Margaret L. Heidrick

Effect of dietary 2-mercaptoethanol on the life span, immune system, tumor incidence and lipid peroxidation damage in spleen lymphocytes of aging BC3F1 mice

The age-related decline in immune function, which is thought to be responsible for the increased incidence with age of certain diseases, including cancer, has been attributed primarily to a loss of T-lymphocyte function. As free radical reactions may contribute to cellular deterioration and loss of cell function with age, we investigated the effect of adding an immunopotentiating antioxidant, 2-mercaptoethanol (2-ME), to the diet of BC3F1 mice in a longitudinal study. For the study, young mice were divided into two groups, one of which received the 2-ME-supplemented diet. Approximately every 3 months for 2.5 years, mice from each group were sacrificed and the spleen lymphocytes assessed for immune function (proliferative response to concanavalin A, phytohemagglutinin, and lipopolysaccharide and the humoral response to sheep red blood cells). The accumulation of fluorescent products indicative of free radical damage was measured in the spleen lymphocytes and the cytochrome P-450 content and activity assessed in the liver. The effect of the 2-ME-supplemented diet on the mean and maximum life span and tumor incidence was also determined. The results showed that the animals fed the 2-ME diet had an increased mean and maximum life span and a postponed onset and decreased incidence of tumors. In general the T-cell-dependent immune responses were higher in the 2-ME-fed mice compared to the controls when the animals were young. No difference was observed between the two groups during mid-life. The responses declined in both groups during the latter half of the life span, but the responses of the 2-ME-fed animals declined to a lesser extent. The accumulation of fluorescent products of lipid peroxidation damage was also delayed in the lymphocytes of the 2-ME-fed mice. Cytochrome P-450 content and activity in the liver was not different in the two groups. The results suggest that the antioxidant activity of 2-ME delayed the accumulation of free radical damage in spleen lymphocytes, which resulted in a delay in the decline of immune function and was associated with the decreased tumor incidence and increased life span.

Free Radical Theory of Aging: Effect of Free‐Radical‐Reaction Inhibitors on the Immune Response†

Immune responses decline with age. Endogenous free radical reactions, implicated in aging, may contribute to this decline. To evaluate this possibility, either α‐tocopherol acetate (vitamin E) or a quinoline derivative (Santoquin), both free‐radical‐reaction inhibitors, were added to the diet of C3HeB/FeJ female mice from age 6 weeks through 88 weeks at a level of 0.25 percent by weight of the diet. Both these antioxidants enhanced immune responses up to age 88 weeks, and probably also for the remainder of the lifespan; the experiment was stopped at age 88 weeks, as all the mice had been used for assay. Vitamin E had a greater effect than Santoquin on humoral responses, whereas cell‐mediated responses were enhanced more by Santoquin. Prompted by the foregoing study, levamisole or one of a number of different free radical inhibitors was added to the diet of groups of young mice for one month, after which the humoral response of spleen cells were determined. The five free radical inhibitors found to be as effective as levamisole were: 2‐mercaptoethanol, butylated hydroxytoluene, 2‐mercaptoethylamine, Santoquin, and sodium hypophosphite. These studies suggest, but do not prove, that some endogenous free radical reactions contribute to the decline of the immune response with age. This decline can be ameliorated by adding inhibitors of free radical reactions to the diet.

Restoration of impaired immune functions in aging animals, IV. Action of 2-mercaptoethanol in enhancing age-reduced immune responsiveness

The enhancing effect of 2-mercaptoethanol (2-ME) on the immune responses of young and old unseparated spleen cells and purified populations of T-cells, B-cells and macrophages was studied. While 2-ME enhances both the normal responses of young cells and the age-reduced responses of old cells, the sulfhydryl compound has a relatively greater enhancing effect on old spleen cells in the humoral response to sheep red blood cells and the blastogenic response to concanavalin A (Con A). The greater enhancing effect in the humoral response appears to be due to 2-ME overcoming a suppressive effect of old T-cells which develops with increasing age. The greater enhancing effect in Con A stimulation appears to be an effect on the Con A responsive sub-population of T-cells and not on the synergistic effect of the presence of B-cells.

Susceptibility to lipid peroxidation and accumulation of fluorescent products with age if greater in T-cells than B-cells

The age-related loss of immune function, which is primarily due to loss of T-lymphocyte function, is also associated with accumulation in spleen lymphocytes of autofluorescent products indicative of peroxidation damage. In this study, we examined T-cell membranes for age-related changes which could be related to lipid peroxidation. Using fluorescence spectroscopy of CHCl3:CH3OH membrane extracts, we observed that old T-cells have a 2-fold greater accumulation of fluorescent products than old B-cells and that young T-cells, when exposed to free radicals in an in vitro system, accumulate significantly more fluorescent products over time than young B-cells. We used fluorescence polarization to show that young T-cell membranes are more fluid than young B-cell membranes. However, T-cell membrane fluidity decreases with age, whereas B-cell membrane fluidity does not change; in old mice, T-cell membranes are significantly less fluid than old B-cell membranes. Using two-dimensional electrophoresis, we showed that membrane extracts of old T-cells contain many more proteins than extracts of young T-cells. Our results indicate that age-related changes occur in T-cell membranes which could be due to their increased susceptibility to lipid peroxidation and these changes may contribute to the age-related decline in immune function.

Metabolism of 3′,5′-cyclic AMP by Strains L cells

Abstract 1. The metabolism of [8− 14 C]3′,5′-cyclic AMP by Strain L cells was studied. The intracellular and extracellular labeled metabolites were separated and identified by ion-exchange column and paper chromatography and quantitated by liquid scintilation counting. 2. 2. The principal metabolites were: intracellular: AMP, ADP, and ATP; extracellular: inosine. 3. 3. Cyclic nucleotide phosphodiesterase was present in the Strain L cells but not in the extracellular medium. 4. 4. The results suggest the inhibition of cell growth by 3′,5′-cyclic AMP is due to the intact cyclic nucleotide since no significant amounts of toxic metabolites were found.

Immunomodulatory action of levamisole--I. Structural analysis and immunomodulating activity of levamisole degradation products.

In our laboratory we observed that solutions of levamisole (LMS) stored at 4 degrees C consistently enhanced the lymphocyte proliferation response to concanavalin A (Con A) more than freshly prepared solutions did. To determine if the increased immunopotentiation observed with the stored solutions of LMS was due to products formed from LMS, we assessed the stability of LMS when stored at 4 or 37 degrees C at pH 6, 7, 7.5 and 8. Analysis of the various solutions by high pressure liquid chromatography demonstrated that LMS decomposes during storage in neutral and alkaline conditions to form three products. The formation of the products was accelerated by increasing the temperature from 4 to 37 degrees C. The three degradation products were purified by preparative high pressure liquid chromatography and their structures determined by mass spectrometry, infrared spectrometry and homo- and heteronuclear two dimensional nuclear magnetic resonance spectroscopy. The degradation products, denoted as No. 1, No. 2 and No. 3, based on their high pressure liquid chromatography retention times, were identified as: No. 1, 3-(2-mercaptoethyl)-5-phenylimidazolidine-2-one; No. 2, 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole and No. 3, bis [3-(2-oxo-5-phenylimidazolidin-1-yl) ethyl] disulfide. Product 2 significantly enhanced murine lymphocyte proliferation responses to concanavalin A (Con A) at concentrations between 0.5 and 10.0 micrograms/ml (whereas the optimum concentration of LMS is 10-100 fold higher (50-100 micrograms/ml)). Products 1, 2 and 3 significantly inhibited the lymphocyte proliferative response at concentrations greater than 2.2, 10.0 and 10.0 micrograms/ml, respectively. These studies indicate that under relatively mild conditions, including physiological conditions, LMS may decompose to products which inhibit or enhance lymphocyte responses to Con A.

Adenosine deaminase and purine nucleoside phosphorylase activity in spleen cells of aged mice

The activities of two enzymes of purine metabolism, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), were determined in spleen lymphocytes from mice of various ages. We found that in the older animals, which have depressed responses to concanavalin A and phytohemagglutinin, there is a decrease in the activity of PNP but normal activity of ADA. The decline of PNP activity was seen at 7.5 months of age and appears to be concurrent with a decline in T-cell function. These results suggest that a decrease in PNP activity may be a contributing factor in the immunodeficient state of the aged.

Immunomodulatory action of levamisole - II. Enhancement of concanavalin a response by levamisole is associated with an oxidation degradation product of levamisole formed during lymphocyte culture

Previously we determined that levamisole (LMS), when stored for a period of time, breaks down to three degradation products at neutral and alkaline pH. At low concentrations (10(-6) M), Product 1 inhibits the lymphocyte response to concanavalin A (Con A). Product 2 enhances the response and Product 3 has no effect. At higher concentrations (10(-5) M) all three products inhibit the response. To determine if these products are formed in culture media under culture conditions (e.g. in RPMI-1640 bicarbonate buffered medium, 37 degrees C, pH 7.0-7.5, during a 72 h culture period), we added freshly prepared LMS solutions to culture media with and without lymphocytes present and maintained the pH at 7.0, 7.25 or 7.5 by varying the amount of CO2 present. Periodically over a 72 h period, aliquots of the media were removed and analyzed for the presence of LMS and the three degradation products. Within 4 h, two of the degradation product began to form in culture media with or without lymphocytes present. Product No. 1, 3-(2-mercaptoethyl)-5-phenylimidazolidine-2-one or dl-2-oxy-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI), which inhibits the lymphocyte response to concanavalin A (Con A) at concentrations above 0.4 micrograms/ml, was formed at pH 7.0, 7.25 and 7.5, but the compound did not reach inhibitory concentrations in the lymphocyte cultures during the 72 h culture period. Product No. 2, 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole, which enhances the Con A response between concentrations of 0.5 and 10 micrograms/ml, was detected at concentrations between 2.5 and 3.5 micrograms/ml at pH 7.25 and 7.5. Product 2 was not detected in cultures at pH 7.0 and subsequently when we cultured lymphocytes with freshly prepared LMS and maintained the pH at 7.0, no significant enhancement of the Con A response was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibody and Tumor Promotion

Summary Following treatment of Strain A mice with either croton oil or TPA, autoantibodies were detected by passive hemagglutination in the sera of the mice. Autoantibody to skin was induced in Strain A mice by the repeated injection of syngeneic skin and liver. Higher levels of antibody were achieved by injection of xenogeneic skin. Mice initiated with DMBA were injected with syngeneic liver and skin. The injected mice developed significantly higher levels of tumors than did noninjected controls. Xenogeneic skin injections produced high levels of autoantibody but did not promote tumor development. The results suggest that autoantibody may play a role in the promotion of tumors.

Influence of fibroblastic collagen and mucopolysaccharides on HeLa cell colonial morphology.

Abstract Human fibroblasts in vitro (a) synthesize collagen and acid mucopolysaccharides and (b) when grown together with HeLa cells, induce a change in the colonial morphology of the latter. This study demonstrates that the induction of colonial changes by the fibroblasts is not necessarily causally related to the production of collagen and acid mucopolysaccharides.