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Department of Biochemistry and Molecular Biology and Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1319
3To whom correspondence should be addressed. E-mail: fraker{at}msu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bone marrow • hematopoiesis • lymphopoiesis • myelopoiesis • zinc deficiency • erythropoiesis • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). ZD and PEM lead to compromised immunity with increased susceptibility to disease. This also accompanies many chronic diseases such as cancer, acquired immunodeficiency syndrome, renal disease, sickle cell anemia or chronic gastrointestinal disorders, and can increase mortality among these patients (1
–4
). Inanition accompanies more severe ZD states, making PEM a potential secondary problem in ZD. Thus, it is important to understand why suboptimal trace metal and protein energy nutriture has such a profound effect on the immune system.
The best-characterized nutritional-immunological paradigm is found in zinc deficiency in mice (1
,2
). A plethora of papers describe the effects of zinc deficiency on the immune system of ZD mice (2
). These include rapid thymic atrophy, accelerated lymphopenia with concomitant loss of antibody and cell mediated responses, substantial reduction in the number of peripheral blood lymphocytes and splenocytes without alteration of the phenotypic distribution of T and B cells of the spleen (5
) and correlation of splenic lymphopenia with reduced antigenic response, although residual splenocytes show normal mitogen response and antibody and interleukin II production (6
). The same fundamental changes in immune status have been noted in humans and primates (1
,2
). This suggested to us that suboptimal zinc nutriture was probably altering the capacity of marrow and thymus to produce adequate numbers of new lymphocytes.
For these reasons, we studied the effects of ZD on marrow B cell lymphopoiesis and found that ZD profoundly reduced B-cell lymphopoiesis in the marrow where 40–70% losses among pre-B cells occurred after 30 d (7
,8
). Substantial losses were also noted among immature or immunoglobulin (Ig)M-bearing B-cells, whereas the earliest B-cell progenitors, the pro-B cells, were somewhat resistant to the deficiency (7
,8
). In the thymus, a large portion of the CD4+CD8+ pre-T cells were lost, with far better survival noted for pro-T cells and mature helper and cytolytic T-cells (9
). Some of the pro-B and pro-T cells, as well as the mature T-cells and B-cells, express higher levels of the antiapoptoic protein Bcl-2, which would account for the resistance to increased apoptosis that we found in ZD mice (10
,11
). These same ZD mice showed substantial losses among pre-B and pre-T cells, which express little Bcl-2 (9
,12
). In sum, ZD rapidly depleted the marrow and thymus of precursor and immature lymphocytes, providing clear evidence that the deficiency altered lymphopoiesis.
As data concerning marrow lymphopoiesis were gathered, initial studies of the myeloid compartment using CD11b indicated a substantial increase in these cells. This was also observed in flow cytometric scatter profiles of marrow from ZD mice (7
). Clearly, ZD was affecting a variety of hematopoietic compartments.
For the study of hematopoiesis, we utilized a marker system that employed CD31 (ER-MP12) and Ly-6C (ER-MP20) to subdivide nucleated marrow cells into several compartments, i.e., cells of the erythrocyte lineage, lymphocyte lineage, granulocytic lineage, monocytic lineage and committed progenitor cells of various lineages (13
,14
). The initial characterization by these investigators utilized flow cytometric cell sorting, cell staining and morphology to identify the compartments. The fidelity of these markers was further verified utilizing population-specific antibodies that included TER 119 to identify cells of the erythroid lineage, B220+ or CD45R to affirm the integrity of lymphocyte compartment, Gr-1 or Ly-6G to identify cells of the granulocytic lineage and CD11b as an additional marker for identification of cells of the myeloid lineage (15
–18
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Six-wk-old A/J mice weighing 17.1 ± 0.4g (Jackson Labs, Bar Harbor, ME) were distributed into three dietary groups. The zinc-adequate group (ZA) consumed diet containing 28 mg Zn/kg and the zinc-deficient group (ZD) consumed diet containing <1.0 mg Zn/kg ad libitum. A third group was food-restricted (RZA) and consumed the ZA diet in the amount consumed by the ZD group the previous day. This is a control for inanition and reduced energy intake that accompany zinc deficiency (19
). Preparation of the biotin-fortified egg white diet containing either AIN-93 or AIN-76 mineral and vitamin mixtures (Bio-Serve, Frenchtown, NJ) and the feeding protocol were described previously in detail (6
–8
). Hanging steel cages, acid-washed water bottles and acidified Milli-Q water (Millipore, Bedford, MA) containing <0.7 µmol Zn/L were used as described previously (6
). After 34 d, ZD mice were divided into moderately zinc-deficient (MZD) or severely zinc-deficient (SZD) mice. MZD mice weighed 73–75% of ZA mice and exhibited modest parakeratosis of the eyes and tails. The latter is a change in the skin characteristic of ZD in both rodents and humans (6
–8
). SZD mice weighed 68–71% of ZA mice and bore more extensive parakeratosis (6
–8
). Blood was collected from the subclavian artery of anesthetized mice and processed individually for serum zinc analysis. The zinc concentrations of the diets and sera were determined by atomic absorption as previously described (Varian Spectra AA 20 Plus, Springvale, CA) (6
,19
). All procedures used herein were approved by the Michigan State University Laboratory Animal Research Committee.
Preparation and immunophenotyping of mouse bone marrow cell suspensions.
Bone marrow from 6–8 mice per dietary group was flushed from femora and tibiae and gently aspirated into a single cell suspension in Hank’s balanced salt solution (HBSS) containing 10 mmol/L HEPES and 4% heat inactivated fetal bovine serum (FBS), pH 7.4. RBC were removed by lysis as previously described (7
,8
). Marrow viability was 92–95% as determined by trypan blue exclusion at the completion of isolation. Marrow was divided into aliquots (1 x 106 cells/sample) for phenotyping. All analyses were performed on individual mice. Samples for B-cell phenotyping were stained as described below before fixation. Samples to be stained with CD31 and Ly-6C were fixed with 1.25% formaldehyde in PBS (pH 7.4) for 40 min, washed and stored at 4°C for up to 2 d in HBSS containing 23 mmol/L sodium azide, 10 mmol/L HEPES and 2% FBS. Comparison of postfixation phenotyping with prefixation phenotyping for CD31 and Ly-6C in the combinations described below showed no alteration in phenotypic results (data not shown). This allowed equivalent phenotyping conditions for the large number of samples in this group.
Five hematopoietic populations were identified using three-color immunophenotyping, which included biotinyl labeled anti-CD31 (ER-MP12)and fluorescein isothiocyanate (FITC)-labeled anti-Ly-6C (ER-MP20). Major lineages of cells in the marrow populations express varying amounts of CD31 and Ly-6C (13
,14
). The populations identified were erythroid lineage cells (CD31-Ly-6C-), lymphocytes (CD31+Ly-6C-), granulocytes (CD31-Ly-6C+), heterogeneous monocytes (CD31-Ly-6C++) and committed progenitor cells (CD31+Ly-6C+). Phycoerythrin (PE)-conjugated antibodies were used individually to confirm, as a third color, the fidelity of the lineages in the marrow identified using CD31 and Ly-6C. The PE-antibodies used were anti-TER 119 for erythroid lineage cells (15
), CD45R or B220 for B-cells (8
,16
,20
), Ly-6G (Gr-1) for granulocytes (17
) and CD11b for cells of the myeloid lineage (18
). Double gating via scatter and PE histogram was used to demonstrate the accuracy of CD31/Ly-6C populations by region.
For the above three-color phenotyping, labeling of the bone marrow was performed using biotinyl-CD31, one of the PE-labeled antibodies and FITC-Ly-6C at 4°C using 30-min incubations. All samples were first incubated with 10 µg of rat Ig (Sigma, St. Louis, MO) to block nonspecific binding. In the second step, biotinyl-CD31 plus one of the conjugated PE antibodies were added to appropriate samples. In the third step, FITC-Ly-6C and Strep Av-Red 670 were added to appropriate samples. After each step, the cells were washed in label buffer (HBSS containing 23 mmol/L sodium azide, 10 mmol/L HEPES and 2% FBS). Control single and dual antibody combinations (FITC-Ly-6C/PE or PE/biotinyl-CD31-Red 670) were prepared in parallel from ZA mice as were three-color (FITC-IgG2a/PE-IgG2a or 2b/biotinyl-IgG2a) isotype-matched negative controls. The latter samples were used to determine background labeling. After labeling, the samples were stored at 4°C in label buffer until analysis the same day.
Bone marrow B-cell subpopulations were identified using FITC-CD43, PE-CD45R and biotinyl-IgM to identify pro-B cells (CD45R+CD43+IgM-), pre-B cells (CD45R+CD43-IgM-) and immature/mature B-cells (CD45R+CD43-IgM+) as previously described (20
,21
). The three antibodies were added simultaneously to appropriate samples after incubation with rat IgG to block random antibody binding as previously described (7
,8
). After one wash, StrepAv-Red 670 was used to detect biotinyl-IgM; 30-min labeling at 4°C was used throughout and washes took place in label buffer. Samples were fixed in 1.25% formaldehyde in PBS, pH7.4, stored overnight at 4°C and analyzed by flow cytometry the next day.
The antibody sources were as follows: biotinyl-CD31 (ER-MP12, IgG2a) and FITC-Ly-6C (ER-MP20, IgG2a) Bachem Bioscience, King of Prussia, PA; PE-CD45R (RA36B2, IgG2a), PE-CD11b (M1/70, IgG2b), PE-Ly-6G (RB6–8C5, IgG2b), PE-TER 119 (IgG2b), FITC-CD43 (S7, IgG2a) PharMingen, Los Angeles, CA; biotinyl-anti-IgM (goat, F(ab')2 µ chain specific) Jackson ImmunoResearch, West Grove, PA; and StrepAv-Red 670, Life Technologies (GibcoBRL), Grand Island, NY. All FITC, PE and biotinylated isotype rat immunoglobulins were from PharMingen.
All antibodies were titered under the protocol used for phenotyping and used at optimal concentrations. The antibody concentrations used for samples of one million cells were as follows: 0.75 µg biotinyl-CD31, 0.6 µg FITC-Ly-6C, 0.8 µg PE-CD45R, 0.6 µg PE-CD11b, 0.3 µg PE-Ly-6G, 0.6 µg PE-TER 119, 1.0 µg FITC-CD43, 1.1 µg biotinyl-anti-IgM and 0.5 µg StrepAv-Red 670.
Flow cytometric analysis.
A Becton Dickinson Vantage flow cytometer equipped with a G3 Mac PC computer and CellQuest software (San Jose, CA) and an I-90 Coherent argon laser were used for phenotypic analysis. FITC, PE and Red 670 were excited at 488 nm with 100 mW laser power and their respective fluorescence emission was detected at 530 ± 15, 575 ± 13 and 670 ± 14 nm. Linear signal amplification was used for forward and side scatter data and a log scale was used for all fluorochromes. Single- and two-color positive and isotype negative samples for all antibodies were used to determine the lower limit of positive fluorescence for each fluorochrome (FITC, PE, RED 670) and to set electronic color compensation between fluorochromes (FITC/PE and PE/RED 670). A gate was drawn in the scatter cytogram to exclude debris and aggregates from phenotypic composition using CD31 and Ly-6C when all five populations were determined simultaneously (Fig. 3
, 5)
. A scatter gate and a gate selecting the PE-positive population of known identity were used when confirming the identity of CD31/Ly-6C subpopulations (Fig. 4)
.
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Statistical analysis.
Data were analyzed using SigmaStat (SPSS, Chicago, IL). One-way ANOVA was performed on all data. Differences between ZA and other dietary groups were determined using Dunnett’s method. Kruskall-Wallis one-way ANOVA on ranks was used when data failed the normality test used by the SigmaStat program. Differences between ZA and the other groups were considered significant at P < 0.05. Unless stated otherwise, values in the text are means ± SD, n = 6–8. All data presented herein are representative of at least three experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The young adult A/J mice initially weighed 17.1 ± 0.4 g. At the end of the 34-d study, the RZA mice weighed 93% as much as the ZA controls. ZD mice remained near their initial weight for 2 wk then gradually lost weight as the study proceeded. After 34 d, ZD mice were divided into MZD (73–75% weight of ZA mice) or SZD (68–71% weight of ZA mice) (Fig. 1
) as described in the methods and previous studies (6
–8
). Results were independent of whether diets contained AIN-76 or AIN-93 vitamin and mineral mixtures.
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24 µmol Zn/L) (Fig. 1)
. Serum zinc did not differ between the RZA and ZA mice. As in previous experiments, serum zinc was not as sensitive an indicator of zinc status as were immune parameters (2
,4
,7
,8
). For example, the thymuses of MZD mice weighed 40%, whereas the thymuses of SZD mice weighed 17% of those of the ZA mice (Fig. 1)
. This degree of thymic atrophy has been associated with an 80% loss in thymic cell number (9
,12
)
Surprisingly, neither marginal nor severe zinc deficiency altered the total, or absolute number of nucleated cells in the marrow (Fig. 2
). Previously published results (7
,8
) and several additional experiments (data not shown) demonstrated that ZD does not reduce the marrow cellularity of tibia and femora in typical 28- to 34-d dietary studies (7
,8
). However, we will demonstrate that the composition of the marrow changed significantly in ZD mice.
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Figure 3
presents a representative contour plots of the five major hemopoietic marrow populations showing differences between the ZA group and the other groups. We verified the identity of these populations using population-specific antibodies gating first by light scatter (R1) and then by a population-specific marker (R2) to provide the CD31 and Ly-6C expression of the known population in a two parameter cytogram (Fig. 4
). CD31 and Ly-6C accurately identified cells as erythroid lineage (R3, CD31-Ly-6C-), B-cell (R4, CD31+Ly-6C-), granulocyte (R5, CD31-Ly-6C+), heterogeneous monocyte (R6, CD31-Ly-6C++) and mixed progenitor (R7, CD31+Ly-6C+) (Fig. 4)
. False positives were < 3% in all cases (Ter 119 < 3%) (CD11b and Ly-6G, <2.5%) (CD45R <2%). This phenotypic protocol was executed in all experiments reported herein and was highly reproducible.
Effect of ZD on the major cell lineages of the bone marrow.
The effects of ZD on the major lineages of cells produced in the marrow are shown in Figure 5
. It is a composite of data gathered for 6–8 mice/dietary group using the multiparameter flow cytometric protocol in Figure 3
and 4
and is representative of three dietary studies, all of which had similar outcomes. The erythroid compartment comprised 18–19% of the nucleated cells of the marrow in ZA and RZA mice. However, the proportion of erythroid lineage cells declined >25% in MZD mice to 13.7% of the nucleated marrow cells, whereas SZD mice showed a decline of nearly 60% in erythroid lineage cells to 9.1% of the nucleated marrow cells. These substantial losses of cells of the erythroid lineage indicate altered erythropoiesis and offer an explanation for the anemia associated with zinc deficiency in humans (4
,22
). These data suggest that ZD in humans and animals can alter the production of RBC in the marrow.
Little change was noted in the lymphoid compartment of RZA mice compared with ZA mice. However, the lymphoid compartment declined 50% in MZD mice and >70% in SZD mice, in agreement with previous studies (7
). These are large declines in both the proportion and number of cells of the lymphoid lineage and explain why lymphopenia is a classical characteristic of ZD (6
–9
).
Changes in the myeloid compartments were extremely interesting and somewhat unexpected. Granulocytes increased in MZD and SZD mice from 40% in ZA mice to 54 and 62% of the marrow, respectively. This represented 36 and 57% increases in granulocytes both in proportion and absolute cell numbers in the marrow of MZD and SZD mice, respectively. There were 75 and 79% increases in the proportion of monocytes in the marrow of MZD and SZD mice, respectively. Because ZD did not alter the total number of nucleated cells in the marrow (Fig. 2)
, granulocyte and monocyte lineage cells increased in absolute numbers in the presence of increasing zinc deficiency during the time erythroid and lymphoid lineage cells were significantly depleted.
So-called committed progenitors are a mixture of cells of various lineages at the earliest stages of development (Fig. 4)
(13
,14
). A modest increase occurred in this compartment for RZA and MZD mice with a 50% increase in progenitors in SZD mice. Taken together, it is clear that ZD affected the numbers and proportions of the various lineages of cells in the marrow.
Effect of ZD on the phenotypic distribution of subsets of cells of the B-lineage.
ZD affected B cell lymphopoiesis (Fig. 6
), consistent with previous studies (7
,8
). The critical finding was loss of pre-B cells from the CD45R+ or B-cell compartment of the marrow in MZD and SZD mice. Within the B-cell compartment, a modest 15% of pre-B cells were lost in MZD mice, but losses were 75% for SZD mice (Fig. 6)
. Pro-B cells increased in proportion within the residual B-cell compartment of ZD mice. In SZD mice, the percentage of pro-B cells was more than double that of ZA mice. The proportion of IgM+ cells, which would include immature (IgM+IgD-) and mature B-cells (IgM+IgD+), did not differ from the ZA mice in the experimental groups. Previous studies in which more markers were used showed that this population was predominantly mature B-cells (7
). Thus, ZD dramatically reduced the lymphoid compartment of the marrow (Fig. 5)
and markedly changed the composition of the surviving B-cells within the B-cell compartment.
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CD11b was used to evaluate the phenotypic distribution of cells within the myeloid compartment (Fig. 4)
because cells of this lineage increased in the percentage and absolute number in the marrow as ZD advanced (Fig. 5)
. The proportion of heterogeneous monocytes within the myeloid compartment was 70% greater in both MZD and SZD mice than in ZA mice, and the committed progenitor population was 60 and 73% greater in MZD and SZD mice, respectively, indicating that the proliferative myeloid lineages were functional (Fig. 7
). Monocytes and committed progenitors were <10% (4.2 ± 0.4) and 2% of the myeloid group, respectively. The proportion of granulocytes, the major myeloid population, shows a slight but biologically insignificant decrease in zinc deficient mice as a result of expansion of proliferative myeloid populations. Clearly, the response of myelopoiesis and lymphopoiesis in the adult marrow is markedly different in ZA and ZD mice, suggesting that the immune system may be able to adapt to nutritional deficiencies in ways that were not appreciated heretofore.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In addition, an increase in committed progenitors of mixed lineage was also noted. This dichotomy in effect of ZD on these major lineage classes suggests that ZD reprograms myelopoiesis, lymphopoiesis and possibly erythropoiesis.
Prasad et al. (22
) were among the first to identify zinc deficiency in humans. In addition to showing that suboptimal zinc reduced growth and development of the reproductive system, they demonstrated that anemia also accompanied ZD in humans (22
). The data herein show that cells of the erythroid lineage were significantly depleted during the 34-d study. Moreover, the reduction in erythroblasts correlated with the degree of zinc deficiency in mice as assessed by thymic atrophy, body weight and parakeratosis rather than serum zinc (Figs. 1
, 5)
. Clearly, Zn deficiency alters the production of RBC in higher animals, beginning with alterations in erythropoiesis in the marrow that may involve a stimulation of self-renewal and an arrest of differentiation within the erythroid lineage (23
).
Multiparameter flow cytometry indicated that ZD substantially reduced the proportion and/or number of cells within B-lineage subsets of the lymphoid lineage, consistent with previous studies (6
–9
). The high losses of pre-B cells, along with previous data showing preferential losses of pre-T cells in the atrophying thymus of ZD mice, provide the underlying cause of the reduction in peripheral lymphocytes noted in ZD humans and rodents (9
). This reduction in absolute number of mature but naïve lymphocytes in the peripheral system clearly contributes to the reduction in cell and antibody mediated responses (1
–3
). Recently, it has been shown that increased apoptosis among the pre-T cells accounts, in part, for these losses during ZD (9
,12
).
It is important to understand the underlying causes of the ZD-associated lymphopenia. The probable mechanism is the activation of the hypothalamus-pituitary-adrenal stress axis by ZD and PEM, which results in chronic production of glucocorticoids. We have shown that the concentrations of corticosterone generated during ZD were able to induce apoptosis in pre-B cells (24
,25
). Glucocorticoids are potent inducers of cell death in pre-T cells or thymocytes, as well (26
). Moreover, adrenalectomies or reduction in ability of ZD mice to produce corticosterone provided substantial protection to pre-T and pre-B cells and the immune system in general (27
,28
). Although the precise role of these glucocorticoids in changes in the marrow remains to be defined, it is clear that the glucocorticoid-induced apoptosis plays a prime role in the loss of pre-B and pre-T cells during ZD (9
,12
,28
). Because of the parallels to PEM, we suspect that similar mechanisms are at work in this deficiency (3
,29
). These steroids, along with limited available zinc, appear to combine to drastically alter lymphopoiesis and reduce replenishment of lymphocytes in the peripheral immune system during ZD.
Perhaps the most surprising finding was the apparent increase in the proportion and absolute numbers of granulocytic and monocytic cells in the marrow of both MZD and SZD mice in three dietary studies. This expansion of the myeloid compartment was regulated because the lineage members remained in nearly the same proportions during the expansion (Fig. 7)
. Short-term glucocorticoid implants in mice that generated serum concentrations of corticosterone similar to that in ZD mice caused a similar enhancement in granulocytes in the marrow (30
). However, expansion of myeloid progenitors and extensive loss of erythroid lineage cells were not noted. The retention of the erythroid lineage and absence of expansion of proliferative myeloid populations suggest that 36 h was insufficient to affect hematopoiesis. The expansion in granulocytes may be explained by evidence that they are less sensitive to glucocorticoid-induced apoptosis and thus accumulate as lymphocytes were lost (31
).
We noted several years ago that the ratio of neutrophils to lymphocytes escalated in the peripheral blood of ZD mice (2
). Because mice are examined periodically for subacute infections and no infection was present, it would seem that the increasing proportion of neutrophils in blood was a result of ongoing deficiencies in zinc and changes in the hematopoietic processes. Moreover, it is interesting to consider that for over a decade immunologists have known that cultures of primary murine marrow will yield primarily myelopoietic cells if small amounts of glucocorticoids are added (32
). It would, therefore, seem that ZD is a natural disturbance that gives an outcome within the bone marrow that is somewhat analogous to that observed in cell cultures.
These observations also suggest that there is an important adaptation at work during malnutrition that is at least partially mediated by glucocorticoids. The immune system, when considered in totality, is a large and expensive system to maintain, especially when nutrients become limiting. We know that major metabolic changes occur in the passage from the well-fed to the starved state; these changes are designed to protect the truly vital tissues of the body as long as possible. Less is known about how the immune system adapts to these changes, although the high turnover rate and nutritional cost of maintaining the lymphoid system as ZD progresses must be considered. Perhaps part of the reason for induction of the chronic production of glucocorticoids is to preserve the first line of defense or innate immunity while down-regulating the secondary line of defense. Adequate production of phagocytic cells would allow for the continued removal of pathogens, debris, damaged and apoptotic cells. Perhaps the lymphocytic system is down-regulated within the marrow to conserve nutrients. Further adaptations may be possible because we have noted that the half-life of myeloid cells may be extended in ZD mice and that the residual or remaining lymphocytes appear to be somewhat hyperresponsive (6
,9
). Indeed, a number of investigators have noted that one cell type of the myeloid series, namely, the human neutrophil, has an extended half-life when exposed to glucocorticoids (31
). All of this suggests that ZD and perhaps PEM do not simply randomly dismantle the immune system as previously thought. Rather the changes in the immune system may be built-in fail-safes or adaptations put in place to provide as much innate immune protection as possible in the face of declining nutriture.
Future experiments will attempt to examine the changes in marrow function as altered by ZD more extensively. It is important, for example, to know more about the functional capacity of phagocytic cells in the ZD mice. Can they carry out phagocytosis, chemotaxis, respiratory burst or wound repair in a normal way? What is the rate of production of cells of the lymphoid series? Is lymphopoiesis gradually down-regulated or are cells produced at near normal rates only to die apoptotically? Myeloid cells become a greater portion of the marrow, but is their rate of production actually altered? Using DNA dyes in conjunction with CD31 and Ly-6C, we previously found a >50% increase in the myeloid progenitors in the S and G2/M phase of the cell cycle (33
). Whether there could be an actual increased rate of production as zinc becomes limiting remains to be seen. There are also probably major changes in the cytokine production by stromal cells in the microenvironment of cells of the marrow of ZD mice. These changes could promote more myelopoiesis while suppressing production of cytokines required for lymphopoiesis. It would be useful to know what changes in Bcl-2, Bax, heat shock protein or redox enzymes are made by myeloid cells in the marrow and periphery to protect themselves from ZD and associated glucocorticoids. Finally, changes in the glucocorticoid receptor numbers and/or modulation of chaperones such as BAG-1 may alter the activity of the steroid receptor such that some cells such as myeloid cells are protected (34
). Much remains to be learned about the mechanism of adaptation of the two major branches of the immune system to ZD and other nutritional deficiencies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by a grant from the National Institutes of Health, DK 52289–23.
4 Abbreviations used: FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HBSS, Hank’s balanced salt solution; Ig, immunoglobulin; MZD, moderately zinc-deficient; PE, phycoerythrin; PEM, protein-energy malnutrition; RZA, restricted-fed zinc-adequate; SZD, severely zinc-deficient; ZA, zinc-adequate; ZD, zinc-deficient.
Manuscript received 12 June 2002. Initial review completed 10 July 2002. Revision accepted 26 August 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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