Louis E. King

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.

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

Abstract. 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 Go/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.

Alterations in cell membrane properties caused by perfluorinated compounds.

The recent detection of perfluorinated compounds (PFCs) in wildlife from even remote locations has spurred interest in the environmental occurrence and effects of these chemicals. While the global distribution of PFCs is increasingly understood, there is still little information available on their effects on wildlife. The amphiphillic nature of PFCs suggests that their effects could be primarily on cell membranes. In this study we measured the effects of PFCs on membrane fluidity and mitochondrial membrane potential using flow cytometry and effects on membrane permeability using cell bioassay procedures (H4IIE, MCF-7, PLHC-1). Of the PFCs tested, only perfluorooctane sulfonic acid (PFOS) increased the permeability of cell membranes to the hydrophobic ligands used. Three PFCs were tested in the membrane fluidity assay: PFOS, perfluorohexane sulfonic acid (PFHS), and perfluorobutane sulfonic acid (PFBS). PFOS increased membrane fluidity in fish leukocytes in a dose-dependent fashion, while PFHS and PFBS had no effect in the concentration range tested. The lowest effective concentrations for the membrane fluidity effects of PFOS were 5-15 mg/l. Effects on mitochondrial membrane potential occurred in the same concentration range as effects on membrane fluidity. This suggests that PFOS effects membrane properties at concentrations below those associated with other adverse effects.

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.

A role for leptin in sustaining lymphopoiesis and myelopoiesis.

Although leptin is known for its regulation of food intake, it has many emerging roles in immune function. To better define the role of leptin in hematopoietic processes, a leptin-deficient obese mouse (ob/ob) and C57BL/6 lean wild-type controls were compared. Despite their large size and consumption of substantial amounts of nutrients, the ob/ob mice had only 60% as many nucleated cells in their marrow as controls. The greatest impact of leptin deficiency was on the B cell compartment that had 70% fewer cells, reducing the absolute number of pre-B and immature B cells to 21% and 12% of normal, respectively, and indicating a significant reduction in lymphopoiesis in ob/ob mice. Whereas the proportion of myeloids remained nearly normal in the obese mice, they also exhibited a reduction of 40% and 25%, respectively, in absolute numbers of granulocytes and monocytes. Seven days of provision of recombinant leptin promoted substantial lymphopoiesis, increasing the numbers of B cells in the marrow of the obese mice twofold, while doubling and tripling, respectively, the numbers of pre-B and immature B cells. Twelve days of supplementation brought these subpopulations to near-normal proportions. Leptin treatment also facilitated myelopoiesis such that the marrow of the obese mice contained normal numbers of monocytes and granulocytes after 7 days. Taken together, the data support an important role for leptin in sustaining lymphopoiesis and myelopoiesis.

Versatility of merocyanine 540 for the flow cytometric detection of apoptosis in human and murine cells

Since apoptosis plays many roles in development, immune function, and disease, there is an ongoing need to identify inexpensive and reliable fluorochromes for the quantitation of apoptosis. Merocyanine 540 (MC540) binds to the outer membrane of cells and readily fluoresces in the highly disordered membranes of apoptotic cells making them readily detectable by flow cytometry. Protocols for the effective labeling and gating of MC540br apoptotic cells are provided. For example, MC540br cells from dexamethasone (Dex) treated thymocytes were found to be equivalent in proportion to apoptotic cells noted in the propidium iodide (PI) stained and annexin-V stained populations. Sorting of the MC540br cells followed by counterstaining with PI demonstrated that these cells resided in the low DNA fluorescent or sub-G1 region and were small in size based on light scatter. Dexamethasone, etoposide, irradiation, and a calcium ionophore were used to induce cell death with equivalent numbers of apoptotic cells obtained with MC540 and PI. Moreover, apoptotic human bone marrow (BM) B cells, neutrophils, Jurkat T cells, and testicular cells could readily be identified with MC540. The latter is particularly noteworthy since some of the standard methods for identifying cell death have not worked well with human cells. The versatility of this dye is such that it was also possible to phenotypically label cells stained with MC540 to analyze apoptosis in heterogenous populations of cells. Finally, the rate of detection of apoptotic cells after treatment of thymocytes with dexamethasone at 2, 4, 6, and 8 h with MC540 was shown to be equivalent to PI and annexin-V. Taken together, the data demonstrate that when proper precautions are taken, MC540 is a reliable, versatile, and inexpensive fluorochrome that can be used to identify apoptotic cells of human or murine origin even in heterogenous populations that require multicolor labeling.

Natural glucocorticoids induce expansion of all developmental stages of murine bone marrow granulocytes without inhibiting function

Natural glucocorticoids (Gc) produced during stress have profound effects on the immune system. It is well known that Gc induce apoptosis in precursor T and B cells, markedly altering lymphopoiesis. However, it has been noted that marrow myeloid cells expanded both in proportion and absolute numbers in the mouse after Gc exposure. Mice were implanted with a corticosterone (CS) tablet that increased serum Gc and caused atrophied thymuses, both classic signs of activation of the stress axis. Blood neutrophil counts were elevated (4.8×), whereas lymphocyte counts declined. Flow cytometric analysis of the marrow revealed that the phenotypic distribution of the various major classes of cells was shifted by Gc exposure. As expected, marrow lymphocyte numbers declined >40% after 3 days of exposure to Gc. Conversely, in the myeloid compartment, both monocytes and granulocytes increased in number by >40%. Further, all granulocyte developmental stages showed large increases in both total number and percentage of cells. To investigate the functional capacity of mature granulocytes from Gc-treated mice, an improved granulocyte isolation method was developed. Gc exposure had little effect on the ability of granulocytes to produce superoxide or undergo chemotaxis or phagocytose bacteria. These results indicate that Gc treatment shifts bone marrow composition and provides evidence that granulocytes and their progenitors are selectively preserved under stressful conditions without losing function.

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)

Chronic zinc deficiency in mice disrupted T cell lymphopoiesis and erythropoiesis while B cell lymphopoiesis and myelopoiesis were maintained.

Objective: The purpose of this study was to determine the impact of chronic zinc deficiency (ChrZD) on T and B cell lymphopoiesis, myelopoiesis and erythropoiesis in mice. Methods: Young adult mice were fed a zinc adequate (ZA) or ChrZD synthetic diet for 34, 45, and 50 days. The cellular composition of the thymus and marrow were determined to assess the impact of ChrZD on lymphopoietic and hematopoietic processes using flow cytometry. Body weights, serum zinc and corticosterone (Cs) were monitored. Results: For ChrZD mice growth was reduced 10% and serum zinc declined 15% by d 34 compared to ZA mice. By d 50 a 25% decrease in growth and 70% depression in serum zinc was noted though there was never any significant reduction in diet intake. Corticosterone rose 2.5 fold by d 34 and remained elevated in ChrZD mice indicating induction of the stress axis. At d 34 the thymus of ChrZD mice was normal but by d 50 there was a 50% cell loss and a 10% reduction in the proportion of Pre-T cells. Most importantly there was a 60% increase in Pre-T cells undergoing apoptosis in ChrZD mice. Pro-T, T helper, and T cytolytic populations were more resistant to ChrZD. Bone marrow cellularity and granulocyte, monocyte, and lymphocyte compartments remained unchanged in ChrZD mice. However, the erythroid compartment was reduced by 35% at d 50. Conclusions: The thymus was the most sensitive primary tissue to ChrZD. By d 50 it had atrophied by 36% with significant loss of Pre-T cells via apoptosis such that T-cell lymhopoiesis was disrupted. Significant reductions were also noted in the erythropoietic population by d 50. Conversely the marrow maintained myelopoiesis and B cell lymphopoiesis for the 50 d period indicating greater ability to survive a chronic zinc deficiency and exposure to Cs. The anemia and T cell lymphopenia associated with ChrZD in both rodents and humans may result from a greater sensitivity of their precursor cells to zinc deficiency and elevated Cs.

Glucocorticoid-induced apoptosis in early B cells from human bone marrow.

The sensitivity of normal human lymphoid precursor cells to glucocorticoid-induced apoptosis is a subject of controversy. The in vitro response of cells of the B lineage (CD19+) from the marrow of 22 adult subjects to glucocorticoids was evaluated herein using both natural steroids and dexamethasone (Dex). When exposed to 1 μM Dex, 32% of the subjects exhibited high losses of CD19+ B cells in the range of 45%. The remaining subjects exhibited more modest losses in CD19+ cells of 26%–40%. Surprisingly, cortisol, a naturally produced glucocorticoid, produced B lineage losses nearly equivalent to Dex, which reached maximum by 12 hr. It was subsequently noted that the variances in losses of CD19+ cells among the subjects correlated closely with the proportion of early CD10+ CD19+ B cells present in the initial population. The latter cells exhibited a high degree of sensitivity to glucocorticoids, with losses of 60%–80% noted. Mature B cells bearing IgD, on the other hand, were fairly resistant to glucocorticoids. Merocyanine 540, a membrane dye that fluoresces in the disordered membrane of apoptotic cells, confirmed that early or progenitor B cells in human bone marrow were indeed undergoing glucocorticoid-induced apoptosis, which could be blocked by the glucocorticoid antagonist RU38486. These data provide evidence that human marrow B cells, especially early B-cell progenitors, are quite sensitive to glucocorticoids and readily undergo apoptosis within a few hours of exposure to the steroids.