Carnitine and acylcarnitine levels of human peripheral blood lymphocytes and mononuclear phagocytes.

Lymphocytes and mononuclear phagocytes are essential to host defense, yet little is known about their metabolic requirements. To determine the involvement of carnitine in the intermediary metabolism of these cells, the amounts of free carnitine and individual acylcarnitines were determined for human peripheral blood lymphocytes and mononuclear phagocytes. Lymphocytes from healthy young adults contain 79 +/- 6 and 56 +/- 5 nmol/10(9) cells total and free carnitine, respectively, showing a 29% acylation. When expressed per mg cell protein, they contain 3.41 +/- 0.54 nmol total and 2.44 +/- 0.40 nmol free carnitine. By comparison, phagocytes contained approximately 4-fold more total carnitine per cell (301 nmol/10(9) cells) and had a much higher level of acylation (69%). Acetylcarnitine was the predominant acylcarnitine in both lymphocytes (15.4 nmol/10(9) cells) and phagocytes (98.3 nmol/10(9) cells) accounting for 72-73% of total acylcarnitines. Long-chain acylcarnitines constituted 3-4% of total acylcarnitines. These data suggest that carnitine is involved in the metabolism of both short and long-chain acyl-CoAs within lymphocytes and phagocytes, but its specific roles in these two cell types have not been determined.

A distinct role for apoptosis in the changes in lymphopoiesis and myelopoiesis created by deficiencies in zinc.

Reduced numbers of lymphocytes in the peripheral immune system appeared to be a significant cause of the loss in host defense capacity in humans and animals that are zinc deficient (ZD). A series of studies verified that ZD substantially reduced the lymphocyte compartment of both the marrow and thymus in young adult mice, with large losses noted among the pre-B and pre-T cells. Suboptimal nutriture along with chronic production of glucocorticoids generated during ZD had accelerated apoptosis among these precursor lymphocytes two- to threefold. Thus, the primary cause of the lymphopenia created by ZD was reduced production of lymphocytes and heightened cell death among precursor cells. The data will also show that myelopoiesis in the marrow was protected and enhanced numbers of myeloid progenitor cells were found in S and G2/M. Thus, as zinc became limiting the second line of defense appeared to be down-regulated via reduction of lymphopoiesis while cells of the myeloid lineage were protected to maintain the first line of defense that provides innate immunity. This may represent an important adaptation of the immune system to suboptimal nutriture that deserves further exploration.

Optimization of peroxide production by resident macrophages.

Previous reports indicated that resident macrophages produced low or nondetectable quantities of H2O2 (1-10 nmol H2O2/mg resident macrophage protein) when measured by the phenol red assay described by Pick and Mizel [5]. However, modifications of the assay that included addition of calcium to assay solutions, alterations in cell-harvesting methods, increasing the concentration of cells analyzed, incubating the cells in ambient air, and enhancing the interactions between macrophages and particulate stimulants by centrifugation resulted in increased production of peroxide. Each variable was instrumental in improving the assay conditions and the combination of changes dramatically increased the amount of peroxide (30-60 nmol H2O2/mg resident macrophage protein) obtained from murine resident macrophages, making it possible to better assess the role of these cells in microbicidal killing.

Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry.

The ability of glucocorticoids to induce apoptosis or programmed cell death in mouse thymocytes is well-established. Measurement of apoptosis-associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. Since these methods measure DNA fragmentation in whole cell lysates rather than intact cells, they have severe limitations, particularly with heterogeneous cell populations. When mouse thymocytes were incubated with glucocorticoids, fixed, stained with propidium iodide and analysed flow cytometrically for cell cycle distribution, a distinct subpopulation of cells was observed to form below the G0/G1 region, denoted as the A0 region. The presence of cells in this region was consistent with the presence of internucleosomal DNA fragments as determined by gel electrophoresis. Inhibitors of transcription, translation and endonuclease activity, and a glucocorticoid receptor antagonist prevented accumulation of cells in this region. Irradiation of mouse thymocytes also produced a population in the A0 region. Cells in this region are believed to have undergone glucocorticoid-induced DNA fragmentation. This method represents a useful alternative to whole cell lysate assays, since apoptosis can be evaluated on an individual cell basis.

Improved plaque production for short-term bone marrow and fetal liver cultures.

The addition of 0.5% globulin-free (GF-BSA) or 0.5% delipidated BSA (D-BSA) to short-term murine bone marrow (BM) (cultures) increased the number of plaque-forming cells (PFC) responding to trinitrophenylated lipopolysaccharide (TNP-LPS) 2-5-fold (1.1 X 10(4)-2.7 X 10(4) PFC per 10 X 10(6) nucleated BM cells). Although it was necessary to continue to supplement these cultures with 5% fetal calf serum (FCS), the inclusion of the aforementioned BSA preparations provided enhanced PFC production for all lots of FCS tested. Similarly, these preparations of BSA made it feasible to also culture BM in autologous mouse sera (MS) or in medium without 2-mercaptoethanol (2-ME) if in the latter case the D-BSA was pretreated with 2-ME. Thus, the inclusion of GF-BSA or D-BSA in short term cultures of BM not only substantially increased the number of Ig-secreting B cells produced in response to TNP-LPS but seemed to eliminate the need to screen for supportive batches of FCS or MS. These preparations of BSA also facilitated hapten specific PFC responses of fetal liver cultures.

Flow cytometric analysis of the expression of murine B and T surface markers from birth to adulthood.

The proportion of nucleated splenocytes bearing B-lymphocyte markers B220, surface IgM (sIgM) and sIgD, as well as the T-lymphocyte markers Thy 1.2, CD5, CD8a and CD4 were quantitated by flow cytometric analysis (FACS) throughout postpartum development in the A/J mouse. Full expression of B lymphocyte markers was achieved much sooner than expression of T lymphocyte markers. This was especially true for B220, which was found on 8% of all splenocytes at day 5 and reached adult levels (47-50%) by weaning at day 22. Expression of sIgM and sIgD were 13% and 9%, respectively, of all splenocytes at day 5 with mature levels not expressed until day 35 postpartum (approximately 36% of cells were positive for these markers). T lymphocyte markers, on the other hand, did not reach full expression until sexual maturity. For example, Thy 1.2 expression was 8% on day 5 and did not reach mature levels (28-30%) until day 56. CD5 closely paralleled Thy 1.2 expression rising from only 2% on day 5 to 27% by day 56. Likewise, CD8a and CD4 marker development paralleled one another with CD8a rising from 1% on day 5 to 10% by day 56 and CD4 rising from 5% on day 5 to 19% by day 56. These data demonstrate the variability in the time of appearance and rate of maturation of the various lymphocyte cell surface markers during postpartum development. They also serve as a reference to identify alterations in lymphocyte development created by immunodeficiency diseases.

Zinc: health effects and research priorities for the 1990s.

This review critically summarizes the literature on the spectrum of health effects of zinc status, ranging from symptoms of zinc deficiency to excess exposure. Studies on zinc intake are reviewed in relation to optimum requirements as a function of age and sex. Current knowledge on the biochemical properties of zinc which are critical to the essential role of this metal in biological systems is summarized. Dietary and physiological factors influencing the bioavailability and utilization of zinc are considered with special attention to interactions with iron and copper status. The effects of zinc deficiency and toxicity are reviewed with respect to specific organs, immunological and reproductive function, and genotoxicity and carcinogenicity. Finally, key questions are identified where research is needed, such as the risks to human health of altered environmental distribution of zinc, assessment of zinc status in humans, effects of zinc status in relation to other essential metals on immune function, reproduction, neurological function, and the cardiovascular system, and mechanistic studies to further elucidate the biological effects of zinc at the molecular level.

Zinc deficiency and immune function.

Deficiency in zinc, an essential trace element, is a frequent human dietary problem in the United States and is also associated with such disease states as alcoholism, renal disease, burns, gastrointestinal tract disorders, and acrodermatitis enteropathica. Skin lesions and poor wound healing are observed in severe forms of the deficiency. However, modest deficits in zinc cause lymphopenia and reduced immune capacity among affected humans. With the mouse used as a model because it has an immune system analogues to that of humans, the effects of zinc deficiency on immune function have been well characterized. A suboptimal intake of zinc causes marked atrophy of the thymus, a 50% reduction in leukocytes, a rise in corticosterone levels, and a 40% to 70% reduction in antibody-mediated, cell-mediated, and delayed-type hypersensitivity responses.

The many roles of apoptosis in immunity as modified by aging and nutritional status.

Apoptosis plays a vital role in the elimination of anti-self clones, down regulation of immune responses and the killing of virally infected and malignant cells. There is ample evidence that as we age the immune system not only becomes less potent, but dysregulated which includes apoptotic dependent functions. Reductions in the production of naive T and B-cells, reduced cytolytic killing capacity, accumulation of larger numbers of malignant cells, enhanced inflammatory responses, etc., in the aged suggest that apoptosis is dysregulated. Changes in nutritional status can also alter apoptosis. A short period of zinc deficiency (ZD) in young adult mice greatly accelerated apoptosis among pre-B and pre-T cells by 50% to 300% providing a mechanistic explanation for the lymphopenia and thymic atrophy long associated with this and other nutritional deficiencies. Since apoptosis has been shown to be altered by aging and nutritional status, it seemed important to determine how ZD affected these processes in the aged mouse. It was quickly discovered that the pre-B cells were reduced by 80% in the 28 month aged mouse making further studies problematic. In marked contrast to suboptimal zinc, caloric restriction (CR) which when initiated in younger mice delayed the onset of autoimmunity and immunosenescence. CR appeared to also slow the aging of mitochondria and, thereby, reduced the release of reactive oxygen species that damage cells. Thus, it is probable that CR also helped maintain the integrity of mitochondria and apoptotic processes as mice aged. Though CR is not a very practical nutritional model for humans, the outcome of these studies reinforce the potential value of anti-oxidants in our diets. In contrast to their normal nutritional role some nutrients especially small amounts of free metals can induce apoptosis. There is considerable zinc in neurons. As will be discussed, a number of investigators think that this zinc is released during Alzheimer's, Parkinsons's, or brain injury and accelerates apoptosis in surrounding tissues causing greater damage. Data are discussed that indicate nanomoles of free zinc is, indeed, a potent inducer of apoptosis in a variety of tissues. In sum, there is no doubt that nutritional status as well as individual nutrients can modulate apoptosis and that their impact on cell death may become greater in the aged.

Possible roles for zinc in destruction of Trypanosoma cruzi by toxic oxygen metabolites produced by mononuclear phagocytes.

The effects of a single nutrient deficiency on immune function is now most extensively characterized using the dietary zinc deficient murine model. Deficiencies in zinc have rapid adverse effects on host defenses of humans and rodents. This impaired defense seems to be, in part, the result of a reduction in number of lymphocytes available for surveillance since residual lymphocytes are able to carry out many normal functions. In vitro, the lymphocytes were able to proliferate at a normal rate as well as produce antibodies or interleukin 2 in response to mitogens or antigens even when cultured in autologous serum to reduce the possibility of restoration of zinc deficient functions. Conversely, mononuclear phagocytes (MNP) from deficient mice had a significantly reduced capacity to associate with and kill the parasite Trypanosoma cruzi (T. cruzi) which causes Chaga's disease. Moreover, indicating the specificity of the deficient function, a short incubation of ZnCl2 but not other metals completely restored the capacity of MNP from deficient mice to take up and kill T. cruzi. Dependency on H2O2 production by the MNP's oxygen burst for killing of T. cruzi.suggested that MNP from zinc deficient mice might produce smaller amounts of H2O2. The possibility that zinc might play an integral role in the oxygen burst seemed evident from the ability of zinc to quickly restore the killing capacity of MNP from the zinc deficient mice. Further, the renewed interest in the role of metals in the production of highly reactive oxidants in biological systems prompted a literature search to identify enzymes and/or reactions known to be involved in the generation of oxygen radicals or toxic oxygen metabolites that might be zinc dependent. The literature review provided herein indicates many possible roles for zinc in the generation of toxic oxygen species. The data indicated that normal levels of H2O2 are produced by MNP from zinc deficient mice. The amount of H2O2/mg macrophage protein is normal in response to phorbol or opsonized zymosan but reduced in response to direct stimulation by T. cruzi. However, the reduced H2O2 production by T. cruzi-stimulated zinc deficient MNP was due to reduced stimulation as a result of fewer T. cruzi associated with the MNP. Thus, H2O2 levels/parasite were the same as zinc adequate controls. Yet, this does not preclude the possibility that reduced killing of T. cruzi by MNP from zinc deficient mice may be due to a function for zinc in the actual killing process or in the production of some other agent important in the killing of T. cruzi.

Zinc pyrithione induces apoptosis and increases expression of Bim.

We demonstrate herein that zinc pyrithione can induce apoptosis at nanomolar concentrations. Zinc pyrithione was a potent inducer of cell death causing greater than 40-60% apoptosis among murine thymocytes, murine splenic lymphocytes and human Ramos B and human Jurkat T cells. Conversely, the addition of a zinc chelator protected thymocytes against zinc pyrithione induced apoptosis indicating these responses were specific for zinc. Zinc-induced apoptosis was dependent on transcription and translation which suggested possible regulation by a proapoptotic protein. Indeed, zinc induced a 1.9 and 3.4 fold increase respectively in expression of the BimEL and BimL isoforms and also stimulated production of the most potent isoform, BimS. This increase in Bim isoform expression was dependent on transcription being blocked by treatment with actinomycin D. Overexpression of Bcl-2 or Bcl-xL provided substantial protection of Ramos B and Jurkat T cells against zinc-induced apoptosis. Zinc also activated the caspase cascade demonstrated by cleavage of caspase 9. Addition of specific inhibitors for caspase 9 and caspase 3 also blocked zinc-induced apoptosis. The data herein adds to the growing evidence that free or unbound zinc could be harmful to cells of the immune system.

The role of corticosterone in the loss in immune function in the zinc-deficient A/J mouse.

Previous studies from this laboratory have shown that zinc deficiency causes rapid atrophy of the thymus with subsequent loss of T-cell helper function in the young adult A/J mouse. The purpose of this investigation was to determine if zinc deficiency constituted a chronic stress on the mouse leading to the elevation of glucocorticoid levels which is known to destroy thymic lymphocytes. The results of these experiments indicate that zinc-deficient mice indeed have increased levels of plasma corticosterone (115 mug/100 ml plasma) compared to mice fed zinc-adequate diets (40 mug/100 ml plasma). A significant reduction in T-cell helper function, which occurred 4 days after this rise in steroid concentration, suggests that corticosterone may contribute to the loss in immunity; however, about half of the total loss in T-cell helper function occurred prior to the increase in plasma corticosterone and was due to other factors associated with the lowered zinc levels.

Suppression of the antigenic response of murine bone marrow B cells by physiological concentrations of glucocorticoids.

Data presented here indicate that the immature B cells of murine bone marrow (BM) may be as sensitive to glucocorticoids (GC) as are immature thymocytes, since physiological levels of the steroids significantly inhibited the response of these cells to trinitrophenylated lipopolysaccharide (TNP-LPS) in short-term culture. The in vitro response of B cells of the marrow to TNP-LPS was reduced more than 50% by concentrations of corticosterone and cortisol analogous to that found in plasma during stress and trauma. The more potent synthetic GC, dexamethasone (DX), caused a 50-80% decrease in plaque-producing cells at concentrations of 10(-6) and 10(-8) M. The same pattern of inhibition was noticed regardless of whether DX was added 24 hr prior or up to 48 hr after addition of antigen to culture. However, no inhibition in the response of B cells was noted when DX was added 72-96 hr after stimulation of the cultures. These effects were found to be specific for GC since neither testosterone nor progesterone at physiological concentrations inhibited the response, while the glucocorticoid receptor antagonist RU 38486 provided protection. A greater than 80% reduction in the proportion of B cells present in the DX-treated cultures was noted after 5 days, corresponding to the 80% inhibition of plaque-forming cell production observed at that time. This reduction in B cells was rapid since almost 40% of the B220+ cells were depleted within 12 hr of DX addition. These data indicate that physiological levels of GC can readily inhibit the capacity of BM to respond to antigen by depleting the cultures of immature B cells.

Methionine-enkephalin alteration of mitogenic and mixed lymphocyte culture responses in zinc-deficient mice.

The effects of methionine-enkephalin (Met-Enk) on mitogenic and mixed lymphocyte culture (MLC) proliferation of splenocytes from Zn-deficient, restricted and control mice were evaluated. The data from this experiment show that Met-Enk can suppress the responses of splenocyte from the 3 groups to concanavalin A (Con A), but less inhibition was observed in the Zn-deficient group. Met-Enk can also enhance the responses to pokeweed mitogen (PWM) and decrease the response to lipopolysaccharide (LPS) in all groups. Alteration of proliferative responses to Con A and PWM were reversible in the presence of naloxone 10 mumol/L indicating that the effect of Met-Enk on cellular proliferation was mediated by the opioid receptor. In the proliferation of MLC, the response of lymphocytes from Zn-deficient mice was increased in the absence of Met-Enk and Met-Enk can suppress this increased response. It is therefore concluded that Met-Enk can modify the pattern of mitogenic responses and the alteration in Con A and MLC responses can be influenced by zinc deficiency.

Flow cytometric analysis of the phenotypic distribution of splenic lymphocytes in zinc-deficient adult mice.

Many immunodeficiency states create an imbalance in the ratio of T cells to B cells or in the subsets of lymphocytes found within these two major classes. A 30-d period of suboptimal intake of zinc often caused a 50% decrease in the total number of splenic lymphocytes in young adult mice. One- and two-color flow cytometric analysis was made of the residual splenocytes of the zinc-deficient mice to determine whether the deficiency had also altered the composition or phenotypic distribution of any of the major subsets of T cells or B cells. Marginally zinc-deficient mice had a normal ratio of T cells to B cells with no notable change in the subsets of lymphocytes making up these two classes. Severely zinc-deficient mice that exhibited a significant degree of parakeratosis demonstrated a 20% increase in the overall ratio of T helper to T suppressor/cytotoxic cells as well as a modest decline in the percentage of B cells (5-8%). The latter decrease was not in the B cells bearing immunoglobulin M or D but in the minor subsets bearing other immunoglobulin isotypes. Thus it seems that, whereas zinc deficiency caused significant reductions in the total numbers of splenic lymphocytes, it caused no significant change in the composition of the splenic lymphocytes except in the most severe cases.

Zinc reversibly inhibits steroid binding to murine glucocorticoid receptor.

Previous work has demonstrated that several transition metals and their anions, including cadmium, arsenite, and selenite, can inhibit glucocorticoid binding to glucocorticoid receptors in vitro. In this study, we demonstrated that in vitro zinc can also inhibit the binding of glucocorticoids to their receptor at relatively modest concentrations (10 to 100 microM). This inhibition was demonstrated in both crude and immunopurified receptor preparations and was reversible following removal of zinc. Inhibition could also be reversed by addition of the reducing agent dithiothreitol (DTT). This suggested that zinc might be acting by interacting with the vicinal dithiols in the steroid binding region of the receptor as previously described for other transition metals and anions. The ability of a biologically important trace metal to block steroid binding suggests a role for zinc in the regulation of glucocorticoid receptor-ligand interactions and may explain the ability of zinc to block glucocorticoid-induced apoptosis.

A reappraisal of the role of zinc in life and death decisions of cells.

There is a great deal of interest in chemicals and biochemicals that can modulate apoptosis. As will be discussed, zinc, an essential trace element, can induce as well as block apoptosis. High concentrations of extracellular zinc (500-1000 microM) have frequently been used to block apoptosis or programmed cell death in a variety of systems. Early investigators provided evidence that this concentration of zinc could block DNA fragmentation that is often associated with apoptosis. Since zinc plays a role in many aspects of cell function, there are probably many sites in a death pathway that zinc could potentially modulate. In the case of glucocorticoid-mediated apoptotic death, new evidence presented herein indicates that high zinc can also block the binding of steroids to the glucocorticoid receptor thereby inhibiting the death signal itself. In this case, zinc probably binds to the vicinal cysteines in the receptor ligand binding site thereby blocking binding of glucocorticoid. Indeed, glucocorticoid-induced apoptosis in thymocytes has become one of the most frequently studied systems and is a focal point of this review. Studies herein will show that unlike zinc other trace-like metals such as nickel, copper, cadmium, and gold do not afford thymocytes protection against the DNA fragmentation induced by glucocorticoid-mediated cell death. Interestingly, in attempting to determine if lower or more physiological concentrations of zinc could provide protection against apoptosis, it was found that 80-200 microM zinc could actually induce death in 40% of CD4+ CD8+ alpha beta TCR10CD3(10) thymocytes. From these experiments one might have been optimistic that zinc could, indeed, be a modulator of cell death. However, this thought has been overshadowed by growing evidence that zinc does not provide long-term protection to so-called surviving cells.

Variations in the cell cycle status of lymphopoietic and myelopoietic cells created by zinc deficiency.

Zinc deficiency causes thymic atrophy and lymphopenia. It was recently shown that zinc deficiency causes sizable losses among the precursor lymphocytes, such that this compartment was depleted 40%-50% in the marrow of young adult mice. However, the myeloid compartments increased substantially both in proportion and absolute number as zinc deficiency advanced. Zinc deficiency caused no change in the cell cycle status of precursor B cells and only modest changes in cycling pro-B cells. Conversely, cells of the myeloid series, especially monocytes, exhibited as much as a 40% increase in the proportion of cells in S and G(2)/M, while myeloid progenitors had an overall 56% increase in cells in the proliferative phase as zinc deficiency advanced. Whether zinc deficiency alters the rate of production of myeloid and lymphopoietic cells or alters the degree of apoptosis or both awaits further study.