HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset.

BACKGROUND: T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2-3 fold over controls in percentage of cells producing inflammatory cytokines IFN-gamma and TNF-alpha. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1-2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4-5 fold) in IFN-gamma and TNF-alpha mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-gamma and TNF-alpha (2.3-4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80-86% of the IFN-gamma and TNF-alpha production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-gamma production, showed a small but significant increase in TNF-alpha production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-gamma and TNF-alpha. Thus, the capacity for increased production of cytokines IFN-gamma and TNF-alpha in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual.

Enhanced interferon-gamma by CD8+ CD28- lymphocytes from HIV+ patients.

Flow cytometric analysis of T cells from HIV+ and normal individuals activated for 15 hr showed that the percentage of cells producing interferon-gamma (INFgamma) was enhanced approximately threefold (39 compared to 14%) in the HIV+ CD8+ population. Activation modes, other than anti-CD3 with PMA, were ineffective, and in no case did the percentage of HIV+ CD4+ T cells show increased INFgamma production over controls. Enhanced INFgamma production was not induced by either anti-CD3 or PMA alone, or anti-CD3 or ConA with anti-CD28, or enhanced by N-acetylcysteine. In contrast to INFgamma production, the percentage of CD4+ T cells producing interleukin-2 (Il-2) greatly exceeded that of the CD8+ T cells. The results from flow cytometry analyses of HIV+ CD8+ T cells was supported by quantitative analysis of INFgamma mRNA (by PCR) and INFgamma secretion by ELISA. These methods showed a sixfold and three- to fivefold increase, respectively, on a per cell basis. As HIV infection progresses, as shown by loss of CD4+ T cells, the proportion of CD8+ CD28- T cells increases, and it is this T cell subset that is responsible for 80% or more of the enhanced INFgamma production. The enhanced INFgamma in HIV+ patients derives from two factors: the increase in CD8+ CD28- cells to 70% and the percentage producing INFgamma (60%, compared to 21% for CD8+ CD28+ cells). Our findings of a substantial increase in INFgamma production in HIV infection arising from the increased number of CD8+ CD28- T cells are compatible with clinical studies which show elevated INFgamma in HIV+ serum and INFgamma producing CD8+ T cells dominating HIV+ lymph nodes. We also found a significantly decreased proliferative response of the HIV+-derived CD8+ T cell fraction with coactivator anti-CD-28, in contrast to PMA (with anti-CD3), which is probably a reflection of the diminished population of CD8+ CD28+ T cells in HIV+ donors compared to normal donors (30.7 compared to 67.9%).

The suppression of T cell function and NF(kappa)B expression by serine protease inhibitors is blocked by N-acetylcysteine.

Direct evidence that N-acetylcysteine (NAC) enhances the immune response of peripheral blood T cells at the level of NF(kappa)B is presented. In addition, NAC blocks the suppression of T cell mitogenesis and cytokine production by protease inhibitors such as N-tosylphenylalanine chloromethyl ketone (TPCK). The proliferative responses of purified CD4+ or CD8+ T cells are suppressed more strongly by TPCK when anti-CD28 rather than the phorbol ester PMA is used as the mitogenic coactivator. Cytokine (IL-2, IL-6, INF-gamma) production is inhibited 95-100% by concentrations of TPCK that totally suppress the mitogenesis of CD4+ or CD8+ cells. Using electrophoretic mobility shift assays, we find that TPCK virtually abolishes (to less than 1%) the levels of NF(kappa)B (but not Oct-1) found in nuclear and whole cell extracts of activated T cells. Strikingly, the immunosuppressive effects of TPCK are blocked when T cells are pretreated for 15 min with 5 mM NAC. NAC not only blocks the effect of TPCK but enhances mitogenesis and cytokine production (>2.5-fold in some cases) upon activation of unsuppressed T cells. Our data support the notion that NF(kappa)B and I(kappa)B proteases play obligate roles in T cell activation and mitogenesis, roles that are enhanced significantly by NAC.

Anti-CD2-induced tyrosine phosphorylation of T cell polypeptides is independent of the PMA-induced modification of p56lck.

The molecular basis for T cell activation involves the phosphorylation of of polypeptides at both serines and tyrosines. We find that with human peripheral T cells the serine phosphorylation of p56lck is independent of the more rapid tyrosine phosphorylation of other polypeptides via stimulation of the CD2 receptor with anti-CD2 (anti-T11(2) and anti-T11(3) mAb's). Triton X-100 soluble polypeptides were analyzed by Western blotting with the subsequent immunodetection by anti-phosphotyrosine or anti-lck antibodies. While polypeptides from resting T cells showed very low levels of endogenous tyrosine phosphorylation, incubation with anti-CD2 for periods as short as 30 sec resulted in the tyrosine phosphorylation of a 75-kDa polypeptide (p75). Polypeptide bands were also observed at 27 and 54 kDa, but these were artifacts from the reaction of anti-CD2 with the horse anti-mouse secondary antibody used in our detection system. Preincubation of T cells with phenylarsine oxide amplified the anti-CD2-induced tyrosine phosphorylation of the p75 and revealed additional phosphotyrosine polypeptides of 120, 100, and 33 kDa. The mitogenic combination of phorbol 12-myristate 13-acetate (PMA) with anti-CD2 changed neither the intensity nor the pattern of the tyrosine phosphorylation observed with anti-CD2 alone. The tyrosine phosphorylation of the p75 was not induced by concanavalin A (Con A) or PMA. While PMA alone failed to stimulate tyrosine phosphorylation above resting levels, PMA induced the nearly complete conversion of p56lck into p60lck (the lck-shift) at 30 min; no lck-shift was observed at 30 and 90 sec. Neither anti-CD2 nor Con A induced the lck-shift. Whereas PMA with either anti-CD2 or Con A was required for mitogenesis, only anti-CD2 led to tyrosine phosphorylation, and only PMA induced the lck-shift.

Two-dimensional analysis of autophosphorylated chick brain synaptic subfractions.

Chick brain synaptosomes were autophosphorylated upon incubation with [32PO4] and glucose, exposed to depolarizing ionic conditions, subsequently lysed, and respective synaptic subfractions were isolated. Analysis of these subfractions by two-dimensional electrophoresis revealed complex and unique phosphopeptide patterns. The phosphorylation levels of several soluble and membrane polypeptides appeared to change upon exposure to the depolarizing conditions.

An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization.

An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.

Autophosphorylation of chick brain synaptic polypeptides. Phosphorylation of proteins during incubation of intact synaptosomes with 32PO4.

Chick brain synaptosomes incorporated phosphate into proteins when incubated in physiological buffer containing energy sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that three synaptosomal polypeptides were significantly phosphorylated after 15 sec incubation while at least fifteen polypeptides were active kinase substrates after 15 min incubation. Labeled synaptosomes were hypotonically lysed and separated by centrifugation into soluble, membrane, and mitochondrial fractions. Every fraction exhibited significant phosphate incorporation. Electrophoresis revealed that each fraction had several unique phosphorylated polypeptides and a distinctive phosphorylation pattern. The same polypeptides appear to be labeled whether MgATP was added to synaptic plasma membranes or synaptic plasma membranes were isolated after synaptosomal autophosphorylation.

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.