Yvonne G. Newhouse

Expression and catalytic activities of protein tyrosine kinases (PTKs) Fyn and Lck in peripheral blood T cells from elderly humans stimulated through the T cell receptor (TCR)/CD3 complex

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.

Human B cel proliferative responses during aging. Reduced RNA synthesis and DNA replication after signal transduction by surface immunoglobulins compared to B cell antigenic determinants CD20 and CD40

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.

Differential expression of the α- and β-isoforms of protein kinase C in peripheral blood T and B cells from young and elderly adults

Abstract The expression of α- and β-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 PKCα which was

Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of ζ-chains and p59fyn/p56lck in human T cells

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.

Phosphorylation and coupling of ζ-chains to activated T-cell receptor (TCR)/CD3 complexes from peripheral blood T-cells of elderly humans

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.

The Impaired Ability Of Human Monocytes To Stimulate Autologous And Allogeneic Mixed Lymphocyte Reactions After Exposure To Cyclosporine: Associated Alterations Of Hla-dr Expression And Physical Characteristics Of Monocytes

Human monocytes (Mø) preexposed to cyclosporine (CsA) concentrations ranging between 1.0 and 10.0 μg/ml were impaired in their ability to stimulate autologous and allogeneic mixed lymphocyte reactions (MLR) when they were compared with control Mø unexposed to CsA. Mø preexposed to CsA and Mø preexposed to PGE2 displayed reduced expression of HLA-DR. Indomethacin protected Mø 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 Mø membranes because decreases in the indirect light scattering properties of Mø were detected with the various CsA concentrations tested. Higher CsA concentrations significantly reduced the cellular volumes of Mø. The reductions of cellular volume were considerably less than the decreases in indirect light scatter. These data show that CsA interacts directly with Mø, 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.

Inhibition of human B lymphocyte colony responses by endogenous synthesized hydrogen peroxide and prostaglandins

Abstract The possible mediators responsible for inhibition of human B-cell colony formation stimulated in vitro by Staph protein A (SpA) were investigated. Exogenous PGE1 and PGE2, but not PGF2a, markedly diminished colony growth in cultures with optimal B cells and monocyte ratios. Colony responses were quite sensitive to prostaglandin E series (PGEs) inhibition ranging from 2 to 50% of controls at concentrations between 1000 and 1 nM, respectively. Similarly, concentrations of exogenous hydrogen peroxide (H2O2) between 10−2 and 10−5M inhibited colony responses. The suppression by PGEs and H2O2 represented an absolute decrease of the colony numbers with the relative frequency of colonies containing cells characteristic of B cells being identical to controls. Lipopolysaccharide (LPS) was also found to inhibit the responses of cultures with optimal B cell-monocyte ratios, but the inhibition could be reversed by either indomethacin or catalase. The diminished colony responses in high-density cultures with supraoptimal monocyte numbers were significantly augmented by either indomethacin or catalase. In contrast, identical experiments in SpA-stimulated liquid cultures failed to demonstrate a modulation of proliferative responses by exogenous or endogenously produced mediators. These data suggest that human B-cell colony precursors are quite sensitive to the inhibitory effects of exogenous and endogenous PGEs and H2O2. Additionally, the inhibition of colony responses by LPS does not represent a direct effect on colony precursors, but is indirectly mediated by the endogenous production of H2O2 and PGEs presumably derived from monocytes.

Cyclic AMP modulation of human B cell proliferative responses: Role of cAMP-dependent protein kinases in enhancing B cell responses to phorbol diesters and lonomycin☆

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.

Function of T cells from elderly humans: reductions of membrane events and proliferative responses mediated via T3 determinants and diminished elaboration of soluble T-cell factors for B-cell growth

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.

Human B-lymphocyte colony responses: Suboptimal colony responsiveness in aged humans associated with defective function of B cells and monocytes

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.