Usha Ponnappan

Department of Geriatrics, Medicine, Microbiology and Immunology, University of Arkansas for Medical Sciences, and GRECC, John L. McClellan Memorial VA hospital, VA Medical Research, GC143, 151/LR, 4300 West 7^th street, Little Rock, Arkansas 72205

Received 1/15/97 Accepted 1/29/98

5.0. NF KAPPA B AND IMMUNE SENESCENCE

Several studies have demonstrated transcription factor NF kappa B to be central in T cell activation through the regulation of IL-2 and IL-2R-alpha genes (161-163,170) both of which significantly decline with advancing age. Given its important role, any alteration in the induction and regulation of NF kappa B is likely to affect T cell function. Results from our laboratory demonstrate that NF kappa B induction is significantly decreased in activated T cells from the elderly regardless of the activator or signaling pathway used, indicating this to be an age-related phenomenon rather than an activation dependent event (76). Consistent with these results, Whisler et al. (78), have also shown NF kappa B to be decreased in T cells from three out of five elderly donors in response to PMA and ionomycin, anti-CD3, either alone or in combination with PMA or PHA. This is however, in contrast to those reported by Albright et al. (171) which demonstrate no age-related decrease in the induction of NF kappa B in T cells from aged mice following activation with anti-CD3. The decrease in nuclear induction of NF kappa B observed during aging is consistent with the observation of lowered IL-2R-alpha detected following activation in T cells from the elderly (20,24,61). As NF kappa B is a critical regulator of IL-2R-alpha expression, a decrease in the induction of NF kappa B is likely to negatively impact on IL-2R-alpha expression following activation.

Studies on other transcription factors provide evidence that physiological aging and in vitro cellular aging does in fact down regulate some transcription factors. Senescent fibroblasts, which have been studied as a cellular model of aging, exhibit decreases in the levels of transcription factors such as AP-1 and E2F, but not NF kappa B (79,172,173). A defect in the induction of heat shock transcription factor (HSF) was reported following heat shock treatment of hepatocytes from aging rats and this was postulated to contribute to the age-related decrease in the expression of heat shock protein 70 (HSP70) (174,175). In addition, studies on other transcription factors in immune cells have demonstrated decreased NFAT in activated T cells from both aging rats and humans (176,78) and the induction of AP-1 has been demonstrated to be defective following activation of T lymphocytes from both mice and humans (77,171). Thus, dysregulation of transcription factors appears to accompany aging in several cell types and may contribute to the age-related changes in cellular function.

Aging in mice and humans has been shown to be accompanied by a shift in the ratio of memory (CD45RO⁺) T cells to naive (CD45RA⁺) T cells, and this shift has often been postulated to underlie age-related immune hyporesponsiveness. Experiments designed to test the inducibility of NF kappa B in memory and naive T cell subsets, produced results which demonstrated that regardless of donor age, memory T cells were not altered in their responsiveness to TNF when compared to similarly treated naive T cells and that an underlying defect in the induction of NF kappa B existed in both memory and naive T cells from elderly donors (177). This suggests that the observed age-related decrease in the levels of NF kappa B cannot be attributed solely to the skewing of the T cell population towards the memory phenotype in the elderly. It was previously believed that activation induced conversion of T cells from the CD45RA⁺ to the CD45RO⁺ phenotype was unidirectional and irreversible thus defining CD45RA⁺ as naive and CD45RO⁺ as memory T cells. Recent studies, however, have brought into question the validity of using CD45RO and CD45RA as markers to define naive and memory T cells. Using a polyclonal human T cell line, Rothstein et al. (55) showed that although initially down regulated after stimulation, CD45RA was reexpressed when cells returned to a resting state. Bell and Sparshott (56) transferred CD45R⁺ (naive) or R-(memory) allotype marked T cells into athymic nude rats and demonstrated that both subsets could generate cells of the opposite phenotype suggesting that the CD45 markers can interconvert. It has therefore been suggested that the expression of CD45RO or CD45RA may actually reflect the activation state of the cell or may confer functionally distinct properties on T cells rather than being indicative of antigenic exposure (127). The conflict surrounding the identification of memory and naive T cells makes it difficult to assess their role in aging. Until another stable surface marker is identified for the isolation of naive and memory subsets, in humans, we may have to contend with identifying them as CD45RO⁺ and CD45RA⁺ subsets. Different activators utilizing different signaling pathways resulted in lowered NF kappa B induction in the elderly which suggested a defect in a common mediator and/or event regulating the induction of NF kappa B. Experiments examining the constitutive levels of NF kappa B present in the cytoplasm of resting T cells from young and elderly donors demonstrated that differential levels of precursor NF kappa B available for induction could not account for the lowered amount of NF kappa B induced following T cell activation (76), similar to the results reported by Richardson et al. (175), who demonstrated that alterations in the constitutive levels of HSF was not responsible for age-related decline in the induction of HSF. Although mediating different functions, both HSF and NF kappa B require post translational events for their activation. This suggests that aging may affect post translational processes required for activation of transcription factors rather than affecting constitutive levels.

Failure to detect any accumulation of NF kappa B binding activity in cytosols of activated T cells from the elderly suggested that modifications of NF kappa B necessary for nuclear translocation or processes involved in the nuclear translocation of NF kappa B per se remained intact with advancing age. Employing antibody supershift assays we demonstrated that p50 and p65 subunits were the primary components of the inducible NF kappa B heterodimer in both anti-CD3 and TNF-alpha activated T cells from both young and elderly donors (76). This result is consistent with several studies that identify p50 and p65 as being the primary inducible active NF kappa B dimer involved in the rapid and early activation response not only in T cells but also in many other cell types (86-89, 115). Although Pimentel-Muinos et al. have shown c-rel to be a component of induced NF kappa B in human T cells (115), c-Rel was not detected in our system. Pimental-Muinos et al. demonstrated c-rel to be a component of long term (7-40 hours) activated NF kappa B rather than being a part of the rapid and transiently induced NF kappa B response. In our studies, T cells were treated with activators for short periods of time (less than 4 hours) measuring the early and rapid phase of NF kappa B induction, rather than the sustained long term phase. From the studies of Pimental-Muinos et al it can be predicted that c-rel may be induced at later time points.

From our observation of lowered activation-induced degradation of I kappa B-alpha it became evident that defects in I kappa B-alpha degradation were central to the decreased induction of NF kappa B, and thus immune decline observed in the elderly (194). This result is consistent with reports of lowered I kappa B-alpha degradation in situations that induce lowered levels of NF-kappa B such as those observed following treatment with the anti inflammatory agents, salicylate or tepoxalin (178-181).

Further dissection of the underlying mechanisms for this lowered degradation of I kappa B-alpha led us to demonstrate a significant decline in proteasomal activity, specifically the chymotryptic activity (194). Alterations in proteasomal degradation have been demonstrated in other cell systems during aging (182,183). Ubiquitinated proteins, which are degraded by the proteasome, accumulate in the brains of aging mice and in neural tissues of patients with age-associated neurodegenerative diseases (183). As chymotrypsin-like activity is the most sensitive proteolytic activity in the proteasome, it is not surprising that it is this activity that appears to be most affected with advancing age (184). Although, alteration in proteasomal activity may not be an universal occurrence, this pathway may decline in certain tissues under particular conditions (reviewed in 195).

Information on the regulation of proteasomal activity in human T cells is limited, however, in other cell types and species enzymatic activity of the proteasome appears to be affected by several regulatory elements that either enhance or inhibit it. Rechsteiner et. al. (185) have demonstrated that a protein complex called the 11S regulator (REG) associates with the proteasome and can greatly enhance peptide cleavage activity. Furthermore, calcium appears to bind to the 11S regulator to inhibit its stimulatory activity. There are also several ATPases which have also been shown to associate with the proteasome and are required for proteasomal activity (153). Interferon-gamma treatment induces two subunits of the proteasome, LMP2 and LMP7, which modify the peptidase activities of the proteasome for enhanced proteolysis of peptides for antigen presentation by Class I MHC (186-188). Thus it is possible that the age-related decrease in proteasome activity may be due to alterations in any or all of these types of regulatory elements or events.