Age-related decline in the lethal hit but not the binding stage of cytotoxic T-cell activity in mice

The activity of allogeneic cytotoxic T lymphocytes (CTL) declines with aging, as measured at the population level in chromium release assays, and is associated with a decreased proliferative response to the inducing alloantigens. At the single cell level, no difference was noted between target binding cells obtained from alloantigen-stimulated spleen cells of young and old mice. However, a consistent, large, and significant difference was observed between CTL generated from the spleens of young and old mice in the percentage of bound effector cells with lysed targets, i.e., the percentage of killer cells. These results show age-related changes in the lethal hit stage and not the binding stage of CTL activity. This change could be used as a probe to identify the important mechanism(s) which mediates CTL activity.     

Non-specific immune suppression by CD8+ T cells in Brugia pahangi-infected rats

Non-specific suppression of the immune response was investigated in Brugia pahangi-infected Lewis rats. The proliferative response of peripheral blood lymphocytes or splenic non-adherent cells to mitogens was significantly reduced by B. pahangi infection. The degree of hyporesponsiveness of splenic non-adherent cells to mitogens was comparable between microfilaremic and non-microfilaremic animals. The suppressed proliferative response of splenic non-adherent cells was restored by blocking with anti-CD8 monoclonal antibody. After separation of T cells into CD4+ and CD8+ subpopulations, only CD8+ T cells from B. pahangi-infected rats suppressed the proliferative response of normal spleen cells to concanavalin A. CD8+ T cells from normal rats had no suppressive effect. On the other hand, the proliferative response of CD4+ T cells to concanavalin A was comparable between normal and infected rats. These results suggest that CD8+ T cells participate in the non-specific suppression of immune response in experimental filariasis.     

The immunological properties of the main outer membrane protein of Legionella

The immunogenic properties of Legionella outer membrane main protein (OMMP) were studied by its effect on the proliferative activity of lymphocytes in guinea pigs. Preliminary immunization with OMMP activated only the specific and nonspecific proliferation of spleen cells. After infection with Legionella, secondary immune response developed in the spleen and lungs of previously immunized animals, in contrast to intact ones, and the nonspecific proliferative activity of lymphocytes in the spleen and lungs of previously immunized animals considerably increased. These results are indicative of the fact that Legionella OMMP, similarly to other Legionella antigens and immunomodulators, may be used for the formation of protective immunity.     

Immune parameters of untrained or exercise-trained rats after exhaustive exercise

The effect of a single exhaustive swimming exercise bout on immune competence of untrained or exercise-trained female Wistar rats was compared with the competence of control sedentary rats. After the exhaustive exercise bout, the blastogenic response to concanavalin A by spleen cells of untrained rats was extensively suppressed, whereas the response of the trained rats was only marginally suppressed. The suppressed immune competence of the untrained rats after the exhaustive exercise was associated with an increase in immune-suppressive activity of splenic lymphocytes. The macrophages of the untrained rats and of the control sedentary rats were slightly immune suppressive to normal spleen cells through a prostaglandin-dependent mechanism. The addition of prostaglandin E2 (PGE2) to the blastogenesis cultures revealed that the spleen cells of untrained rats were unusually sensitive to the suppressive effects of PGE2. In contrast to the untrained rats, the marginal level of immune suppression in trained rats after the exhaustive exercise was associated with a lesser degree of lymphocyte-suppressive activity, an immune stimulatory activity by the splenic macrophages, and an insensitivity of the splenic lymphocytes to the suppressive effects of PGE2.     

The proliferative response of rat T cells to calcium ionophores increases with age

Splenocytes and T cells from both old and young rats proliferate to A23187 and ionomycin, and this response increases 3- to 10-fold in aged animals. Augmented responsiveness to ionomycin occurs in the absence of any defect in Con A-induced proliferation of T lymphocytes of aged rats and is dependent upon the addition of thiol compounds to the tissue culture medium. Augmented proliferative responses to ionomycin precede the significant but much smaller decline (30 to 40%) in Con A-induced proliferative responsiveness of splenocytes, which is evident only when rats reach 24 months of age. Heightened proliferation to calcium ionophores is not caused by a greater ability of T lymphocytes from aged rats to increase [Ca2+]i in response to ionomycin. The increased proliferative response of lymphocytes from aged rats to ionomycin occurs in the absence of detectable amounts of secreted IL 2 or IL 4. The ionophore response is a much more sensitive biomarker of age than the decline in Con A-induced proliferative responses of lymphocytes and identifies an activity of T lymphocytes that increases rather than decreases during the aging process.     

Protective immune response to experimental infection with Sarcocystis neurona in 57BL/6 mice

Immunocompetent C57BL/6 mice were infected with Sarcocystis neurona merozoites to assess the protective immune response to active infection. Using a direct agglutination test, all infected mice seroconverted to S. neurona merozoite antigens by day 14 postinfection (PI). Further, mice developed splenomegaly and bilateral symmetrical lymphadenopathy by day 14 PI, which appeared to be resolving by day 28 PI. Histologic analysis revealed a marked increase in germinal center formation in the spleen and lymph nodes by day 14 PI. Corresponding to gross and histopathological changes, the percentage of B-cells decreased significantly by day 14 PI but then increased significantly and persisted at day 28 PI in the blood, spleen, and multiple lymph nodes. There was a sharp nonspecific significant decrease in CD4 percentages by day 14 PI in the blood, spleen, and lymph nodes. Early-activation CD8 lymphocytes (CD62/CD8) were significantly down-regulated coinciding with a significant compensatory up-regulation of memory (CD44/CD8) lymphocytes in multiple organs. We propose that the protective cell-mediated immune response to S. neurona involves both CD4 and CD8 cells, with CD8 lymphocytes appearing to play a more critical role.     

Stimulation and suppression of cellular immunity in mice during liver regeneration

Augmentation of proliferative activity of spleen cells from (CBA X C57BL/6)F1 mice was demonstrated in allogeneic mixed lymphocyte reaction on day 3 after partial hepatectomy, with the decrease of proliferative response observed on day 10 after operation. The decrease was due to the activity of suppressor cells in the spleen of hybrid mice. The immune response of mice in graft-versus-host reaction as well as cytotoxicity of natural killer cells and cytotoxic T lymphocytes were also reduced on days 10-11 after partial hepatectomy.     

Metabolic and morphologic properties of single muscle fibers in the rat after spaceflight, Cosmos 1887

The adaptation of a slow (soleus, Sol) and a fast (medial gastrocnemius, MG) skeletal muscle to spaceflight was studied in five young male rats. The flight period was 12.5 days and the rats were killed approximately 48 h after returning to 1 g. Five other rats that were housed in cages similar to those used by the flight rats were maintained at 1 g for the same period of time to serve as ground-based controls. Fibers were classified as dark or light staining for myosin adenosine triphosphatase (ATPase). On the average, the fibers in the Sol of the flight rats atrophied twice as much as those in the MG. Further, the fibers located in the deep (close to the bone and having the highest percentage of light ATPase and high oxidative fibers in the muscle cross section) region of the MG atrophied more than the fibers located in the superficial (away from the bone and having the lowest percentage of light ATPase and high oxidative fibers in the muscle cross-section) region of the muscle. Based on quantitative histochemical assays of single muscle fibers, succinate dehydrogenase (SDH) activity per unit volume was unchanged in fibers of the Sol and MG. However, in the Sol, but not the MG, the total amount of SDH activity in a 10-microns-thick section of a fiber decreased significantly in response to spaceflight. Based on population distributions, it appears that the alpha-glycerophosphate dehydrogenase (GPD) activities were elevated in the dark ATPase fibers in the Sol, whereas the light fibers in the Sol and both fiber types in the MG did not appear to change. The ratio of GPD to SDH activities increased in the dark (but not light) fibers of the Sol and was unaffected in the MG. Immunohistochemical analyses indicate that approximately 40% of the fibers in the Sol of flight rats expressed a fast myosin heavy chain compared with 22% in control rats. Further, 31% of the fibers in the Sol of flight rats expressed both fast and slow myosin heavy chains compared with 8% in control rats. Immunohistochemical changes in the MG were minimal. These data suggest that the magnitude and direction of enzymatic activity and cell volume changes are dependent on the muscle, the region of the muscle, and the type of myosin expressed in the fibers. Further, the ability of fibers to maintain normal or even elevated activities per unit volume of some metabolic enzymes is remarkable considering the marked and rapid decrease in fiber volume.     

Low proliferation capacity of lymphocytes from alloxan-diabetic rats: involvement of high glucose and tyrosine phosphorylation of Shc and IRS-1

The proliferation capacity of lymphocytes obtained from mesenteric lymph nodes of control and alloxan-diabetic (40 mg/kg) rats in response to concanavalin A (ConA) and lipopolysaccharide (LPS) stimuli was examined. Proliferation response of lymphocytes from diabetic rats was significantly reduced under Con A (43%) and LPS (46%) stimulation as compared with the control group. Insulin (166 microM) promoted a marked increase of lymphocyte proliferation (7.5-fold) in the control group and this response was much lower (2.6-fold) in lymphocyte from diabetic rats. Cells were also cultured in medium containing glucose at 5, 10 or 20 mM. High glucose concentration (20 mM) caused a marked inhibition of lymphocyte proliferation reaching the values of the diabetic group. In lymphocytes from control rats, the degree of Shc tyrosine phosphorylation was gradually increased, whereas that of cells from diabetic rats was much lower in response to insulin. In lymphocytes obtained from control rats, the tyrosine phosphorylation of IRS-1 was time-dependent on insulin. In cells from diabetic rats, the basal tyrosine phosphorylation of IRS-1 was higher than that of control rats, however, there was no further phosphorylation after insulin addition. We conclude that the response of lymphocyte proliferation from diabetic rats to Con A and LPS stimuli is decreased but insulin was able to promote a significant proliferative effect on these cells. Also, high glycemia in addition to the lack of insulin participates in the reduced proliferation capacity of lymphocytes from diabetic rats.     

Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.     

Effects of a β3-adrenergic agonist on the immune response in diet-induced (cafeteria) obese animals

Molecules with affinity for beta3-adrenoceptors are not only effective anti-obesity agents in rodent models, but may play a role in the regulation of the immune response. The aim of the current investigation was to analyse the effects of trecadrine on the immune response in diet-induced (cafeteria) obese rats. Male Wistar rats were divided into 2 groups, the control group (C, n=9) was fed with the standard pelleted chow laboratory diet, while the other group was fed with a high-fat (cafeteria) diet. Cafeteria-fed rats were divided into two new subgroups (n=9 each), which received either i.p. saline (obese, O) or trecadrine (1mg/kg/day) (obese+trecadrine, O+T) daily for 5 weeks. Lymphocyte subpopulations and the proliferative response were determined by validated procedures. The administration of trecadrine was able to prevent the onset of obesity in cafeteria-fed rats. Trecadrine-treatment to obese animals appeared to improve the number of lymphocyte subpopulations (CD4+ and CD8+) as compared to those animals only receiving the high-fat diet, being the values of the trecadrine-treated animals on the high-fat diet similar to the control rats. However, the lymphoproliferative response when stimulated with several mitogens was markedly reduced by the cafeteria intake and was further decreased by the beta3-adrenergic administration. The spleen mRNA expression level of UCP2, PPARgamma and Ob-Rb were not affected by the trecadrine treatment. Summing up, at the immune system level, trecadrine administration increased the proportion of CD4+ spleen lymphocytes, although it was not able to restore the lymphocyte proliferative response which was depressed.     

Energy restriction restores the impaired immune response in overweight (cafeteria) rats

Impaired immune function linked to obesity has been shown in both human and animal studies. The purpose of this work was to evaluate the effects of a 4-week energy restriction (50% of total energy intake) on immune function in previously diet-induced (cafeteria) overweight rats. Flow cytometric analysis revealed that the number of spleen T helper cells were significantly (P < 0.05) elevated in control and overweight energy-restricted rats as compared with groups fed ad libitum groups. The proliferative response of splenocytes to phytohaemaglutinin and concanavalin A from overweight rats after energy restriction was significantly (P < 0.05) higher compared to overweight nonrestricted rats. The cytotoxic activity of natural killer cells tended to be lower in overweight rats compared to controls. Finally, control rats under the dietary deprivation period presented higher levels of uncoupling protein 2 mRNA and lower levels of leptin receptor mRNA compared with the reference control group. These results suggest that energy restriction is able to restore, at least in part, the impaired immune response commonly observed in overweight animals.     

Music,immunity and cancer

The effects of music on the immune system and cancer development were evaluated in rodents subjected to sound stress. Animals were exposed daily to broad band noise around midnight and/or music for 5 hours on the following morning. Thymus and spleen cellularity, peripheral T lymphocyte population, the proliferative response of spleen cells to mitogen concanavalin A and natural killer cell activity were calculated in BALB/c mice. Sprague Dawley rats were injected i.v. with Walker 256 carcinosarcoma cells; 8 days later the rats were sacrificed and the number of metastatic nodules on the surface of the lungs was calculated macroscopically. Music reduced the suppressive effects of stress on immune parameters in mice and decreased the enhancing effects of stress on the development of lung metastases provoked by carcinosarcoma cells. Music enhanced the immune parameters and the anti-tumor response in unstressed rodents. Our data at present demonstrates that music can effectively reverse adverse effects of stress on the number and capacities of lymphocytes that are required for an optimal immunological response against cancer in rodents.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

The effect of exogenous whole-body hyperthermia on humoral immunity]

We investigated the proliferative responses of spleen cells (SC) to polyclonal mitogens lipopolysaccharide (LPS) and pokeweed mitogen (PWM), immune responses to sheep red cells (SRC) in mice undergoing hyperthermia. There were increased proliferative responses of lymphocytes to PWM if we used mice having rectal temperature 42 degrees C. Thermal shock in mice was accompanied by suppression of immune response. If we used mice suffering from hyperthermia (43-44 degrees C) for 20 minutes; there were decreased proliferative responses of lymphocytes to PWM or LPS for 10-30 days. We observed low immune response to sheep red cells in mice for 5-20 days. The changes of immune response were not revealed on the 40th day after induction of hyperthermia in mice.     

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.     

Effect of repeated stress and administration of phenobarbital on the lymphoid tissue and on protein metabolism in the lymphocytes of infant and adult rats

Further study of the response to chronic stress stimulation in the early postnatal phase showed that the i.p. injection of physiological saline (stress stimulation) induced lymphopenia, a 50% decrease in the incorporation of 3H-leucine into isolated lymphocytes and a decrease in the weight of the thymus in 7-day-old male rats. No such changes were observed in adult animals. If repeated doses of phenobarbital were administered to stressed young rats, however, lymphopenia did not occur and the rate of the incorporation of 3H-leucine into isolated lymphocytes was not different from the control value; the protein content of the lymphocytes was significantly raised, however. In adult animals, phenobarbital increased the rate of incorporation of 3H-leucine into the lymphocytes. The repeated administration of phenobarbital reduced the weight of the thymus in both young and adult animals, but a decrease in spleen weight was recorded only in the young animals. A single i.p. injection of ACTH or dexamethasone caused lymphopenia and slowed down the incorporation of 3H-leucine into the lymphocytes of both young and adult animals. The results show that the striking decrease observed in the rate of the liver metabolism of corticosterone in suckling young rats not injured by repeated stress stimulation is accompanied by significant changes in the lymphoid tissue.     

Specific unresponsiveness to skin allografts in anti-lymphocyte serum-treated, marrow-injected mice: participation of donor marrow-derived suppressor T cells

Sequential changes of cell-mediated immune reactivities were examined in anti-lymphocyte serum-(ALS) treated, C3H/He (C3H; H-2k) bone marrow-injected (C57BL/6 X A)F1 (B6AF1; H-2b/k.d) mice bearing enhanced C3H skin grafts. Spleen cells of these mice exhibited marked suppression of the proliferative response to phytohemagglutinin and concanavalin A. When the spleen cells were assayed for the direct lymphocyte-mediated cytotoxicity against H-2k targets, their lytic activity remained low until the time of graft rejection, in contrast to the increasingly high cytotoxic activity exhibited by spleen cells of control B6AF1 mice given only ALS and C3H skin grafts. When spleen cells of marrow-injected B6AF1 mice were cultured with mitomycin-C treated C3H spleen cells, the proliferative response was significantly suppressed the throughout the course, despite the early appearance of high "secondary-type" cytotoxic activity. Co-culture experiments demonstrated the presence of C3H antigen-specific suppressor cells in the ALS-treated, marrow-injected mice bearing intact allografts. Treatment of spleen cells with anti-H-2, anti-Thy 1 and anti-I-J sera and C revealed that the suppressor cells present late in the marrow-injected mice were T cells of donor C3H bone marrow cell origin.     

Dietary folate improves age-related decreases in lymphocyte function

Although low folate status is thought to be fairly common in the older population, its implication on immunity has not been adequately investigated. Using 11-month-old and 23-month-old male rats (Fisher 344), the present study was undertaken to examine the modifying effects of feeding a control diet (NIH-07) supplemented with folate (35.7 mg/kg) for 3 weeks on the immune cells of spleen and mesenteric lymph node (MLN) origin. The serum concentrations of folate along with vitamin B(12) were elevated in response to the folate supplementation (P<.05). These results were accompanied by an improved proliferative response (stimulation index) to mitogens in both the spleen and MLNs (P<.05). The proportion of T cells in the MLNs, but not in the spleen, was significantly increased in rats fed a diet supplemented with folate. In the spleen, the folate-supplemented diet prevented the age-associated decrease (P<.05) in the production of interferon (IFN)alpha by unstimulated cells and the decrease in T-helper (Th)1/Th2-type response after stimulation with phorbol myristate acetate and ionomycin. In the MLNs, on the other hand, the folate-supplemented diet failed to influence any age-related increase in interleukin (IL)-2, tumor necrosis factor alpha and IFNgamma following stimulation but did result in a significantly increased production of IL-4 (P<.05). Overall, this study provides data suggesting that aging is associated with changes in the proportion of T cells, the ability of immune cells to proliferate and the production of cytokines after stimulation. Supplementing a folate-sufficient diet with additional folate improves proliferative response to mitogens, the distribution of T cells in the MLNs and the age-related changes in cytokine production in the spleen. These results suggest that the dietary folate requirement may be higher in the older population than in the younger population to support immune functions.     

Assessment of the immune responsiveness of mice exposed to a 1.5-Tesla stationary magnetic field

Humoral and cell-mediated immune responses were assayed following a 6-day exposure of LAF1/J mice to a 1.50 Tesla (1 T = 10(4) Gauss) stationary magnetic field. In tests of the immune response to sheep erythrocytes, the number of Jerne plaques formed by spleen lymphocytes and the level of serum IgM were not significantly different for the exposed mice in comparison with control animals. Tests for mitogen-induced lymphocyte proliferation also demonstrated no significant differences in the response of spleen lymphocytes from exposed and control groups of mice.