Age-related decline in cell-mediated immunity in the C58 leukemic mouse strain

The leukemia-prone C58 strain of mouse was examined for age-related changes in cellular immune function. Proliferative responses of lymphocytes to autologous and allogeneic stimulator cells [autologous mixed lymphocyte response (AMLR) and mixed lymphocyte response (MLR), respectively] and to mitogens were tested both prior to and around the usual age of disease onset which occurs at 7-8 months. Leukemia in these animals was defined by elevated peripheral blood and splenic white blood cell counts. The AMLR declined greater than 30% by 6-7 months of age and was virtually absent by 8 months of age even in animals that were not overtly leukemic. The MLR declined precipitously (greater than 95%) at 9 months of age. Both declines occurred at a younger age in C58 mice than in nonleukemic strains. Mixing experiments with cells from young and old animals indicate a defect in the Ly 1+23-, L3T4+ responding T cells. No evidence indicating a role for suppressor cell activity in this decline of cell-mediated immunity could be found. Deficiencies in cytokine (IL-2 and IL-1) production were not observed except in the oldest mice tested. Around the usual time of disease onset, splenic natural killer (NK) cell activity declines sharply even in nonleukemic mice. Cell-mixing experiments showed no evidence of suppressor cell activity by spleen cells from older mice, leukemic or nonleukemic, on the NK cell activity of young adult animals. Interferon alpha, beta treatment enhanced the NK activity of cells from old mice but did not restore the level of activity seen in young mice. Evidence has therefore been found for a premature decline in cellular immune function in two responses with proposed immunoregulatory roles, the AMLR and NK cell activity. It is possible that their decline could play a predisposing role in the onset of this retroviral leukemia or that these cell populations may be the target of the retrovirus.     

Hormonal Control of Messenger Ribonucleic Acid Metabolism in Barley Aleurone Layers

Ribonucleic acid containing poly(adenylic acid) [poly(A)-RNA] is present in barley aleurone layers. This poly(A)-RNA becomes labeled with radioactive precursors of RNA during the incubation of isolated aleurone layers with or without gibberellic acid. However, the rate of synthesis of poly(A)-RNA is enhanced by gibberellic acid. This enhancement begins within 3-4 hr of addition of the hormone and reaches a maximum, which is about 50-60% over the control, 10-12 hr after addition of the hormone. Cordycepin inhibits total RNA as well as poly(A)-RNA synthesis in barley aleurone layers. However, cordycepin inhibits the hormone-controlled synthesis of alpha-amylase (EC 3.2.1.1) only if it is added 12 hr or less after gibberellic acid. The insensitivity of alpha-amylase production to cordycepin after 12 hr of gibberellic acid treatment suggests that alpha-amylase is translated from stable messenger RNA.     

Age-associated impairment of murine natural killer activity.

Natural cell-mediated cytotoxicity may be critical in the resistance displayed by animals and humans to tumors and various pathogenic microorganisms. Because the frequency of tumors and infections increases markedly in aging populations, we have compared the natural killer (NK) competence of lymphoid tissues (spleen, bone marrow) and of peritoneal cells of young adult and aged mice. Spontaneous NK activity was much lower, and the rate of target cell lysis was much less, in aged mice. The level of NK activity was only modestly increased in old, compared to young, mice when they were exposed to Trypanosoma musculi, an organism that provides strong stimulation of NK activity. Restricted NK activity of aged mice was not attributable to suppressor cells. The NK effector cells in old mice were characterized as being nonadherent to plastic, completely susceptible to lysis by complement plus an antiserum against specificity NK 1.2, and only slightly affected by treatment with antiserum against specificity Thy-1.2. Two indirect methods were employed to assess the relative frequency of splenic NK cells at the time of maximal stimulation by T. musculi: a cytotoxicity assay with antiserum against NK 1.2 and a binding assay involving monolayers of YAC-1 tumor target cells. Similar results were obtained in both assays, indicating that at maximal stimulation about 10% of the total spleen cells of both young and old mice were NK cells. We conclude (cautiously) that the functional efficiency of aged NK cells is impaired and that this defect may account, in part, for reduced ability of aged individuals to resist certain types of cancer and certain pathogenic microorganisms.     

Role of suppressor cells in experimental pyelonephritis

The effect of suppressor cells on the synthesis of antibody and development of antigen-responsive lymphocytes was studied in rabbits with pyelonephritis produced with Escherichia coli O6:K13:H1. The responsiveness of splenic lymphocytes to phytohemagglutinin (PHA), which was reduced early in infection, was restored by preincubation of cells with indomethacin. The response of lymphocytes to E. coli antigen was also suppressed early. Indomethacin increased response to PHA but did not affect response to antigen. Rabbits given indomethacin from days 1-3 of infection had a delay in suppressor cell activity until day 9, but this early inhibition had no effect on synthesis of immunoglobulin or antibody or on serum levels of IgM or IgG. Thus, activity of splenic suppressor cells in pyelonephritis can be diminished by indomethacin, an inhibitor of prostaglandin synthetase, but the suppressor cells do not appear to influence the immune response or activities of bone marrow-derived lymphocytes such as antibody formation in experimental pyelonephritis.     

Hormone-induced increase in levels of functional mRNA and alpha-amylase mRNA in barley aleurones

Incubation of barley aleurone cells with gibberellic acid produces a progressive increase in the RNA content of the cells. The activity of poly(A)-containing RNA, measured in an in vitro wheat germ protein-synthesizing system, reaches a maximum ≈12 hr after hormone addition and declines thereafter. The structurally intact functional mRNA content in these cells, measured as poly(A)-RNA with 5′ “caps,” also shows a maximum at 12 hr and correlates with the translational capacity of poly(A)-RNA. Activation of mRNA by guanylylation or methylation after addition of gibberellic acid is ruled out. Available evidence indicates that gibberellic acid stimulates protein synthesis by increasing the synthesis of mRNA. Studies with cycloheximide suggest that the induction of synthesis of α-amylase mRNA by gibberellic acid requires protein synthesis after hormone addition.     

老年小鼠淋巴结T淋巴细胞的功能变化

目的：探讨老年小鼠淋巴结T淋巴细胞的功能。方法：采用^3H-TdR掺入法测定淋巴细胞的增殖;^3H-缬氨酸掺入法测定总蛋白合成;以依赖株细胞增殖反应测定法检测IL－2活性;^3H-缬氨酸掺入IL－2单克隆抗体－IL－2复合物,检测IL－2蛋白合成能力。结果：20月龄老年小鼠和27月龄老年小鼠与4月龄青年小鼠相比,其淋巴结细胞的增殖能力分别下降65．6％和32．8％;总蛋白合成下降40．2％和48．9％;IL－2活性下降36．6％和71．5％;IL－2蛋白合成能力下降32．9％和57．1％。结论：老年小鼠淋巴结细胞对ConA刺激的增殖能力、总蛋白合成水平、IL－2活性及IL－2蛋白合成水平与青年小鼠相比显著下降。     

Translation of RNA that contains polyadenylate from yeast mitochondria in an Escherichia coli ribosomal system.

RNA that contains poly(A) [poly(A)-RNA] has been isolated from yeast mitochondria by poly(U) Sepharose-4B column chromatography. Pulse-labeled poly(A)-RNA shows 8-10 discrete peaks by acrylamide gel electrophoresis. The specific activity of mitochondrial poly(A)-RAN is six to eight times greater than that of mitochondrial rRNA after pulse labeling of protoplasts with [3H-]uridine. Ethidium bromide inhibits incorporation by over 90%. The total mitochondrial RNA preparation was contaminated with 5-15% cytoplasmic rRNA as determined by gel electrophoresis, but RNA exhaustion hybridization experiments indicated little or no cytoplasmic contamination of the mitochondrial poly(A)-RNA. The poly(A)-RNA stimulates [3H]leucine incorporation into protein in an E. coli cell-free system. A fraction of the labeled product is precipitated with antibody directed toward yeast cytochrome oxidase, but not with antibody directed toward bovine serum albumin. Sodium dodecyl sulfate gel electrophoresis of the immunoprecipitated material reveals labeled peptides having the mobility of the three larger cytochrome oxidase peptides, which are known to be translated by mitochondrial ribosomes.     

Immune response to Plasmodium berghei sporozoite antigens. I. Evaluation of murine T cell repertoire following immunization with irradiated sporozoites

The Plasmodium berghei sporozoite antigen-specific T cell repertoire was analyzed in C57BL/6 (H-2b), BALB/c (H-2d) and C3H/HeN (H-2k) mice following immunization with irradiated sporozoites. Proliferative responses were correlated with the protective status of each strain. Proliferative reactivities to sporozoite antigens were compared in cultures containing either CD4+ T cells, CD8+ T cells, or total splenic lymphocytes. CD8+ T cells had no proliferative activity to sporozoite antigens; CD4+ T cells and splenic lymphocytes responded to the priming antigen, but the responses varied according to the mouse strain tested. The proliferative activity diminished at the onset of protection, presumably due to the induction of regulatory or non-proliferative T cell subsets. Sporozoite-immune lymphocytes did not respond to P. berghei circumsporozoite synthetic peptides. The restricted utilization of T cell epitopes during anti-sporozoite responses can be interpreted as resulting in part from a limited processing of the CS protein antigen.     

Cell-free translation of a biologically active, antigen-specific suppressor T cell factor.

In vitro synthesis of an antigen-specific T cell suppressor factor (TsF) has been accomplished by using partially purified poly(A)-containing RNA in a rabbit reticulocyte lysate cell-free translation system. The poly(A)-containing mRNA was isolated from a cloned T cell hybridoma that constitutively produces a TsF specific for the synthetic polypeptide antigen poly-(LGlu60LAla30LTyr10) (GAT). The RNA was fractionated by size and translated in vitro. The 16S RNA fraction stimulated synthesis of a biologically active protein that specifically suppressed both the GAT-specific antibody response by spleen cells in vitro and the proliferation response to GAT by lymph node T cells from GAT-primed mice. Further, the suppressor factor had a binding site for GAT, a determinant encoded by the I subregion of the major histocompatibility complex (MHC), and an apparent Mr 19,000 estimated by functional assays on protein separated by NadodSO4/polyacrylamide gel electrophoresis. These results indicate that virtually no posttranslational modifications (other than proteolytic cleavage) are necessary to obtain biologically active TsF. Hence, the presence of carbohydrate or other chemical groups does not contribute to either the serological properties of GAT-TsF or its biological properties.     

The cellular basis of impaired T lymphocyte functions in the elderly

Immunologic changes associated with aging were studied by various immunologic tests in 24 aged persons (age range, 76-83) and 25 young persons (age range, 20-40). The responses to phytohemagglutinin (PHA) and concanavalin A (Con A) were depressed in the aged subjects compared to the young ones (p less than 0.05), whereas the responses to pokeweed mitogen (PWM) were similar. The activity of adhereent and non-adherent cells was assessed in various combinations. The adherent cells of aged persons were indistinguishable from those of young persons in their ability to response to Con A. Lymphocytes from the aged synthesized larger in vitro amounts of immunoglobulin than did lymphocytes from the young, when stimulated with PWM. Con A-stimulated T lymphocytes derived from aged subjects showed a variable loss of suppressor activity. The mixed lymphocyte culture reaction with mitomycin-treated allogeneic and autologous cells was also impaired in aged subjects. Such an impaired response in the aged is related to higher incidences of malignant lesions and auto-antibodies.     

In vitro transcription of heat-shock-specific RNA from chromatin of Drosophila melanogaster cells.

Polyadenylylated RNA synthesized after heat shock was isolated from polysomes of cultured cells of Drosophila melanogaster and used as template to prepare cDNA. An excess of poly(A)-RNA from heat-shocked cells hybridized to 80% of the cDNA, whereas cytoplasmic poly(A)-RNA from cells grown at 25 degrees could drive only half of the cDNA probe into hybrid. These sequences were removed from the cDNA population by annealing to poly(A)-RNA from cells grown at 25 degrees. The unreacted material represented only heat-shock-induced mRNA sequences, as shown by a second cycle of hybridization. Isolated chromatin was transcribed in vitro at 25 degrees with Escherichia coli RNA polymerase, with mercurated UTP as precursor. RNA transcribed from chromatin that was prepared from cells 1 hr after the temperature was shifted to 37 degrees hybridized with 100-fold faster kinetics to the heat-shock-specific cDNA probe than did RNA transcribed from chromatin of cells grown at 25 degrees. Therefore, heat shock results in a change in chromatin structure recognizable by E. coli RNA polymerase.     

Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland.

The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.     

Gibberellic Acid Causes Increased Synthesis of RNA Which Contains Poly(A) in Barley Aleurone Tissue

Incubation of isolated barley aleurone layers with gibberellic acid for 16 hr caused a 50% increase in the synthesis of RNA that contains poly(A) sequences [poly(A)-RNA], but had no measurable effect on the syntheses of the major RNA species. The syntheses of both the poly(A) and the heteropolymeric fractions of the poly(A)-RNA were increased.The poly(A) sequences were separated into two classes by size, one containing an average of 250 nucleotides and the other about 70 nucleotides. The two classes occurred in a molar ratio of about 1:1. Gibberellic acid increased the syntheses of both sequences to the same extent.We interpret these results to mean that gibberellic acid increases specifically the synthesis of mRNA in this tissue.     

Interferon treatment of mice: enhanced expression of histocompatibility antigens on lymphoid cells.

Treatment of young and mature mice with potent mouse interferon preparations results in a marked enhancement of the expression of histocompatibility antigens on the surface of thymocytes and splenic lymphocytes as measured by an enhanced absorption of alloantiserum. We postulate that such modifications of the cell surface may reflect an effect of interferon on lymphocyte maturation and may be relevant to the effect of interferon on lymphocyte function.     

The effect of antigen priming on immunological response regulation in aging.

The primary anti-sheep red blood cells response and the response of antigen primed CBA mice aged 3–29 months were compared. There was a 30-fold decline with age in the primary immunological response, tested by the direct plaque-forming cell number. In young and young adult mice, priming resulted in a fall of antibody production and of number of Ig-positve spleen lymphocytes. During the lifespan the response of primed mice increased, reaching and then exceeding the level of primary response in senescence as much as 20-fold. Negative correlation was established between antibody-forming and nuclear spleen cell numbers of old primed mice.The adoptive transfer of spleen cells of primed young adult mice suppressed the antibody production in immunized recipients, whereas that of old primed spleen cells produced no reaction, the response of the recipients of old primed spleen cells being thus higher.The addition of young bone marrow to old primed mice enhanced the response and abolished the negative correlation between plaque-forming cell and nuclear spleen cell numbers.The data obtained showed a decreased suppressor activity of the antigen primed spleen cells with aging. This phenomenon seems to reflect certain features of the immune response regulation in old age.     

Aging and hematopoiesis. VI. Neutrophilia and other leukocyte changes in aged mice

The concentration of blood leukocytes rose progressively as mice aged. All blood leukocytes increased, although a greater degree of increase was seen in neutrophils and monocytes than in lymphocytes and eosinophils. The total number of nucleated cells per marrow cavity of the humerus was also higher in aged than in young adult mice, the increase primarily reflecting peroxidase-positive cells. Both blood and marrow neutrophils of aged mice responded to perturbations induced by bleeding or by endotoxin injection in a manner qualitatively similar to that seen in young adults. When hematopoietic chimeras were produced by marrow transplantation, blood and marrow neutrophils were characteristic of the age of the recipient, not the cells; i.e., young mice kept a "young" neutrophil pattern and old mice kept an "old" neutrophil pattern when given marrow from either old or young mice. Colonies of granulocytes and/or monocyte-macrophages were grown from young marrow cells placed in plasma clots in Millipore chambers in the peritoneal cavity. The number of colonies was the same in young and in old mice, suggesting that long-range humoral stimulation of cell growth was similar in young and old. Thus, neutrophilia due to increased neutrophil production appears to be a normal part of the aging process in the mouse. The increase in neutrophil production may be due to a changing hematopoietic microenvironment.     

The effect of recombinant murine interferon-gamma on the hematopoietic and immunological parameters of mice bearing metastatic Lewis lung carcinoma tumors

The in vitro and in vivo effects of murine recombinant interferon-gamma (rIFN-gamma) on hematopoietic and immune parameters of normal mice and of mice bearing metastatic variant Lewis lung carcinoma (LLC-C3) tumors were assessed. The in vitro addition of rIFN-gamma to bone marrow or spleen cells from normal and LLC-C3-bearing mice reduced their capacity to grow into colonies in soft agar (CFU) and minimized their immune suppressive activities. In vivo studies showed that when LLC-C3 tumor-bearing mice were injected with rIFN-gamma for 2 days prior to sacrifice, there was a reduction in femoral bone marrow cellularity, CFU, and suppressor cell activity. In contrast, spleen cells of tumor-bearing mice that were injected with rIFN-gamma showed reduced blastogenesis, and increased spleen cellularity, CFU, and suppressor cell activity. Thus, short-term rIFN-gamma treatment of LLC-C3-bearing mice may be beneficial with regard to the bone marrow because it caused a decrease in hematopoiesis and suppressor cell activity, whereas it may be detrimental in the spleen because it appeared to stimulate hematopoiesis and increase splenic suppressor cell activity. The dichotomy between the in vitro versus in vivo effects of rIFN-gamma on splenic hematopoiesis and suppressor activity may be due to the stimulation of production of colony-stimulating factor (CSF) activities by spleen cells of rIFN-gamma-treated mice. Our results suggest that the tumor stimulation of hematopoiesis and its associated appearance of immune suppressor cells can be both positively and negatively altered by rIFN-gamma.     

Loss of suppressor t cells in the pathogenesis of the autoimmunity of nzb/w mice

Loss of suppressor T cells was demonstrated in NZB/W mice, an animal model of autoimmunity. As NZB/W mice matured they lost splenic T cells that could be activated by concanavalin A (Con A) to become suppressor cells and lost the ability to produce the regulator of humoral immune responses, soluble immune response suppressor (SIRS). However, NZB/W spleen cells retained the capacity to respond to suppressor signals from Con A pulsed normal spleen cells. Thrice weekly administration of SIRS containing supernatants of Con A pulsed normal spleen cells to young NZB/W mice lead to a striking reduction in the manifestations of autoimmunity.