Ronald L. Whisler

Redox Signals and NF-κB Activation in T Cells

Abstract Accumulating data from a number of laboratories have recently indicated that the response of transcription factor NF-κB to alterations in the redox homeostasis of cells may play an important role in modulating immune function. The activation of NF-κB has been recognized to regulate a number of genes necessary for normal T cell responses including IL-2, IL-6, IL-8, and several T cell surface receptors. Diminished NF-κB activity has been shown to occur in T cells with aging, suggesting that impaired activation of NF-κB might occur during cellular senescence. In addition, aberrancies in NF-κB activity have been implicated in the immunopathogenesis of diseases involving immune or inflammatory processes such as atherosclerosis and HIV-1 infection. The role of H2O2 and other reactive oxygen species (ROS) as an integratory secondary messenger for divergent T cell signals has been complicated by the fact that various T cell lines and peripheral blood T cells differ markedly in the levels of NF-κB activation induced by oxidant stress. Additionally, proposed pathways of NF-κB activation have been based on indirect evidence provided by experiments which used antioxidants to inhibit active NF-κB formation. Further, complete activation of T cells requires at least two signals, one that stimulates an increase in intracellular calcium and one that stimulates enzymatic processes including kinases. Similarly, substantial evidence indicates that full activation of NF-κB requires dual signals. The ability of H2O2 or other ROS to induce T cell signals and functional responses by these two mechanisms is reviewed and the specific response of NF-κB to redox changes in T cells is examined. Data are also presented to suggest that the redox regulation in NF-κB activation may be relevant to immune-related diseases and to aging.

Age-related alterations in the activation and expression of phosphotyrosine kinases and protein kinase C (PKC) among human B cells ☆

Age-related changes in the functional properties of human B cells have been reported by several groups, but little is known about the early biochemical events and signaling pathways that might be altered during aging. The present investigation examined whether differences in the activation of protein tyrosine kinases (PTK) and in the expression of protein kinase C (PKC) enzymatic activity could be identified in B cells from 16 elderly subjects (mean 77 years) compared to B cells from 15 young subjects (mean 33 years). B cells from young subjects stimulated with the surface immunoglobulin (sIg) crosslinkers anti-IgM or Staphylococcus aureus Cowan I (SAC) demonstrated rapid increases in PTK mediated de novo tyrosine phosphorylation of endogenous proteins. In comparison, stimulated B cells from elderly subjects were reduced 22-46% in tyrosine phosphorylations. Quantitation of the enzymatic levels and activation/translocation of PKC activity among resting and sIg stimulated B cells showed that B cells from approximately 50% of elderly subjects had significant reductions compared to B cells from young subjects. Further analyses of the expression of PTK and PKC enzymatic activity by stimulated B cells from elderly subjects demonstrated that aging was associated with greater heterogeneity in PKC expression and that defects in PKC enzymatic activity could coexist with relatively normal PTK activity. Thus, these data suggest that aging can alter the expression of PTK/PKC enzymatic activity in human B cells and that these age-related alterations might perturb the balance between PKC-dependent and -independent signaling pathways.

Age-related defects in Th1 and Th2 cytokine production by human T cells can be dissociated from altered frequencies of CD45RA+ and CD45RO+ T cell subsets

The present study investigated whether age-related changes in the production of Th1 and Th2 cytokines by human T cells might be linked to altered frequencies of naive (CD45RA+) and memory (CD45RO+) T cell subsets. T cells from healthy elderly humans (n = 32) stimulated with anti-CD3epsilon monoclonal antibody OKT3 plus PMA produced significantly lower levels of IL-2 and IFNgamma (Th1 type) and of IL-4 (Th2 type) cytokines compared with T cells from young subjects. Although considerable heterogeneity was observed in the levels of cytokines produced by activated T cells from elderly individuals, linear regression analysis failed to demonstrate any significant shift in Th1 to Th2 type cytokine profiles of human T cells during aging. Sufficient T cells were available from eighteen elderly subjects to quantitate the levels of cytokine production in parallel with flow cytometry analysis of the frequencies of CD45RA+ naive and CD45RO+ memory T cells. Compared with the group of young subjects, the elderly group exhibited significant decreases in the frequencies of naive T cells with reciprocal increases in memory T cells. However, defects in Th1 and Th2 cytokine production were not significantly correlated with altered frequencies of naive/memory T cells among elderly individuals. In addition, those elderly individuals with normal frequencies of naive/memory T cells exhibited decreases in cytokine production comparable to the reductions observed for elderly donors with alterations in the frequencies of naive/memory T cells. These findings suggest that age-related defects in Th1 and Th2 cytokine production cannot be attributed entirely to alterations in the frequencies of naive/memory T cell subsets and point toward intrinsic aberrancies within human T cell cytokine networks during aging.

Characteristics of cyclosporine induction of increased prostaglandin levels from human peripheral blood monocytes

Human monocytes (MØ) exposed to 0.5–20 ug/ml of cyclosporine (CsA) produced levels of prostaglandins of the E series (PGE) that were 2–3-fold greater than control MØ cultured in medium alone. Maximal PGE levels were obtained at 24–48 hr incubation, and the failure to observe a linear increase of PGE levels at higher CsA concentrations appeared partially related to cytotoxic effects. CsA was considerably less effective than phorbol myristate acetate or bacterial lipopolysac-charide in increasing PGE production, but the PGE levels achieved with CsA approximated those known to suppress immune responsiveness. Other experiments showed that, although the increased PGE production with CsA was indomethacin-sensitive, CsA mostly functioned to increase the availability of free arachidonic acid (AA) instead of accelerating AA conversion by the cyclooxygenase pathway. Thus CsA can alter MØ physiology, and these alterations might inhibit quite early events during the induction phase of immune responses.

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.