected M phi from decreased HLA-DR expression at lower CsA concentrations, but was unable to prevent the decrease of HLA-DR with higher concentrations of CsA. CsA appeared capable of perturbing M phi membranes because decreases in the indirect light scattering properties of M phi were detected with the various CsA concentrations tested. Higher CsA concentrations significantly reduced the cellular volumes of M phi. The reductions of cellular volume were considerably less than the decreases in indirect light scatter. These data show that CsA interacts directly with M phi, reducing their functional ability to trigger MLR responses and their phenotypic expression of HLA-DR. The decreased HLA-DR expression is mediated via prostaglandins at low CsA concentrations and the decreased HLA-DR expression is mediated via membrane perturbations unrelated to prostaglandins at high CsA concentrations.
Asunto(s)
Ciclosporinas/farmacología , Antígenos de Histocompatibilidad Clase II/análisis , Prueba de Cultivo Mixto de Linfocitos , Monocitos/efectos de los fármacos , Adulto , Linfocitos B/inmunología , Antígenos HLA-DR , Humanos , Síndromes de Inmunodeficiencia/inducido químicamente , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos/métodos , Monocitos/inmunología , Linfocitos T/inmunologíaRESUMEN
Experiments were performed to compare the activation/proliferation characteristics of B cells from elderly and young humans which were triggered by autologous monocytes pulsed with Staph protein A and to also evaluate the influence of biologically active molecules on B cell responsiveness. B cells from most elderly subjects had lower proliferative responses than did B cells from young subjects. Both interleukin 1 (IL 1) and IL 2 were capable of increasing the diminished B cell responses of certain elderly subjects while the responsiveness of other elderly subjects required either higher concentrations of IL 2 than young subjects or remained unchanged. Although interferon typically failed to influence the responses of young or aged adults, the responses of some aged adults were unexpectedly increased or decreased. Flow microcytofluorometry (FMF) analysis revealed that B cells from certain elderly subjects demonstrated distinct differences in surface IgM/D expression or light scattering characteristics as compared to B cells from young subjects. However, the FMF characteristics of B cells from elderly subjects were not closely linked to the activation/proliferation capabilities. These data suggest that B cells from certain elderly subjects are responsive to biologically active molecules but that immunosenesence of the B cell system perturbs the normal relationship between the phenotypic and functional characteristics of B cells.
Asunto(s)
Envejecimiento , Linfocitos B/fisiología , Interferón gamma/farmacología , Interleucina-1/fisiología , Interleucina-2/fisiología , Activación de Linfocitos , Monocitos/inmunología , Adulto , Anciano , Linfocitos B/clasificación , Linfocitos B/inmunología , Citometría de Flujo , Humanos , Inmunoglobulina D/análisis , Inmunoglobulina M/análisis , Proteína Estafilocócica A/farmacologíaRESUMEN
The abilities of human monocytes differentially expressing HLA-DR and of lipopolysaccharide (LPS) to influence T-cell colony responses were investigated. Optimal T-cell colony responses stimulated by soluble Staph protein A were crucially dependent on monocytes. Also, monocyte facilitation of colony responses was markedly inhibited by 10 micrograms/ml LPS and the addition of indomethacin reversed this inhibition. In contrast the inhibition of T-cell colony responses with 100 micrograms/ml LPS was not reversed with indomethacin and preincubation experiments with high concentrations of LPS showed the inhibition could be mediated through T cells by mechanisms other than prostaglandins. The treatment of monocytes with a monoclonal anti-HLA-DR reagent + C reduced the frequencies of monocytes expressing high levels of HLA-DR approximately fivefold and the resulting monocytes which expressed low levels of HLA-DR also poorly functioned in the promotion of colony responses compared to controls. LPS in the presence of indomethacin improved the ability of monocytes expressing low levels of HLA-DR to promote colony responses. However, these monocytes consistently failed to augment colony responses to those levels observed with untreated monocytes and their failure was not secondary to deficient interleukin 1 release. These results indicate that although LPS can somewhat potentiate the accessory cell function of certain human monocytes, it cannot abrogate an additional requirement for those monocytes expressing high levels of HLA-DR.
Asunto(s)
Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Monocitos/inmunología , Adulto , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II , Humanos , Células Madre/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The phenotypic characteristics of peripheral blood T cell subpopulations regulating human B cell colony growth stimulated by Staph protein A were investigated. Colony growth was facilitated by OKT4 cells, and T cells expressing DR antigens were found to be partially responsible for colony facilitation. A linear increase in the magnitude of colony growth was observed with greater T cell numbers, and maximal colony enhancement occurred when T cells were present during the early stages of colony formation. OKT8 cells did not enhance colony growth and also inhibited the facilitation of colony formation by OKT4 cells. Other experiments showed that the functional activities of OKT4 and OKT8 cells differed in their requirements for DNA synthesis. Although active T cell DNA synthesis was absolutely required for the facilitation of colony growth at all concentrations tested, DNA synthesis was not needed for OKT8 inhibition of OKT4 promotion of colony formation. Thus, distinct T cell subsets whose functional properties differ in their requirements for DNA synthesis regulate human colony growth.
Asunto(s)
Linfocitos B/citología , Hematopoyesis , Activación de Linfocitos , Linfocitos T/inmunología , Adulto , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Células Cultivadas , Genes MHC Clase II , Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Cinética , Mitomicina , Mitomicinas/farmacología , Fenotipo , Linfocitos T/clasificación , Linfocitos T Reguladores/inmunologíaRESUMEN
The ability of human C-reactive protein (CRP) to modulate Staph protein A-(SpA) induced human B cell colony formation in semisolid cultures was investigated. Maximal augmentation was observed with 10 and 25 micrograms/ml CRP and was independent of the SpA concentration used to stimulate colony formation. Optimal facilitation of colony numbers was noted when CRP was present during the initial stages of colony formation and the facilitation represented an early increase in colony numbers. At later incubation intervals, however, the colony responses of cultures containing 25 and 50 micrograms/ml CRP were considerably diminished when compared to controls that appeared secondary to the focal disintegration of colony clusters rather than the actual suppression of certain colony progenitors with delayed colony-forming kinetics. Other experiments showed the 18-hr pre-exposure of cells to CRP facilitated colony formation, and that after preincubation, a small frequency of CRP binding cells could be detected by flow microcytofluorometry. Therefore, these studies indicate that a possible biologic function of CRP is the ability to modulate the activities of the B cell system.