Melatonin: immune modulation of spleen cells in young, middle-aged and senescent mice.

We examined the effects of melatonin on the immune response in aged mice in an in vitro model system. Spleen cells were removed from 2 month old, 11 month old, and 19 month old C57/BL6 male mice and incubated with melatonin concentrations ranging from 50 to 5000 ng melatonin/ml. All age groups demonstrated increased ability to proliferation in response to mitogenic stimulation when melatonin and mitogen were added at the same time, and we demonstrated that 11 month old and 19 month old mice exhibited a greater increase in proliferation response than the young 2 month old mice. We further examined whether this increased responsiveness was due to increased levels of IL-2 and found that while there was a significant age-dependent difference in the levels of IL-2 in the different age groups, melatonin had no effect on IL-2 levels in any age groups despite the fact that proliferation increased in all groups. The underlying mechanism of melatonin action is being investigated further.

Aged lymphocyte proliferation following incorporation and retention of dietary omega-3 fatty acids.

T cell activation involves events at the plasma membrane; therefore, molecules such as long chain omega-3 fatty acids that alter the structure of the plasma membrane may affect the activation of aged T cells. In this project we investigated whether the incorporation of omega-3 fatty acids (from fish oil), in the presence of vitamin E, improves age-diminished T cell proliferation. Young and old mice were fed diets rich in either fish (menhaden) oil or saturated fat for various lengths of time. Splenocytes were harvested from these mice and stimulated in culture with either mitogen or the antigen keyhole limpet hemocyanin (for a secondary response); proliferation was estimated by [3H]thymidine incorporation. We found no discernible effect of dietary omega-3 fatty acids (with vitamin E supplementation) on lymphocyte proliferation stimulated by the mitogens concanavalin A or phytohemagglutinin. We did, however, find that the saturated fat diet and the menhaden oil diet in young mice lowered protein kinase C activities in the particulate fractions of spleen cells when compared to chow-fed mice. Middle-aged and old mice were less affected by the experimental diets than young mice, but they demonstrated decreased protein kinase C activity as well. These alterations did not affect the ability of splenocytes to respond to mitogenic stimulation. Fatty acid analysis revealed that lymphocytes from mice fed saturated fat for 8.5 months retained significant amounts of the omega-3 fatty acid docosahexaenoic acid, despite the lack of dietary omega-3 fatty acids. However, when aged (but not young) lymphocytes were clonally expanded by antigen in vivo in the presence of dietary omega-3 fatty acids, they produced a greater secondary proliferative response than old lymphocytes expanded during a saturated fat diet. Although our results suggest that omega-3 fatty acids may enhance aged lymphocyte proliferation, the tenacious retention of these fatty acids makes comparison with omega-3-depleted lymphocytes difficult.

Serine/threonine protein kinases and calcium-dependent protease in senescent IMR-90 fibroblasts.

Three enzymes relevant to signal transduction were compared in replicating, quiescent and senescent human diploid fibroblasts (HDF). These were Ca(2+)-dependent thiol protease (calpain), cAMP-dependent protein kinase (Pk-A), and calcium/phospholipid-dependent protein kinase C (Pk-C). The amounts of these enzymes in quiescent HDF were slightly greater or the same as in replicating HDF. In contrast, senescent HDF exhibited higher Pk-C, Pk-A and proteolytic activities than did either replicating or quiescent cells. While the elevated protein kinase activities could be accounted for by the larger size of senescent cells relative to younger cells, the increased calpain activity exceeded this size differential. Immunoblotting studies with antisera to both Pk-C and calpain demonstrated increased enzyme concentrations in parallel with the increased activities. Photolabeling cell extracts with an analog of cAMP, 8-N3-[32P]cAMP, provides an estimate of Pk-A concentration. By this criterion, senescent HDF have more Pk-A molecules than do young cells that are either replicating or quiescent. Only the type I isozyme of Pk-A (Pk-A-I) was observed in any of these cells. Photolabeling with 8-N3-[32P]cAMP demonstrated more degradative fragments of the Pk-A regulatory subunit (RI) in senescent cells also. This is a logical consequence of the increased calpain activity in senescent cells, since RI is a substrate for calpain. These results imply that senescent cells do not fail to enter S phase owing to inadequate concentrations of Pk-A or Pk-C. Rather, the increased quantities of these enzymes in senescent cells may reflect aberrations elsewhere along signal transduction pathways that coordinate cell size with cell proliferation.

Pregnancy-associated, lymphocyte-derived suppressor factor inhibits protein kinase C activity.

For successful allogenic pregnancy to occur, suppression of maternal defense responses toward the fetus are vital. Suppressor factors elaborated by decidual cells or immune cells may facilitate this suppression. In order for appropriate cellular responses to occur an intact signal transduction/second messenger system must be present. The calcium/phospholipid-dependent protein kinase, Pk-C, plays an important role in regulating immune responses, and may also be important in regulating uterine cell responses and implantation events. Pk-C activation is necessary for IL-2 synthesis and IL-2 receptor synthesis through activation of the proto-oncogenes c-jun and c-fos. These proto-oncogene gene products combine to form the heterodimer AP-1 which then activates IL-2 gene transcription for both peptide and receptor. If Pk-C activity becomes abrogated then appropriate cell responsiveness is diminished. We have shown that Pk-C activity is decreased in the particulate fraction of 4-7 day pregnant spleen, thymus and draining lymph node (DLN) cells. Spleen cells did not exhibit any change in cytosolic Pk-C activity, the thymus was found to have a decrease in both cytosol and particulate fractions, and the DLN cells exhibited a translocation effect whereby particulate Pk-C decreased and cytosolic Pk-C activity increased. Supernatant from 3-day cultures of DLN cells from pregnant animals was shown to inhibit proliferation of spleen cells. In addition, the supernatant was able to directly lower Pk-C activity. We hypothesize that DLN cells secrete a factor(s) that is able to suppress immune response through abrogation of Pk-C activity, thereby decreasing AP-1 formation resulting in decreased IL-2 synthesis and IL-2 receptor synthesis.

Evidence for a role of tert-butyl hydroxylation in the induction of pneumotoxicity in mice by butylated hydroxytoluene.

Previous studies have shown that BHT must be biotransformed, probably to a quinone methide, in order to cause pneumotoxicity in mice. When BHT is incubated with mouse hepatic or pulmonary microsomes, a major metabolite that is formed is the tert-butyl-hydroxylated derivative of BHT (BHT-BuOH). Herein we show that BHT-BuOH has a fourfold greater potency than BHT in increasing the lung wt/body wt ratio, decreases lung cytosolic Ca2+-dependent protease activity at 1/10 the dose required for BHT to do this, and causes pulmonary histopathology at 1/20 the dose of BHT. Lung damage occurs earlier and is repaired faster at lower concentrations of BHT-BuOH than of BHT, but the nature of the damage (type I cell death) and regenerative response (type II cell hyperplasia and differentiation) is apparently identical. Neither BHT-BuOH nor BHT cause damage to liver, kidney, or heart as assessed by light microscopy, so they are both specific pulmonary toxicants. We postulate that BHT-BuOH formation is an essential step in the conversion of BHT to the ultimate pneumotoxin, which might be the corresponding quinone methide.

Age-dependent changes in murine protein kinase and protease enzymes.

Hormone responsiveness is mediated by signal transduction mechanisms involving second messengers, such as cAMP and Ca2+, which regulate reversible changes in the phosphorylation state of proteins. During senescence individuals frequently exhibit a diminished responsiveness to hormones. We examined changes in enzymes involved in protein phosphorylation reactions that might account for this decreased adaptiveness in old mice, and observed the following post-maturational changes: (1) cAMP-dependent protein kinase (Pk-A) specific activity decreased in spleen cytosol and in the particulate fractions of lung, spleen and liver of 24-month-old mice as compared to 2-month-old mice. Splenic cytosolic Pk-A activity decreased by 18 months of age, while particulate activity decreased by 6 months; (2) The amount of 8-N3-[32P]cAMP, a photoaffinity analog of cAMP, incorporated into Pk-A regulatory (R)-subunits from spleen and liver particulate fractions decreased, while photolabeling of R-subunit degradative products with this analog in heart and spleen cytosol increased. (3) Age-dependent increases in membrane-associated protease activities were found in all organs, along with a decrease in cytosolic lung calpain activity. These proteolytic changes may account for the enhanced R-subunit degradation and decreased Pk-A activities observed during senescence. (4) Age-dependent alterations in Ca2+/phospholipid-dependent protein kinase (Pk-C) are organ specific: lung, liver, brain, and heart demonstrate no change in Pk-C activity, while spleen exhibits decreased activity. We hypothesize that these age-dependent alterations in kinase and proteolytic activities may be in part responsible for changes in cellular response to hormonal stimulation, differentiation signals, and antigen responsiveness during senescence.

Changes in pulmonary calpain activity following treatment of mice with butylated hydroxytoluene.

The antioxidant, butylated hydroxytoluene (BHT), causes lung toxicity in mice followed by regenerative repair, and can also modulate the development of carcinogen-induced lung adenomas. We are investigating changes in pulmonary biochemistry following BHT treatment in order to understand the mechanisms of BHT-induced pulmonary regenerative repair. BHT administration lowered cytosolic Ca2+-activated neutral protease (calpain) activity, increased the activity of the endogenous calpain inhibitor, calpastatin, increased the extent of photoincorporation of 8-N3-[32P]cAMP into a Mr 37,000 proteolytic product derived from cAMP-dependent protein kinase regulatory (R) subunits, and increased membrane-associated protease activity. All of these changes were dependent on the BHT dosage; the altered proteolytic activities occurred at a dose lower than that which caused observable lung toxicity as assessed by the lung weight/body weight ratio. Decreased cytosolic calpain activity was detectable within 1 day after BHT administration, was lowest at 4-7 days, and had not returned to control levels by Day 21, a time when normal lung morphology had been regained. The decrease in calpain activity cannot fully be accounted for by increased calpastatin activity; upon separation of these proteins by DEAE chromatography, the amount of calpain activity from BHT-treated mice remained lower than the corresponding peak from control mice. Increased photolabeling of the Mr 37,000 protein began at 1 day and continued to increase up to 4 days after BHT. All of the cytosolic changes preceded the increased particulate proteolytic activity by 1-2 days. R-subunits which have dissociated from their catalytic subunits are more susceptible to degradation by calpain, but BHT treatment did not enhance subunit dissociation as determined by the elution profile of 8-N3-[32P]cAMP-labeled R-subunits following DEAE chromatography. A large percentage of the particulate protease activity was inhibited by calpastatin, leupeptin, and E-64, all of which are known to inhibit calpain activity; this suggested that calpain accounted for most of this activity. Changes in the activities of proteases which catalyze limited proteolysis reactions may play an important role in the repair of acute lung injury.

Macrophage activation: dissociation of cytotoxic activity from Ia-A antigen expression.

Peritoneal macrophages were obtained from DBA/2 mice that were untreated or after the injection of bacillus Calmette-Guerin (BCG), thioglycollate broth, proteose-peptone broth, or gamma-irradiated P-815 tumor cells. These macrophages were "activated" to become cytotoxic for a fibroblast cell line (L 929) by the addition of lymphokines (LKs), lipopolysaccharide (LPS), or fibroblast interferon (IFN-beta), and the expression of I region-associated antigens (Ia-Ad) on the macrophages was examined both before and after activation. Thioglycollate-elicited macrophages became Ia-A+ when activated by LKs, but they remained Ia-A- when activated by LPS or IFN-beta. Resident macrophages and proteose-peptone-elicited macrophages remained Ia-A- when activated with LKs. Macrophages from BCG-infected mice were both Ia-A+ and cytotoxic for tumor cells without further treatment. In contrast, macrophages from mice injected with gamma-irradiated P-815 mastocytoma cells were Ia-A+ but not cytotoxic, and these macrophages could not be made cytotoxic by incubation with LKs. The cultured macrophage-like cell lines P388D1 and WEHI-3 became Ia-A+ after incubation with LKs, and this treatment amplified the cytotoxicity of both cell lines. We conclude that a number of factors are important in determining whether Ia-A expression accompanies macrophage activation and that Ia-A is irrelevant as a surface marker for macrophage activation.