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 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.

Apoptosis plays a distinct role in the loss of precursor lymphocytes during zinc deficiency in mice.

Lymphopenia is a characteristic of zinc deficiency, which is associated with massive loss of pre-B and pre-T cells from the primary lymphoid organs of zinc-deficient mice that have elevated serum corticosterone (CS). We examined whether this naturally elevated glucocorticoid level is associated with increased apoptotic loss of pre-T cells in the thymus of A/J and CAF1/J mice. In three experiments, partially atrophied thymuses were removed from 20 marginally zinc-deficient (ZD) young adult mice and cultured for 6 h in parallel with thymocytes prepared from 17 adequately fed mice. Thymocyte immunophenotyping combined with flow cytometric cell cycle analysis was used to identify the degree of apoptotic cell death among thymocytes of the two dietary groups, which were compared in the absence of in vivo phagocytosis. Apoptosis was enhanced 50-300% among pre-T cells (CD4+CD8+) prepared from ZD mice. This resulted in a 38% shrinkage of the thymic pre-T cell compartment, which was associated with an 80% decrease in thymic cell number. Pro-T cells (CD4-CD8-) and mature T cells (CD4+CD8-, CD4-CD8+), which express higher levels of Bcl-2 protein, survived ZD to a greater extent and formed a greater proportion of the remaining thymocyte population in ZD mice. Collectively, these data show that heightened degrees of apoptotic cell death induced in vivo by CS-disrupted thymic T cell lymphopoiesis, identifies the means of disruption of marrow B cell lymphopoiesis and explains the appearance of lymphopenia.

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.

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.

The dynamic link between the integrity of the immune system and zinc status.

The results of more than three decades of work indicate that zinc deficiency rapidly diminishes antibody- and cell-mediated responses in both humans and animals. The moderate deficiencies in zinc noted in sickle cell anemia, renal disease, chronic gastrointestinal disorders and acrodermatitis enteropathica; subjects with human immunodeficiency virus; children with diarrhea; and elderly persons can greatly alter host defense systems, leading to increases in opportunistic infections and mortality rates. Conversely, short periods of zinc supplementation substantially improve immune defense in individuals with these diseases. Mouse models demonstrate that 30 d of suboptimal intake of zinc can lead to 30-80% losses in defense capacity. Collectively, the data clearly demonstrate that immune integrity is tightly linked to zinc status. Lymphopenia and thymic atrophy, which were the early hallmarks of zinc deficiency, are now known to be due to high losses of precursor T and B cells in the bone marrow. This ultimately leads to lymphopenia or a failure to replenish the lymphocytic system. Glucocorticoid-mediated apoptosis induced by zinc deficiency causes down-regulation of lymphopoiesis. Indeed, zinc itself can modulate death processes in precursor lymphocytes. Finally, there is substantial evidence that zinc supplementation may well reduce the impact of many of the aforementioned diseases by preventing the dismantling of the immune system. The latter represents an important area for research.

Variance in the resistance of murine early bone marrow B cells to a deficiency in zinc.

Little is known of the effects of nutritional deficiencies on lymphopoietic processes. Nevertheless, deficiencies in zinc adversely affect immune function causing thymic atrophy and lymphopenia in both humans and animals. Previous studies of the effects of zinc deficiency (ZD) on lymphopoiesis in adult mice indicated that a suboptimal intake of zinc caused a 50% or more depletion of the marrow of developing B cells. Thus, interference in the production of lymphocytes by ZD appeared to be a significant factor in the loss of host defence capacity. In the current study three-colour immunofluorescence phenotyping of early bone marrow B lymphocytes (B220+ immunoglobulin-) using flow cytometry demonstrated that a 27-day period of ZD caused a 50-70% decline in pre-B cells (B220+ CD43- immunoglobulin M (IgM)-) for moderate and severely zinc-deficient mice, respectively. Conversely, early pro-B cells (B220+ CD43+ 6C3-) and late pro-B cells (B220+ CD43+ 6C3+) exhibited little or no change in their distribution within the marrow. Indeed, the greater resistance of pro-B cells resulted in a 50% increase in the proportion of this subset within the B-cell compartment of the marrow as the deficiency in zinc advanced. Collectively, the data indicate that the B-cell compartment of the marrow is substantially altered by ZD and the stage specific sensitivity noted among early B cells may be related to chronically elevated levels of glucocorticoids present during ZD or other parameters that affect their survival and resistance to apoptosis.

The response of pulmonary vascular endothelial cells to monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro and in vivo.

Monocrotaline pyrrole (MCTP) causes pulmonary vascular endothelial cell (EC) injury followed by progressive pulmonary vascular leak in vivo and the inhibition of EC proliferation in vitro. It was hypothesized that MCTP inhibits cell proliferation in vitro by interfering with cell cycle progression in a cycle phase-specific manner. Furthermore, it was proposed that early alterations in MCTP-induced lung injury leading to hypertension were associated with a similar inhibition of EC proliferation. Subconfluent cultures of bovine pulmonary artery endothelial cells (BECs) were synchronized with aphidicolin (APH), a reversible G1-S phase inhibitor. Upon removal of APH, BECs were exposed to MCTP (5 micrograms/ml) or its vehicle for a 4-h interval corresponding to either the G1-S, S-G2, or G2 through mitosis (M) phases of the cell cycle. Fluorescence-activated cell sorting (FACS) was used to identify MCTP-induced changes in cell cycle progression in BECs, and the transit of S phase cells through the cycle was characterized through the incorporation of bromodeoxyuridine (BrdU). Synchronized BECs exposed to MCTP between mid-S-G2 or G2 through M were briefly delayed in G2-M at 12 h but underwent cell division by 24 h. By contrast, BECs treated with MCTP immediately after release from APH block became arrested in G2-M at 24 h and showed evidence of continued DNA synthesis and hypertetraploidy, but they did not divide. In vivo, MCTP (3.5 mg/kg i.v.) administration caused an increase in arterial EC BrdU incorporation between Days 3 and 7, but no increase in EC density. During this same interval, pulmonary vascular permeability increased and persisted. In summary, MCTP inhibits cell proliferation in a cell cycle phase-dependent manner in vitro. The results suggest that a similar mechanism could occur in vivo and may be associated with delayed EC repair, a process that could contribute to persistent pulmonary vascular leak.

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.

Preferential induction of apoptosis in mouse CD4+CD8+ alpha beta TCRloCD3 epsilon lo thymocytes by zinc.

High concentrations of zinc salts (500 microM and greater) are known to inhibit apoptosis in a variety of systems. However, closer examination of dose effects revealed that lower concentrations of zinc (80-200 microM) could induce apoptosis in approximately 30-40% of mouse thymocytes following 8 h incubation. The ability of zinc to cause thymocyte apoptosis was detected flow-cytometrically by reduction in propidium iodide DNA fluorescence and forward scatter, both quantitative indicators of apoptotic death. Zinc induced both internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis as determined by gel electrophoresis and electron microscopy, respectively. In addition, transcriptional and translational inhibitors prevented zinc-induced apoptosis, indicating a requirement for de novo mRNA and protein synthesis, another characteristic of apoptotic death. Fluorescent immunophenotype-specific apoptotic analysis indicated that zinc-induced apoptosis occurred primarily in the less mature CD4+CD8+ alpha beta TCRloCD3 epsilon lo thymocyte subset, with lower amounts of death occurring in the other subsets. This lineage specificity was shared with glucocorticoid-induced apoptosis. Taken together, these results indicate that zinc induces true apopotitic death in mouse thymocytes and suggests a role for zinc in the regulation of apoptosis.

Depletion of cells of the B lineage in the bone marrow of zinc-deficient mice.

Though lymphopenia is often noted in malnourished humans and rodents, little is known about the effects of suboptimal nutriture on lymphopoietic processes. Focusing primarily on cells of the B lineage in the marrow of young adult mice, a moderate degree of zinc deficiency (MZD) caused a 43% decline in the proportion of nucleated cells bearing B220 with a 91% decline noted among more severely zinc deficient mice (SZD). Early B cells (B220+Ig-) were highly sensitive to the deficiency, being barely detectable in SZD mice and reduced by almost 60% in MZD mice. Immature B cells (B220+IgM+IgD-) were similarly affected, declining 35% to 80% depending on the degree of the deficiency. In MZD mice, mature B cells (IgM+IgD+) exhibited moderate losses, being somewhat resistant. A more profound loss in this population was noted for SZD mice. Flow cytometric (FACS) scatter profiles indicated that zinc deficiency caused a sharp decline in the proportion of small nucleated cells which in the marrow are thought to contain a high proportion of developing lymphoid cells. There was a concomitant increase in large granular cells that paralleled a substantial increase in the proportion of nucleated cells bearing Mac-1 for both MZD and SZD mice. Given the dramatic depletion of cells of the B lineage in the marrow created by a deficiency in zinc, it is probable that disruptions in lymphopoietic processes in the marrow play a key role in the resulting lymphopenia observed in many types of malnutrition.

Possible roles for glucocorticoids and apoptosis in the suppression of lymphopoiesis during zinc deficiency: a review.

Thymic atrophy and lymphopenia are immunological hallmarks of many forms of malnutrition including deficiencies in zinc. Extreme thymic atrophy (70-80%) along with a 50% loss of splenocytes in mice maintained on a zinc deficient diet (ZD) for 30 days suggested that the deficiency might be altering lymphopoiesis or the production of new lymphocytes by the bone marrow. As shown herein, mice who were marginally zinc deficient being 72-75% the body weight of adequately fed controls, exhibited a 50% decline in pre B-cells and a 25% decline in immature B-cells. The mature B-cells of the marrow appeared fairly resistant to effects of suboptimal zinc intake. Interesting, this pattern was similar to results obtained by treating bone marrow cells with levels of glucocorticoids analogous to those found in nutritionally deficient rodents. Furthermore, these same concentrations of steroids were shown to induce significant levels of apoptosis or cell death among pre and immature B-cells which accounted for their declining numbers subsequent to exposure to glucocorticoid. In order to better ascertain the potential role of glucocorticoids generated during zinc deficiency on lymphopoietic processes, adrenalectomies were performed in an attempt to remove glucocorticoids from the equation. Subsequently, adrenalectomized and sham operated mice were placed on a ZD or zinc adequate diet (ZA). Levels of steroids at the time of sacrifice were elevated six fold in non-adrenalectomized ZD mice compared to ZD adrenalectomized mice. Removal of the adrenal gland protected the thymus of ZD mice from atrophy and also provided substantial protection of lymphopoietic processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of apoptotic events in pure and heterogeneous populations of cells using the flow cytometer.

The rapid and highly quantitative nature of flow cytometric cell cycle analysis for determining the proportion of apoptotic cells in a population makes it the method of choice for a variety of studies requiring quantitative information about cell death. Furthermore, by employing multiparameter analysis including phenotypic labeling, FACS makes it possible to study apoptosis in specific subsets of cells within a heterogeneous population. Live sorting of cells in the apoptotic region offers the possibility of studying the effects of this form of cell death on key biochemical functions of the cell. Nonetheless, further modification of the fixing-staining methods presented here will be needed to make FACS useful for analysis of apoptosis in human cells.

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.

Apoptosis accompanies a change in the phenotypic distribution and functional capacity of murine bone marrow B-cells chronically exposed to prednisolone.

Prednisolone (PD) is commonly used for the treatment of inflammation accompanying diseases such as arthritis, allergy, asthma, and autoimmunity. While it is well documented that PD induces apoptosis in immature T-cells of the mouse, the effects of PD on development of immature B-cells in normal bone marrow (BM) was not known. An implantation system was developed which chronically delivered PD at a rate of a few nanograms per milliliter of plasma to mice. Ten days of exposure to such levels of PD caused splenic and thymic atrophy, which was accompanied by a 50% decrease in the numbers of circulating lymphocytes. Flow cytometric analysis (FACS) of the effects of PD on the BM revealed a threefold decrease in the proportion of B220+IgM- pre-B-cells and immature IgM+IgD- B-cells. However, the mature IgM+IgD+ cells were reasonably resistant to the effects of PD. A 25% decrease in small nucleated cells presumed to be part of the lymphocyte compartment was also noted from the scatter profiles of the marrow of PD-treated mice. These marked changes in BM composition were also accompanied by significant reductions in capacity of the BM to respond to trinitrophenylated-lipopolysaccharide (TNP-LPS) after exposure to PD either in vivo or in vitro. Studies to ascertain whether apoptosis played a role in the decline in the number of developing B-cells of marrow exposed to PD were performed in vitro in order to reduce the possibility of phagocytosis of apoptotic cells. A recent modification of FACS cell cycle analysis, which is highly quantitative and allows rapid analysis of heterogeneous tissues such as the marrow, was used to detect the apoptotic cells. After 16 hr of culture in 10(-7) M PD, approximately 40% of IgM+ and B220+ cells of BM resided to the left of G0/G1 in a region associated with apoptotic cells previously termed the A0 or "hypodiploid" region. Thus, these data indicate that chronic exposure to low levels of PD significantly altered the B-cell compartment of the murine bone marrow both in vivo and in vitro, potentially inducing apoptosis in these cells.

Chronic elevation of plasma corticosterone causes reductions in the number of cycling cells of the B lineage in murine bone marrow and induces apoptosis.

Steroid-containing implants were used to ascertain the effects of chronic elevation of physiological levels of plasma corticosterone (CS) (30-100 micrograms/dl) on lymphopoietic processes in the bone marrow of the mouse. Phenotypic analysis of bone marrow B-lineage lymphocytes using flow cytometry (FACS) indicated a 50% decrease in bone marrow Ig+ cells, and a 70-80% decrease in B220+ cells had occurred 3 days after exposure to steroid. By day 5, the B220+ Ig- precursor B cells in the marrow of mice exposed to CS were nearly depleted, with many of the remaining B cells being B220bright IgM+IgDbright. To determine if the depletion of B cells was due to disruption in cell cycling and/or induction of apoptosis, phenotype-gated FACS cell cycle analysis was utilized. The proportion of B220+ cells in the S phase of the cell cycle declined 75% after 24 hr exposure to CS. A few hours after CS implantation, the appearance of a small but distinct population of B220+ and IgM+ cells in the 'hypodiploid' region of the cell cycle was also noted, which was previously termed the Ao region and corresponded to cells undergoing apoptosis. Thus, the chronic presence of modestly elevated levels of plasma CS analogous to that produced during malnutrition, stress and trauma caused rapid depletion of developing B-lineage cells in the marrow by reducing the number of cycling precursor B cells and inducing apoptosis.

Glucocorticoids and irradiation-induced apoptosis in normal murine bone marrow B-lineage lymphocytes as determined by flow cytometry.

A substantial proportion of murine bone marrow B220+ and IgM+ cells were induced to undergo apoptosis when exposed to glucocorticoids or ionizing radiation in vitro. Two-colour flow cytometric analysis of the cell cycle indicated that a distinct subpopulation of cells formed to the left of G0/G1 in the hypodiploid or Ao region previously shown to contain apoptotic cells with fragmented DNA. Indeed, 45-65% of all B220+ or IgM+ cells of the marrow were found in this apoptotic region 12 hr after treatment with dexamethasone (Dex) or exposure to 500 rads of irradiation. Zinc sulphate, a frequently cited inhibitor of apoptosis, prevented accumulation of cells exposed to glucocorticoids or ionizing radiation in the Ao region as did the glucocorticoid receptor antagonist RU 38486. Although Dex was more potent, corticosterone and cortisol also induced significant degrees of apoptosis in B220+ and IgM+ marrow cells at physiological concentrations. These results demonstrate that freshly isolated B-lineage cells of the murine bone marrow readily undergo apoptosis upon exposure to glucocorticoids and ionizing radiation and suggest that apoptosis may play a role in the regulation of lymphopoiesis. The data also show the value of flow cytometry to the study of apoptosis in subsets of cells within a heterogenous population such as the bone marrow which heretofore was exceedingly difficult to evaluate.

Functional capacity of the residual lymphocytes from zinc-deficient adult mice.

Zn deficiency has been shown to reduce host defence drastically. It was of interest to determine the capacity of the residual lymphocytes from Zn-deficient mice to proliferate and produce lymphokines in response to stimulation since there are many Zn-dependent metalloenzymes that might be altered by the deficiency. To address this question, young adult A/J mice were provided Zn-deficient or Zn-adequate diets or restricted amounts of a Zn-adequate diet for 30 d. Splenocytes from moderately or severely Zn-deficient adult A/J mice gave normal proliferative responses and generated adequate interleukin II (IL-2) activity when stimulated with the mitogen Concanavalin A. However, splenocytes from deficient mice exhibited a higher degree of proliferation (about 150%) and production of IL-2 in response to foreign target cells compared with T-cells prepared from mice provided a Zn-adequate diet. B-cells from deficient mice stimulated in vivo with sheep erythrocytes produced fewer total numbers of plaque-forming cells (PFC) per spleen. Nevertheless, the proportion or number of PFC/10(6) viable splenocytes and the amounts of IgM and IgG antibody produced per PFC were equivalent to those of adequately-fed and restricted-fed controls. The previously described responses were not significantly affected by whether the level of Zn in the culture medium was adequate or limiting. Based on these tests it appeared that the residual splenic lymphocytes of Zn-deficient mice were able to carry out many fundamental immune processes.