Biomedical subjects

E T Bloom

Publications and source records attributed to E T Bloom.

At least 55 records · Page 3Linked to original sources

PubMed1993

Signal transduction via phosphorylated adhesion molecule, LFA-1 beta (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells.

It is well known that IL-2 stimulates natural killer (NK) cells to express lymphokine activated killer (LAK) activity and that this stimulation prompts the acquisition of the ability to lyse previously insensitive target cells. The possible role of adhesion molecules in the IL-2 activation process was probed by focussing on a lymphocyte function-associated antigen (LFA)-1-dependent model system. A mAb to the LFA-1 beta chain abrogated LAK activity, but only moderately suppressed NK activity, suggesting a differential role for LFA-1 beta in LAK compared with NK mediated lysis. Orthophosphate labeling demonstrated that the LFA-1 beta chain was strongly phosphorylated in LAK but not NK cells; in contrast, the alpha chain was phosphorylated similarly in both effector cell types. At least a portion of the phosphorylation of the beta chain was on tyrosine residues, as shown by Western blotting with anti-phosphotyrosine antibody of LFA-1 beta immunoprecipitates. Crosslinking of the LFA-1 beta chain with plastic-adhered antibody stimulated Ca(2+)-dependent release of cytoplasmic lytic granules and induced phosphatidyl inositol turnover in LAK but not NK cells. We conclude that the IL-2-induced phosphorylation of the beta chain of the LFA-1 adhesion molecule in LAK cells and associated alteration in signal transduction may be important in the stimulation of LAK cell activity in NK cells.

Antigens, CD↗

PubMed1992

Pore-forming protein in individual cytotoxic T lymphocytes: the effect of senescence provides a probe for understanding the lytic mechanism.

The senescent decline of cytolytic T lymphocyte (CTL) activity was examined (a) to learn more about the effect of aging on the immune system, and (b) to probe the mechanism of cell-mediated cytolysis. The effect of age on the generation of pore-forming protein (Pfp) was examined at the cellular level in a murine model using CTL stimulated in allogeneic mixed lymphocyte culture (MLC). Pfp expression was analyzed by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). Immunocytochemical analyses of Pfp in MLC-stimulated splenic T cells from a large number of mice revealed that although stimulated cells from aged mice exhibited fewer Pfp-producing cells than those from young, the diminution in the proportion of Pfp+ cells was small compared to the age-related decrease in lytic activities (approximately 2-fold vs. approximately 7.4-fold, respectively). Time-course analysis disclosed similar kinetics for the generation of Pfp+ cells among responding cells from young and aged mice. No significant age-related difference in the proportion of Pfp+ cells was observed in MLC-stimulated lymph node cells despite a large and significant difference in lytic activity (approximately 6.5-fold). Purified CD8+ T cells demonstrated a large age-related difference in CTL activity (approximately 3-11-fold) and accounted for virtually all the Pfp. Although little difference in the proportion of Pfp+ CD8+ T cells could be detected between age groups, stimulated CD8+ cells or whole splenic T cells from old mice consistently exhibited a striking reduction in both the intensity of Pfp staining and the apparent numbers of granules per cell. This difference in Pfp was examined by ELISA and total Pfp levels were found to be approximately 12-fold greater in CTL generated from splenic T cells of young compared to aged mice. The results demonstrate that Pfp levels are reduced in CTL from aged compared to young mice at the level of the individual cells and suggest the possibility that a threshold level of Pfp may be required for potency of effector cell function.

Aging↗

PubMed1991

Functional importance of CD4+ and CD8+ cells in cytotoxic lymphocytes activity and associated gene expression. Impact on the age-related decline in lytic activity.

Previously we found that the age-related decline in cytotoxic lymphocyte (CTL) activity in a murine allogeneic system is associated with declining expression of the perforin and two serine esterase genes by senescent CTL. To identify the cell subsets responsible for the age-related decrement in the generation of CTL, splenic T cells, subsets, and mixtures of subsets from aging and young BALB/c female mice were analyzed for their allo-responsiveness to C57BL/6 spleen cells. Depletion studies revealed that CD8+ cells expressed both the lytic activity and the CTL-associated genes, although both CD8+ and CD4+ cells were required for generation of optimal lytic and proliferative responses. Mixture experiments demonstrated that the reduced lytic activity generated by CD8+ cells from the spleens of old mice is a consequence of alterations in both CD8+ and CD4+ cells. The results of experiments in which CD4+ cells from young and old mice were mixed revealed that the alteration in CD4+ cells is consistent with a loss of function. These findings show that (a) the expression of the perforin and serine esterase genes in primary CTL is associated with CD8+ T cells in old and young mice, and exhibits an age-related decrement consistent with that for lytic activity, and (b) the senescent decline in CTL activity is a consequence of aging decreasing activity in both the CD4+ and CD8+ subsets.

Aging↗

PubMed1990

Age-related decrement in cytotoxic T lymphocyte (CTL) activity is associated with decreased levels of mRNA encoded by two CTL-associated serine esterase genes and the perforin gene in mice.

The age-related decline in cytotoxic T lymphocyte (CTL) activity has been recognized for many years. Age-related alterations in several immunologic events have been suggested to be partly or completely responsible for this decline. We had previously demonstrated (Bloom et al., Cell. Immunol. 1988. 144: 440) in mice that a deterioration in the lytic mechanism may be at least in part responsible for the decline in CTL activity. We now report that this decline correlates with an age-related decrease in serine esterase activity released into the supernatant medium in the process of generating CTL. Northern analyses were then used to examine the effect of age on expression of genes encoding for perforin and two CTL-associated serine esterases. The products of all three of these genes have all been postulated to play roles in CTL-mediated lysis. We show that the expression of all three of these genes appears to decline with age in the process of generating allogeneic CTL. These alterations in gene expression correlated both with diminished cytolytic and released esterase activities generated by mixed leukocyte culture in spleen cells of old mice compared to young. The age-related decline in gene expression could not be attributed to shifts in T cell subsets, but CD8+ cells generated by allogeneic stimulation of nylon wool-passed spleen cells from old mice expressed significantly less cytolytic activity than those from young. This report is the first demonstration of an age-related decrease in expression of a functionally related group of genes. In addition, these findings are compatible with the suggested roles for perforin and serine esterase release in CTL-mediated target cell lysis.

Aging↗

PubMed1990

Prostaglandin E2, monocyte adherence and interleukin-1 in the regulation of human natural killer cell activity by monocytes.

Monocytes have previously been shown both to augment and suppress human natural killer (NK) cell activity depending upon the conditions. An interleukin-1/interleukin-2 (IL-1/IL-2)-dependent mechanism has been shown to be involved in the augmentative effect. In the current study, the role of the method of monocyte isolation was evaluated. Monocytes isolated by Percoll gradient centrifugation were ineffective for modulating NK activity, but monocytes isolated by adherence from most donors exhibited increased augmentation with increased interval of adherence (up to 1 h). However, monocytes isolated by adherence from certain donors reproducibly exhibited increased suppression with increased interval of adherence. The observation of augmentation was correlated with an increase in the balance between IL-1 production and prostaglandin E (PGE) production by the monocytes. The roles of PGE2 and IL-1 were therefore examined by mixing these cytokines with enriched null lymphocyte preparations in the absence or presence of monocytes in the NK assay system. The participation of PGE2 was further examined using monocytes treated with indomethacin (10(-6) M), and the participation of monocyte-membrane-bound IL-1 was evaluated using monocytes fixed with 1% paraformaldehyde. The results revealed that PGE2 production is involved in the suppression of human NK activity by human monocytes, and the functional balance between IL-1 and PGE2 determines whether suppression or augmentation is observed. The data of this and previous studies are consistent with the suggestion that membrane-associated IL-1 is the important IL-1 moiety for the augmentation of human NK activity by monocytes.

Adult↗

PubMed1990

The IL-2 receptor beta subunit is absolutely required for mediating the IL-2-induced activation of NK activity and proliferative activity of human large granular lymphocytes.

TU27 monoclonal antibody reacts with the cellular receptor to the beta subunit of the interleukin-2 receptor (IL-2R) (p70-75). This reagent has been utilized to demonstrate directly that the IL-2R beta is the IL-2-binding protein that mediates the activation of large granular lymphocytes (LGL) to proliferate and increase cytolytic activity in response to IL-2. The results presented here show that (i) the frequency of TU27+ cells paralleled the frequency of CD16+ (Leu-11+) cells; (ii) TU27 completely abrogated the proliferative response of LGL to IL-2, while GL439, an anti-IL-2R alpha (anti-Tac) reagent, had a much smaller effect, and the effect of the two together was no different from the effect of TU27 alone; (iii) TU27 abolished the IL-2-induced activation of natural killer (NK) activity and inhibited the development of LAK activity, while GL439 had no effect; and (iv) TU27 also inhibited naive NK activity. Therefore, these data clearly show that the IL-2-IL-2R beta interaction is responsible, and probably completely so, for the proliferative and cytolytic-promoting effects of IL-2 on LGL. In addition, they also suggest a role for this interaction in autocrine effects on native NK activity.

Antibodies, Monoclonal↗

PubMed1989

Effect of IL-2 in vitro on CTL generation in spleen cells of young and old mice: asialo GM1+ cells are required for the apparent restoration of the CTL response.

The role of asialo GM1+ (ASGM1+) cells and exogenous IL-2 in the age-related decline in allospecific CTL activity was evaluated. Primary CTL were generated in mixed leukocyte culture (MLC) [BALB/cANN (H-2d) anti C57BL/6N (H-2b)] and tested for allospecific lytic activity against the EL-4 (H-2b) cell culture line, and for non-MHC-restricted activity against WEHI-3 (H-2d) and YAC-1 (H-2a). Cultures included responder cell populations which had been treated with antibody to ASGM1 plus complement or complement alone, and irradiated stimulator cells, in the presence or absence of rIL-2 or crude IL-2-containing supernatants. The amount of rIL-2 used to accommodate the age-related decline in IL-2 production was determined empirically to be 500 U by assessing IL-2 production in MLCs containing responder cells from young versus old animals. rIL-2 appeared to restore the allospecific CTL activity generated by spleen cells of old mice to the level of that of young. However, treatment with anti-ASGM1 antibody revealed that this restoration was due to an effect of the IL-2 on ASGM1+ cells. The allospecific target cells, EL-4, were not sensitive to lymphokine-activated killer (LAK) cells induced by IL-2 alone under the conditions used. It is suggested that the apparent restoration was due to increased LAK-like (or MHC-nonrestricted) activity mediated by an ASGM1+ cell in the CTL precursor population.

Age Factors↗

PubMed1989

The effect of exercise on natural killer cell activity in young and old subjects.

Alterations in immune function have been commonly reported in elderly persons. We have examined the effect of age on the responsiveness of natural killer (NK) cells to in vitro stimulation with recombinant interleukin-2 (rIL-2) and in vivo stimulation with exercise in 17 healthy subjects (8 young and 9 old). The old subjects were found to have NK cell numbers and function that were not significantly different from the young subjects at baseline. They also responded as well as the young subjects to rIL-2 stimulation of NK cells in vitro. In response to maximal bicycle ergometry exercise, there was a marked rise in NK activity in the old (7.52 +/- 1.71 LU/10(6) pre-exercise vs 15.20 +/- 3.27 LU/10(6) post-exercise, p less than .03) and the young (6.29 +/- .48 LU 10(6) vs 14.56 +/- 1.86 LU 10(6), p less than .005) subjects. Lymphocytes bearing the NK marker Leu 11a also rose significantly post-exercise in both old and young subjects. We conclude that healthy elderly subjects increase their NK activity in response to the acute stressor, exercise, at least as effectively as do young subjects.

Adult↗

PubMed1989

Increase in Tac mRNA expression in natural killer-like cell line in response to interleukin-1 and interleukin-2 shown at the population and cellular level by in situ hybridization.

Our previous results have shown that the increased expression of the Tac component of the interleukin-2 (IL-2) receptor at the surface of a natural killer-like cell line (YTN10) in response to interleukin-1 (IL-1) is associated with an eightfold increase in Tac mRNA production. Tac expression was detected by flow cytometry and anti-Tac binding studies, and mRNA synthesis was assessed by Northern and dot blots. These findings have now been extended utilizing recombinant IL-1 and autoradiographic techniques in experiments using both the parent YT and YTN10 cells. In situ hybridization experiments have shown that the increase in mRNA expression is manifested both in the percentage of cells expressing it and in the level of expression per cell. Consistent results were obtained in both sublines. In addition, exposure of both sublines to recombinant IL-2 also induced increased Tac mRNA synthesis, while inducing only a marginal increase in Tac expression at the membrane level.

Cell Line↗

PubMed1988

Lymphokine-activated killer cells derived from murine bone marrow: age-associated difference in precursor cell populations demonstrated by response to interferon.

We have previously demonstrated that the majority of precursor cells of lymphokine-activated killer (LAK) cells in spleens from both young and old mice reside in the natural killer (NK) cell or pre-NK cell populations. In the present study, we examined the induction of LAK cell activity in bone marrow cells from young and old mice and its modulation by interferon (IFN). Bone marrow cells from either young (6-9 weeks) or old (20-26 months) mice were cultured with 1000 U/ml of human recombinant interleukin 2 (rIL-2) for 5 days. The cells were harvested and tested for their cytotoxicity against NK-resistant fresh tumor cells (MCA-102) in a 51Cr release assay. As with spleen cells, bone marrow cells from old and young mice developed comparable levels of activity. Phenotypic analysis showed that virtually all of the LAK precursor cells were asialo GM1+ and most of the effector cells expressed Thy-1, regardless of the age of the mice. Inclusion of IFN-gamma during the culture with rIL-2 augmented the activity induced in bone marrow of young mice, but, in contrast, inhibited the generation of LAK activity from bone marrow cells of old mice. These results demonstrate a differential sensitivity of LAK precursor (or pre-NK) cells of old and young mice to interferon.

Aging↗

PubMed1988

Increased binding of IL-2 and increased IL-2 receptor mRNA synthesis are expressed by an NK-like cell line in response to IL-1.

An NK-like cell line (YTN10 cells) was stimulated with IL-1, and the IL-2R, as detected by flow cytometry analysis using fluorescein-labeled anti-IL-2R (Tac) antibody, increased displaying a dose-response curve in the shape of an inverted "U." This was associated with increases in 1) the number of high affinity binding sites for IL-2 (Kd approximately 45 pM) which increased from 1270 to 4220 sites per cell, and 2) the number of binding sites for anti-Tac antibody, which increased from 999 to 6763 sites per cell. They were both detected by using radiolabeled ligand-binding methods which are one to three orders of magnitude more sensitive than flow cytometry analysis. No binding sites with low affinity for IL-2 could be detected on untreated or treated cells. However, a change was noted in the number of IL-2 binding sites with intermediate affinity (Kd approximately 1000 pM), which increased from 54,000 to 67,000 sites per cell. This binding affinity is equivalent to that of the p70 IL-2-binding protein detected on YT cells and on large granular lymphocytes by other investigators. This suggests that the increase in the Tac peptide was the limiting factor in the combination of the Tac and p70 components to form the high affinity IL-2R. The corollary is that all new Tac molecules were incorporated into these R. The IL-1 treatment also induced an increase in production of Tac mRNA as detected by Northern blots. Comparison of the relative amounts of Tac mRNA by dot blots verified the quantitative difference between treated and untreated cells. These data demonstrate that the IL-1-induced positive regulation of the Tac protein and high affinity IL-2R on YTN10 cells may operate at the level of gene transcription.

Antigens, Surface↗

PubMed1988

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.

Aging↗

PubMed1988

Age-related alteration in the composition of immunocompetent blood cells in atomic bomb survivors.

A total of 1328 atomic bomb survivors in Hiroshima were studied to determine alterations in the number of blood lymphocytes belonging to T-cell subpopulations, the number of CD19 antigen-positive B cells and the number of Leu 7 and CD16 antigen-positive lymphocytes. Overall, with increasing age, significant decreasing trends in the numbers of some lymphocytes in T-cell subpopulations and of B cells were observed. Furthermore, the number of blood lymphocytes positive for CD5 antigen was significantly lower in the people exposed to radiation (greater than 1 Gy) in the older age group (more than 30 years old at the time of the bombing). A similar tendency for decreases in the numbers of CD4, CD8, and CD19 antigen-positive cells was observed in these older survivors, although the differences were not statistically significant. These results suggest that aging of the T-cell related immune system is accelerated in the irradiated people of advanced age. This may be explained by the age-related decrease in thymic function in those subjects who were older at the time of the bombing resulting in a decreased functional ability of the immune system after radiation injury. On the contrary, the number of Leu 7 or CD16 antigen-positive cells was found to be increased significantly in the older age group compared to the younger group, although there was little dependence on dose.

Age Factors↗

PubMed1988

Immunological responses of aging Japanese A-bomb survivors.

Immune response parameters were studied on 1341 A-bomb survivors residing in Hiroshima, Japan. Mononuclear cells were isolated from venous blood and tested for interleukin-2 production; lymphocytes were purified and tested for natural killer (NK) cell activity and interferon (IFN) production; and serum was tested for IFN and circulating immune complex (CIC) levels. Statistical analyses were performed for each type of assay using a linear models procedure including sex, age at the time of the bomb, radiation exposure, all the interaction variables, and the categorical variable day-of-assay in the model. The findings showed that (1) none of the immunologic variables were significantly affected by radiation exposure; (2) NK activity and CIC levels were positively associated with age; and (3) NK activity was on average higher for males than females. The data exemplify the difficulty in reaching firm conclusions concerning associations with radiation exposure when the dependent variable exhibits a large degree of interindividual and day-of-assay variability.

Aging↗

PubMed1988

Endogenous opioids and the exercise-induced augmentation of natural killer cell activity.

Based on prior observations that both beta-endorphin and exercise stimulate natural killer (NK) cell activity, we have examined the hypothesis that the release of endogenous opioids during the stress of acute exercise may mediate this NK cell augmentation. Eight healthy young women underwent a maximal bicycle ergometer exercise test with prior in vivo administration of a placebo and an opioid antagonist, naloxone (100 micrograms/kg), in a randomized, blind protocol. Exercise after the placebo injection was accompanied by a dramatic rise in NK activity, as well as an increase in the percentage of lymphocytes bearing the NK cell surface markers Leu 11a and Leu 19. Significant stimulation of NK activity was observed with beta-endorphin in vitro before exercise, but after exercise, beta-endorphin had a nonsignificant inhibitory effect. When these experiments were carried out in the presence of naloxone in vivo, the rise in NK activity after exercise was no longer significant. Naloxone did not significantly alter the rise in Leu 11a+ or Leu 19+ cell after exercise, as compared with the placebo. Finally, when naloxone was given to the subjects beforehand, exercise no longer completely blocked the in vitro beta-endorphin stimulation of NK cells. In conclusion, our observations that the exercise-induced augmentation of NK activity and the lack of effect of beta-endorphin in vitro on NK activity after exercise are both significantly attenuated by prior administration of naloxone suggest that the opioid system may play a major role in the modulation of NK cells during physiologic stress.

Adult↗

PubMed1987

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity.

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.

Adult↗

PubMed1987

Lymphokine-activated killer cells and aging in mice: significance for defining the precursor cell.

The present study was undertaken to define the cell populations which mediate lymphokine-activated killer (LAK) cell activity in mice. Because old mice exhibit markedly decreased to nondetectable natural killer (NK) cell activity, this age-associated change provided an advantageous system to examine the contribution of NK and T cells to LAK activity. Spleen cells from either young (6-9 weeks) or old (20-26 months) mice were cultured with 1000 units/ml of recombinant interleukin 2 (rIL 2) for 3-5 days. The cells were then tested in a 51 Cr-release assay for their cytotoxicity against NK-resistant fresh tumor cells (MCA-102). The LAK activity exhibited by spleen cells from old mice following 5 days of culture was equivalent to that developed by spleen cells of young mice. This result was contrary to what would be anticipated if mature NK cells comprise the primary precursors of LAK activity, and required further elucidation. The Thy-1 and asialo GM1 (ASGM1) phenotypes of LAK precursor and effector cells were therefore examined by depletion techniques using the appropriate antibodies plus complement. The results using spleen cells harvested after 5 days of culture with rIL 2 showed that LAK effector cells which developed from spleen cells of both young and old mice were predominantly Thy-1+ (85.3% young; 91.8% old) and some coexpressed ASGM1. Spleen cells were treated prior to culture to study the precursor cells. Development of LAK activity by spleen cells from both young and old mice was greatly reduced by pretreatment with anti-ASGM1 plus complement. However, since spleen cells of old mice exhibit very low mature NK activity, these data suggest that the LAK precursors, at least in old mice, may be ASGM1+ NK precursor cells rather than mature ASGM1+ NK effector cells. In addition, treatment with anti-Thy-1 plus complement inhibited generation of a significant proportion of LAK activity only in the spleens of old mice, suggesting a qualitative difference in LAK precursor cells with age and supporting the heterogeneity of the cells which are capable of developing LAK activity.

Aging↗

PubMed1987

Delayed effects of low-dose radiation on cellular immunity in atomic bomb survivors residing in the United States.

Several parameters of cellular immune function were assessed among persons who survived the 1945 atomic bombs in Hiroshima and Nagasaki but who now reside in the United States. The subjects in this study were exposed to various low doses (T65D) of radiation at the time of the bomb. More than half received an estimated 0 Gy (S0 group). Of those exposed to more radiation (S+ group), nearly 90% received less than 0.50 Gy (50 rad). Lymphocytes were isolated from the peripheral blood of these individuals and were assessed for the following parameters of cellular immunity: mitogenic response to phytohemagglutinin, mitogenic response to allogeneic lymphocytes, natural cell-mediated cytotoxicity (NCMC), and interferon production. In every case, the response of the S+ group was greater than that of the S0 group, although only the difference for NCMC was statistically significant. Results of studies presently being performed on A-bomb survivors residing in Hiroshima do not confirm this difference. Therefore, it is difficult to say whether the increase in natural cytotoxicity observed among the American and not the Japanese A-bomb survivors exposed to very low doses of radiation is a hormetic effect which was modulated by post-radiation environmental conditions or a result of selective migration.

Cytotoxicity, Immunologic↗