![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
The Journal of Nutrition Vol. 128 No. 1 January 1998,
pp. 20-27
, and
* Institute of Parasitology and ** School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, Ste. Anne de Bellevue, QC H9X 3V9, Canada and Center for the Study of Host Resistance, Montreal General Hospital, Montreal, QC H3G 1A4, Canada
 |
ABSTRACT |
|---|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
This study examined whether the impaired immune responses in zinc deficient- and/or energy-restricted mice exposed to a challenge infection of Heligmosomoides polygyrus might be associated with reduced numbers of spleen cells, altered proportions of spleen cell subpopulations and/or altered function of the T cells or antigen-presenting cells (APC). Female BALB/c mice were given free access to either a zinc-sufficient (60 mg zinc/kg diet, Zn+) or a zinc-deficient diet (0.75 mg zinc/kg diet, Zn
) or were pair-fed (PF) the zinc-sufficient diet. Significant differences in parasite burdens were observed. Worm numbers were lowest in Zn+ mice, intermediate in the PF mice and highest in the Zn mice, showing that both zinc deficiency and energy restriction reduced protective immunity against the gastrointestinal nematode H. polygyrus. Although the absolute numbers of spleen cells were reduced in both Zn and energy-restricted (PF) mice, neither deficiency altered the phenotypic distribution of the subpopulations of positive marker cells in the spleen. In vitro functional assays using a 1:1 ratio of APC:T cells showed that T-cell proliferation in response to parasite antigen (Ag) was impaired by a dietary effect of zinc deficiency on T cells and of energy restriction and zinc deficiency on APC function. Consequences of the nutritional deficiencies on cytokine production in response to parasite antigen were more complex: zinc deficiency reduced T-cell function [interleukin-4 and interleukin-5 (IL-4 and IL-5) production], and both nutritional deficits depressed APC functions [IL-4, IL-5, and interferon-
(IFN-) production] and T-cell function (IFN- production). Thus, this study showed that zinc deficiency and energy restriction played identifiably distinct roles in regulating host immune responses against the gastrointestinal nematode H. polygyrus.
Suboptimal intake of zinc has rapid and adverse effects on the defense systems of rodents, primates and humans (Keen and Gershwin 1990 To date, attempts to determine the direct cause of the impaired T-cell-dependent immune responses at the cellular level have been done in zinc-deficient animals only in the absence of infectious agents and have resulted in mixed conclusions. Most researchers agree that a reduction in lymphocyte numbers is a major factor and that this reduction is independent of the specific cell population (Cook-Mills and Fraker 1993 Our laboratory is involved in ongoing research concerning the effects of nutritional deficiencies on host defense mechanisms using the gastrointestinal trichostrongyloid nematode of mice, Heligmosomoides polygyrus. This parasite is used as a model for chronic gastrointestinal helminthiasis (Monroy and Enriquez 1992 In this study, the potential cellular defects of zinc deficiency and energy restriction on different compartments of the immune system were examined in mice with a challenge infection of H. polygyrus. The objective was to obtain evidence that would indicate whether the impaired immune responses that occur during the challenge infection in energy-restricted and/or zinc-deficient mice might be associated with reduced numbers of spleen cells, altered proportions of spleen cell subpopulations and/or altered function of splenic T cells or antigen-presenting cells (APC). To determine if energy restriction and/or zinc deficiency affected T cell function, APC from Zn+ mice were paired with T cells from different dietary groups; T cell proliferation in response to mitogen and parasite antigen was assayed, and cytokine production was quantified in vitro. The same protocol was used to examine the function of APC, except that control T cells from Zn+ mice were paired with APC from different dietary groups. Three Th2 cytokines (IL-4, IL-5 and IL-10) were assayed because of the key role that the Th2 response plays in host-parasite defense mechanisms to H. polygyrus (Urban et al. 1992 Mice and diets.
To achieve our objectives, we required a strain of mouse in which protective immunity exerts its effect within a few weeks of challenge infection rather than days or months, and in which a Th2 response phenotype was expected. The BALB/c mouse was chosen. This mouse strain is classified as an "intermediate responder" to the H. polygrus parasite because worm expulsion normally occurs within 4 wk of challenge infection (Monroy and Enriquez 1992
Infection protocol.
To stimulate a strong host protective immune response against H. polygyrus infection, an anthelmintic-abbreviated immunizing protocol was used. Four weeks after consuming the experimental diets, mice from each dietary group were infected with 100 infective third-stage H. polygyrus larvae (L3). On d 9 and 14, mice were treated orally with pyrantel pamoate (Pfizer Canada, Kirkland, Canada), at a dose of 172 mg/kg body weight, to eliminate adult parasites from the intestine. One week later (d 21), mice were reinfected with 100 L3 parasites.
Liver zinc and copper analysis.
Livers were freeze-dried to constant weight, fat extracted with petroleum ether and wet-ashed in nitric acid. Zinc and copper concentrations were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer 3100; Perkin-Elmer Canada, Montreal, Canada) and expressed as milligrams per kilogram fat-free dry weight.
Parasite and parasite antigen.
Heligmosomoides polygyrus L3 were obtained by culturing the feces of stock CD1 mice (Charles River) on moist filter paper for 7 d. The cultured larvae were suspended in deionized water (100 larvae/20 µL), and administered by gavage to mice using a pipetteman. The accuracy of the dose was estimated by direct counts of the number of larvae in each of five sham doses that were dispensed into plastic petri dishes before the experimental infection.
Spleen cell preparation.
Single cell suspensions were prepared aseptically by passing individual spleens in Hanks' balanced salt solution (HBSS, Gibco, Burlington, Canada) through a fine, stainless steel screen. Red blood cells were lysed with NH4Cl (0.17 mol/L) for 5 min and the cells were washed twice with HBSS. Membrane debris was removed by filtering the cell suspension through sterile gauze. Cell viability was determined by trypan blue exclusion and was always >90%. The total nucleated splenocyte suspension was kept in complete RPMI 1640 medium: RPMI 1640 (Gibco) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco), 2% HEPES buffer (Gibco), 100,000 IU/L penicillin and 100,000 µg/L streptomycin (Gibco).
Flow cytometric analysis.
We also examined whether in addition to reduction in the cell numbers there were alterations in the composition of the lymphocyte populations as indicated by flow cytometric analysis of spleen cells from the different dietary groups. Spleen cell suspensions prepared as above were diluted to a concentration of 2 × 109 cells/L. Aliquots of 0.5 mL were transferred to microtubes and blocked in sorting buffer [PBS, with bovine serum albumin (BSA), 10 g/L, and sodium azide, 5 g/L] with normal goat serum (1:40). After two washes at 300 × g with cold sorting buffer, cells were incubated for 30 min on ice in 50 µL of sorting buffer containing 1 µg monoclonal antibody (mAb)/106 cells of either fluorescein-isothiocyanate (FITC)-conjugated anti-CD3 (clone 145-2C11), anti-B220 (clone RA3-6B2), anti-Mac-1 (clone M1/70), and anti-Iab (clone AF6-120.1) mAb, or FITC-conjugated anti-CD3 combined with phycoerythrin (PE)-conjugated anti-CD4 (clone RM4-5) or anti-CD8 (clone S3-6.7) mAb (Pharmingen, San Diego, CA). The cells were washed twice and resuspended in 150 µL of 5 g/L paraformaldehyde in PBS. One- and two-color flow cytometric analyses were performed immediately after labeling, using a Coulter EPICS Profile II (Coulter, Hialeal, FL).
T cell and APC enrichment.
To obtain cell populations of both T cells and APC from each mouse, a nylon wool column separation technique was used. This method is known to yield a population of spleen cells containing 85-90% T cells, sufficiently enriched for most analytical purposes (Julius et al. 1973 T-cell proliferation.
For the in vitro functional studies of T cell and APC, a 1:1 ratio of APC to T cells was chosen on the basis of the strong proliferative responses seen when such a ratio was used to examine mitogenesis in nutritionally adequate mice (Croft et al. 1992 Cytokine production.
The mixed cell suspensions, containing equal numbers of APC and T cells, were adjusted to a final concentration of 5 × 109 cells/L in the complete RPMI 1640 medium. Aliquots of 1.0-2.0 mL were incubated in 24-well flat-bottom tissue plates (Becton Dickinson Labware) with parasite Ag (at a final concentration of 6.5 mg protein/L) prepared as above, or with Con A (final concentration of 5 mg/L) (Calbiochem). The cell suspensions were incubated at 37°C in 5% CO2 atmosphere with humidity. Forty-eight hours later, the cell suspension was centrifuged at 350 × g, 4°C, for 10 min. The cell-free supernatants were recovered and stored at Cytokine ELISA.
Two-site sandwich ELISA were used for all cytokine determinations. The following paired Abs were used: anti-mouse IL-4 mAb (BVD4-1D11, Pharmingen) and biotinylated anti-mouse IL-4 mAb (BVD6-24G2, Pharmingen) for IL-4 quantification; anti-mouse IL-5 mAb TRFK-5 (Pharmingen) and biotinylated TRFK-4 (Pharmingen) for IL-5; anti-mouse IL-10 mAb JESS-2A5 (Pharmingen) and biotinylated anti-mouse IL-10 mAb SXC-1 (Pharmingen) for IL-10, and anti-mouse IFN- Statistical analysis.
To test for dietary effects, one-way ANOVA on untransformed homogeneous data was performed. All tests of the data were done using SYSTAT version 5.03, Evanston, IL (Wilkinson 1991 Host nutritional status and H. polygyrus numbers.
Dietary zinc deficiency significantly reduced the food intake and growth of the Zn
Spleen cell population.
Energy restriction and zinc deficiency reduced both the size of the spleen relative to body weight and the total number of spleen cells (Table 3). Zinc-deficient mice had significantly fewer spleen cells than both PF and Zn+ mice, and PF mice also had a lower number of the cells than Zn+ mice. Neither zinc deficiency nor energy restriction had a significant effect on the phenotypic distribution of cells bearing the following markers: CD3+, CD3+CD4+, CD3+CD8+, B220+ or Mac 1+ (Table 3). Therefore, a selective shift in immune cell populations was not induced by either zinc deficiency or energy restriction.
APC function.
When Con A was used as the mitogen, similar proliferative responses were observed among the dietary groups, regardless of the source of APC in the culture (Fig. 1A). In contrast, when parasite-specific Ag was used as the stimulus, T-cell proliferation was significantly lower in the presence of APC from both Zn
T-cell function.
To determine whether the function of T cells was affected by dietary zinc deficiency or energy restriction, the following protocol was used. Control APC from Zn+ mice were paired with T cells from the different dietary groups. As shown in Figure 1C, the proliferative responses of T cells to Con A did not differ among groups. However, T cells from Zn This study showed that zinc deficiency and energy restriction played identifiably distinct roles in regulating host immune responses against H. polygyrus. Both zinc deficiency and energy restriction resulted in elevated worm numbers. Both nutritional deficiencies reduced the size of the spleen, resulting in a reduced number of splenocytes. On the basis of our in vitro cell mixing experiments that used a 1:1 ratio of APC:T cells, we concluded that zinc deficiency weakened host protection primarily by causing functional defects in T cells, resulting in impaired T-cell proliferation, and reduced T-cell cytokine production (IL-4). Energy restriction, as well as zinc, impaired host immune response by limiting the capacity of APC to induce T-cell responses (proliferation and production of IL-4, IL-5 and IFN-
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). In addition to the well-documented consequences of zinc deficiency on T cells (Keen and Gershwin 1990), zinc is important in the regulation of certain macrophage functions (James et al. 1987, Wirth et al. 1989), and in the production and development of B cells (King et al. 1995). Importantly, T-cell mediated responses are critical for host protection against parasitic infections (Mitchell 1980), and any impairment of T cells or the cells that present antigen to them, caused by the nutritional deficiencies, is therefore expected to cause a decrease in host protection. Yet, surprisingly few studies have examined the effect of dietary zinc deficiency on host-parasite interactions (El-Hag et al. 1989, Fenwick et al. 1985, Wirth et al. 1989). Even fewer studies have attempted to isolate the effect of zinc deficiency per se from the effect of reduced food intake that accompanies zinc deficiency and that also impairs immune function (Luecke et al. 1978, Minkus et al. 1992).
, Keen and Gershwin 1990). However, there is less agreement regarding the effects of zinc deficiency on lymphocyte function. When tested on a per cell basis, some studies report no change in T-cell function (Cook-Mills and Fraker 1993), whereas others indicate an impaired ability to proliferate in response to mitogens (Zanzonica et al. 1982). James et al. (1987), in particular, reported that zinc deficiency impaired T-cell proliferative response to the mitogen phytohemagglutinin (PHA).5 Defective T-cell-dependent responses may also result from nutritional impairment of antigen presentation to T cells as shown by reduced T-cell mitogenesis when macrophages from zinc-deficient mice were mixed with T cells from control mice (James et al. 1987).
). CD4+ T cells, which are the principal effector T cells mediating host protective immune responses against H. polygyrus infection (Urban et al. 1991a), can be divided into two, or possibly three, functional groups defined by their patterns of cytokine secretion. Th1 cells secrete interleukin-2 (IL-2), interferon-gamma (IFN-), and lymphotoxin, whereas Th2 cells secrete interleukin-4 (IL-4), IL-5, IL-9 and IL-10. Both types express IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and tumour necrosis factor (TNF). A putative third type of Th cells has been identified on the basis of production of TGF-
, which is thought to be involved in mucosal T helper function and down-regulation of Th1 responses (Chen et al. 1994). The cytokines produced by each of the two main CD4+ T subsets, Th1 and Th2, work by down-regulating the proliferation or functional activity of the opposing subset (Mosmann and Moore 1991). Thus, Th1 cells produce IFN-, which preferentially inhibits proliferation of Th2 cells, whereas Th2 cells, which are responsible for generating strong humoral immunity against helminth infections (Sher and Coffman 1992, Urban et al. 1992), synthesize both IL-4, which preferentially stimulates the generation of Th2 cells, and IL-10, which downregulates cytokines of Th1 cells by affecting APC (Sher and Coffman 1992). IgE and IgG1 are regulated by IL-4, eosinophils are regulated by IL-5, and all have been linked with host protective immunity against this parasite H. polygyrus (Urban et al. 1991a, 1991b and 1992). Current evidence suggests that the key protective component of this response is IL-4 (Urban et al. 1993). In adequately nourished mice, primary H. polygyrus infection stimulates a CD4+ T cell response that has the following Th2 characteristics: increased IL-4 gene expression and protein secretion, elevated levels of IL-3, IL-5 and IL-9 mRNA, eosinophilia and a vigorous B-cell response, resulting in substantial increases in serum IgE and IgG1 (Svetic et al. 1993, Urban et al. 1992, Wahid et al. 1993). Recently, we demonstrated that zinc deficiency and energy restriction enhanced the survival of H. polygyrus during both a primary (Shi et al. 1994 and 1995) and a challenge (Shi et al. 1995) infection. During primary infection (Shi et al. 1994), zinc deficiency, independent of energy restriction, resulted in a decreased delayed type hypersensitivity response in vivo and impaired production of IL-4 and IFN- by spleen cells in vitro. The observed decreases in serum levels of total IgE and IgG1, impaired eosinophilia and impaired production of IL-5 were attributed to a combined effect of zinc deficiency and energy restriction.
). In addition, IFN- was measured because it is an important stimulatory cytokine for a Th1 response, and its overexpression could down-regulate the Th2 cytokines required for the resolution of this gastrointestinal nematode infection (Sher and Coffman 1992, Urban et al. 1992).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
, Wahid et al. 1993). The immune response of BALB/c mice has been well studied and shown to be Th2 dominant (Urban et al. 1991a, 1991b and 1992), although infection also can induce production of the Th1 cytokine, IFN- (Shi et al. 1994, Urban et al. 1992).
0.75 mg Zn/kg diet) or pair-fed (PF, 60 mg Zn/kg diet), for which individual mice were matched to a Zn mouse. Each mouse in the PF group was fed the Zn+ diet daily in amounts equal to that consumed in the previous 24 h by its Zn paired mate. Mice were fed a powdered, biotin-fortified, egg-white-based diet, formulated to meet 1978 NRC requirements for the laboratory mouse (Table 1). Zinc was added as zinc sulfate to provide 60 mg zinc/kg diet for the Zn+ diet, an amount twice the recommended level (NRC 1978). All other nutrients were included at levels sufficiently above recommendation to ensure that a 30% reduction in food intake would not generate any other specific nutrient deficiencies in the PF or Zn mice. Diets were offered in mouse powder feeders (Laboratory Products, Montreal, Canada) specifically designed to minimize food spillage. Mice in Zn+ and Zn diet groups were allowed free access to diets. Daily food intake was determined by subtracting the amount of food remaining in the feeder from the amount given the previous day. Body weights were measured weekly, and total body weight gain was determined. All procedures were approved by the McGill Animal Care Committee according to the Canadian Council on Animal Care (1984).
View this table:
Table 1.
Composition of diets1
20°C. At a later time, worms were removed and counted.
). Spleen cell suspensions, pooled from groups of two mice, were adjusted to a density of 1 × 1010 cells/L in complete RPMI 1640 medium, plated in 90-mm tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) and incubated for 2 h, at 37°C in 5% CO2. After incubation, the nonadherent cells were removed by gently washing the surface of each dish with 37°C complete RPMI 1640. The adherent cells were collected using a cell scraper and formed part of the APC source. The nonadherent cells were enriched for T cells by passage through a 10-mL syringe column containing 0.7 g nylon wool (Polisciences, Warrington, PA). The column was incubated for 1 h at 37°C, and T cells were eluted with warmed medium. Antibody staining with anti-CD3 and flow cytometry revealed that cells were ~85% T cells.
). The effectiveness of this treatment was evidenced by the low proliferation of the cells (18.5-29.5 Bq to concanavalin A (Con A) stimulation, and 7.4-11.1 Bq to Ag stimulation). After incubation, the cells were washed three times with an excess of complete RPMI medium, counted and adjusted to a concentration of 5 × 109 cells/L. Flow cytometry analysis showed that there were ~80% Ia+ cells in the APC population.
and 1994).
70°C until they were assayed for IL-4, IL-5, IL-10 and IFN-.
mAb R4-6A2 (Pharmingen) and biotinylated anti-mouse IFN- mAb XMG1.2 (Pharmingen) for IFN- quantification. Streptavidin-horseradish peroxidase conjugate (Gibco) was used as the secondary layer, and the reaction was visualized with ABTS (Bio-Rad). The concentrations of cytokines were calculated from standard curves generated with the use of known concentrations of recombinant murine IL-4 (Genzyme, Markham, Canada), recombinant murine IL-5 (Pharmingen), recombinant murine IL-10 (Pharmingen) and recombinant murine IFN- (Genzyme). The sensitivities of the assays were as follows: IL-4, 0.05-0.1 µg/L; IL-5, 0.03-0.05 µg/L; IL-10, 0.1-0.2 µg/L; IFN-, 0.11-0.22 µg/L.
) with Tukey's post-hoc pairwise comparison (Lund and Lund 1983). All differences were considered significant at the 0.05 level.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
group (Table 2). Zinc deficiency was confirmed by a significantly lower liver zinc concentration in zinc-deficient mice than in either PF or Zn+ mice (Table 2). Pair-feeding also reduced the body weight gain of mice. Therefore, this PF group was truly energy restricted, having a significantly greater energy deficit per gram of body weight compared with either the control or Zn mice, which did not differ (data not shown). By inference, the Zn mice were not additionally energy restricted when their food intake per gram of body weight was compared with the intake of the Zn+ mice, even though, according to the classic pair-feeding protocol, absolute food intake of the isoenergetic diets and therefore total energy intake was significantly lower. We concluded that the Zn mice were singularly zinc deficient. Measurements of worm burdens showed that very few worms remained in zinc-sufficient mice killed 3 wk postchallenge infection (pci); twice as many worms were obtained from PF mice, and twice as many again were obtained from Zn mice (P < 0.05) (Table 2). This showed that protective immunity was reduced by energy restriction and impaired even further by zinc deficiency.
View this table:
Table 2.
Effect of zinc deficiency and energy restriction (PF) on nutritional status of BALB/c mice and parasite numbers at 3 wk postchallenge infection with 100 Heligmosomoides polygyrus L31
View this table:
Table 3.
Effect of zinc deficiency and energy restriction (PF) on relative spleen weight, total numbers and phenotypic distribution of spleen cells of BALB/c mice at 3 wk postchallenge infection with 100 Heligmosomoides polygyrus L31
or PF mice than in cultures with APC from Zn+ mice (Fig. 1B). Thus, these results show that energy restriction and zinc deficiency affected the ability of APC to stimulate proliferation of T cells in response to parasite-specific Ag, when a 1:1 ratio of APC to T cells was used.
View larger version (31K):
[in a new window]
Fig 1.
Effects of zinc deficiency and energy restriction in mice on T-cell proliferative responses to concanavalin A (Con A) (Panels A and C) or parasite antigen (Ag) (Panels B and D). To evaluate antigen-presenting cell (APC) function (Panels A and B hatched), APC from the different dietary groups were cultured with control T cells (Zn+) to test T-cell function (Panels C and D solid). T cells from different dietary groups were cultured with control APC from Zn+ mice. Values are means ± SEM, n = 4, each pooled from 2 mice. Different letters represent significant differences (P < 0.05).
mice compared with those from PF and Zn+ mice, but also a lower level of IL-4 was produced in cultures containing APC from PF mice compared with cultures with APC from Zn+ mice. A significant effect of energy restriction and zinc deficiency on the function of APC was also apparent for IL-5 and IFN- production. APC from the three different dietary groups induced similar levels of IL-10 production by T cells. When cells were stimulated with Con A (Table 4), energy and zinc restriction reduced the ability of APC to stimulate IL-5 and IFN- production, but neither energy restriction nor zinc deficiency altered their ability to stimulate IL-4 or IL-10 production at the 1:1 ratio of APC to T cells that we used. Data from the APC functional studies thus demonstrated that both energy restriction and zinc deficiency impaired APC function.
View this table:
Table 4.
Effects of zinc deficiency and energy restriction (PF) on T-cell cytokine production, assayed using combinations of antigen-presenting cells (APC) and T cells from different sources and stimulated with either concanavalin A (ConA)
or Heligmosomoides polygyrus antigen (Ag)1
mice had a significantly lower proliferative response to parasite-specific Ag compared with T cells from either PF or Zn+ mice (Fig. 1D). These data show that zinc deficiency impaired the proliferative response of T cells to Ag but not to Con A, and that energy restriction had no effect on T-cell proliferation. Both Th1 and Th2 cytokines were produced by T cells in response to parasite-specific Ag, but the magnitude of the responses was reduced by zinc deficiency (Table 4). T cells from Zn mice produced significantly lower amounts of IL-4 than T cells from either the PF or Zn+ group. T cells from Zn mice also produced a significantly lower level of IL-5 compared with T cells from Zn+ mice. Neither zinc deficiency nor energy restriction had any effect on IL-10 production. In contrast, energy restriction and zinc deficiency impaired the ability of T cells to produce IFN-; significantly lower levels of IFN- secretion were observed in cultures with T cells from both Zn or PF groups compared with cultures containing T cells from Zn+ mice. When Con A was used to stimulate cytokine production (Table 4), secretions of IL-4, IL-5 and IFN- did not differ, regardless of the source of T cells. However, significantly lower amounts of IL-10 were produced by Con A-stimulated T cells from both Zn and PF mice compared with T cells from Zn+ mice. Therefore, reduced IL-10 production in response to Con A was associated with energy restriction and with zinc deficiency. In general, under our experimental conditions, zinc deficiency impaired IL-4 and IL-5 production in response to parasite-specific Ag, whereas both energy and zinc restriction impaired IFN- production in response to Ag and IL-10 production in response to Con A.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). In these cell-mixing experiments, there is always the concern that use of a single ratio may be inadequate because the absence of an effect may represent a false negative for that ratio only. For example, T-cell proliferation and IL-2 production were shown to increase as the number of APC relative to a constant number of CD4+ T cells increased to a ratio of 1:1 (Croft 1992 and 1994). Nevertheless, their maximal responses were obtained at the 1:1 ratio, suggesting that this ratio was reasonable for a first attempt at exploring the consequences of nutritional deficiencies on immune function in infected hosts.
). In this study, the numbers of parasites recovered in all three dietary groups were much lower than in our previous study (Shi et al. 1995), perhaps because of differences in the infectivity of the larvae between experiments. Nevertheless, Zn+ mice had the lowest parasite burden, PF mice had an intermediate value and Zn mice had the highest worm numbers. Therefore, as previously reported, host protective immunity was stimulated in the Zn+ mice, but less so in PF and Zn mice.
mice, suggesting that fewer lymphocytes were available for a protective immune response against the parasite infection in these hosts. This observation is consistent with most nutritional studies showing a reduction in lymphocyte numbers in zinc-deficient animals (Cook-Mills and Fraker 1993). Although the precise nature of splenic involvement in immunity against the intestinal nematode is not yet clear, the spleen responds to H. polygyrus infection, as evidenced by the pronounced splenomegaly that accompanies infection (Parker and Inchley 1990, Shi et al. 1994). It has been suggested that this occurs when the mesenteric lymph nodes (MLN) are unable to process all of the parasite antigen (Parker and Inchley 1990). An effective immune response requires not only a sufficient number of normally functioning lymphocytes, but also a balanced composition of lymphocyte populations. Neither zinc deficiency nor energy restriction altered the proportion of total T cells, their CD4+ and CD8+ subsets, B cells or macrophages. This is consistent with data from infected rodents showing that, except in the most severe cases, zinc deficiency does not alter the composition of splenic lymphocytes in the uninfected model (King and Fraker 1991); it is also consistent with studies showing that energy restriction has no effect on the ratio of CD4+ to CD8+ spleen cells, despite the generalized increase in the percentage of cells carrying these markers (Woodward et al. 1995).
), changes in cell membranes and signal transduction pathways (Csermely and Somogyi 1988) or perturbations in parasite antigen and class II receptors or co-stimulatory CD28 and/or CTLA-4 molecules on T cells, and B7 molecules on APC (Lu et al. 1994) are potential underlying mechanisms that could result in the low T-cell proliferation associated with zinc deficiency.
). Although altered immunity has been reported in situations of reduced food intake (Luecke et al. 1978), the mechanisms by which energy restriction affects the immune response have not yet been determined. The results presented in our study suggest that functional impairment of APC may be one of the possible cellular mechanisms for the decreased T cell-dependent immune function commonly seen in the energy-restricted host (Woodward et al. 1995).
reported that zinc deficiency reduced the ability of macrophages to activate T-cell proliferation to PHA, whereas we tested the proliferative response to the mitogen Con A and parasite antigen using a different ratio and different cell populations. James et al. (1987) used pure macrophage populations, whereas we used total APC populations containing perhaps 10-15% macrophages. Therefore, our protocol was more likely to detect nutrient effects on B cells, the predominant cells in the APC population, rather that the relatively smaller population of macrophages in the study of James et al. (1987).
, Urban et al. 1991b and 1995), and our study shows that both zinc deficiency and energy restriction dramatically impaired cytokine secretion by perturbing APC and T-cell function. Con A stimulation after energy restriction reduced the ability of APC to stimulate T-cell production of IL-5 and IFN-, whereas energy restriction reduced the ability of T cells to produce IL-10. Cytokine production in response to parasite antigen was different. IL-4 secretion was reduced not only by the effects of zinc deficiency on T cells, but also by the combined effects of energy restriction and zinc deficiency on APC induction of T cells. Decreased IL-4 levels in the supernatants could reflect effects of zinc on one or more of the steps involved in transcription, translation (Vallee and Falchuk 1993) or in transport of the cytokine to the cell membrane, its passage through the membrane or its release from the cell surface. The effect of zinc on APC stimulation of T cells was consistent with that of James et al. (1987), in which macrophage function was impaired, after 12 wk of zinc deficiency, but differed from that of Cook-Mills et al. (1991) in which APC from Zn mice were able to process and present antigen efficiently. Reduced IL-4 production in the Zn host would be expected to prolong parasite survival and also to cause a decreased IgG1 and IgE response, because IL-4 stimulates B cell class switching to IgG1 and IgE secretion (Mosmann and Moore 1991). Our in vivo data had previously shown that all of these defects occurred in Zn and energy-restricted mice (Shi et al. 1994) after a primary infection. Now we can conclude that similar changes occur during a challenge infection.
), the reduced IL-5 production was presumably the cause of the low eosinophilia that we previously reported in both Zn and PF mice (Shi et al. 1994).
), we attributed these impaired immune responses in part to reduced numbers of lymphocytes caused by both zinc deficiency and energy restriction. In addition, we demonstrated that the reduced capacity of APC to induce T-cell responses in vitro was caused by both energy restriction and zinc deficiency, whereas functional defects in the T cells were caused by zinc deficiency. Although we studied only one ratio of T cell to APC, there is always the possibility that a different ratio could produce different results. We have no evidence that either deficiency altered the proportion of cell subpopulations in the spleen. Our results provide novel information concerning the underlying cellular mechanisms whereby zinc deficiency and energy restriction impair host protective immunity against the intestinal parasite, H. polygyrus, at the cellular level by profoundly perturbing T-cell and APC function.
| |
FOOTNOTES |
|---|
; interferon-gamma; IL, interleukin; L3, third-stage larvae; L4, fourth-stage larvae; mAb, monoclonal antibody; MLN, mesenteric lymph node; pci, post challenge infection; PE, phycoerythrinpo; PF, pair-fed (energy restricted); PHA, phytohemagglutinin; Th, T helper; TNF, tumor necrosis factor; Zn+, zinc sufficient; Zn, zinc deficient.
Manuscript received 19 June 1996. Initial reviews completed 13 August 1996. Revision accepted 27 August 1997.
| |
LITERATURE CITED |
|---|
|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Induction and measurement of cytotoxic T lymphocyte activity. In: Current Protocols in Immunology (Coligan, J. E., Kruisbeek, A. M. Margulies, D. H., Sheyach, E. M., Stroher, W. eds.) Supplement 21, pp. 3.11.1-3.11.20. John Wiley & Sons, Inc., Toronto, Canada.This article has been cited by other articles:
|
|
 |
|
  T. Niiya, Sk. Md. F. Akbar, O. Yoshida, T. Miyake, B. Matsuura, H. Murakami, M. Abe, Y. Hiasa, and M. Onji Impaired Dendritic Cell Function Resulting from Chronic Undernutrition Disrupts the Antigen-Specific Immune Response in Mice J. Nutr., March 1, 2007; 137(3): 671 - 675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hillyer, B. Dao, P. Niemiec, S. Lee, M. Doidge, I. Bemben, T. Neyestani, and B. Woodward Elevated Bioactivity of the Tolerogenic Cytokines, Interleukin-10 and Transforming Growth Factor-{beta}, in the Blood of Acutely Malnourished Weanling Mice Experimental Biology and Medicine, September 1, 2006; 231(8): 1439 - 1447. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J Rahman, P. Sarker, S. K Roy, S. M Ahmad, J. Chisti, T. Azim, M. Mathan, D. Sack, J. Andersson, and R. Raqib Effects of zinc supplementation as adjunct therapy on the systemic immune responses in shigellosis Am. J. Clinical Nutrition, February 1, 2005; 81(2): 495 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Neyestani and B. Woodward Blood Concentrations of Th2-Type Immunoglobulins Are Selectively Increased in Weanling Mice Subjected to Acute Malnutrition Experimental Biology and Medicine, February 1, 2005; 230(2): 128 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Kristan and K. A. Hammond Morphological plasticity varies with duration of infection: evidence from lactating and virgin wild-derived house mice (Mus musculus) infected with an intestinal parasite (Heligmosomoides polygyrus; Nematoda) J. Exp. Biol., June 1, 2004; 207(13): 2351 - 2360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Cousins, R. K. Blanchard, J. B. Moore, L. Cui, C. L. Green, J. P. Liuzzi, J. Cao, and J. A. Bobo Regulation of Zinc Metabolism and Genomic Outcomes J. Nutr., May 1, 2003; 133(5): 1521S - 1526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lanningham-Foster, C. L. Green, B. Langkamp-Henken, B. A. Davis, K. T. Nguyen, B. S. Bender, and R. J. Cousins Overexpression of CRIP in transgenic mice alters cytokine patterns and the immune response Am J Physiol Endocrinol Metab, June 1, 2002; 282(6): E1197 - E1203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Fan, K. Kouda, H. Nakamura, and H. Takeuchi Effects of Dietary Restriction on Spontaneous Dermatitis in NC/Nga Mice Experimental Biology and Medicine, December 1, 2001; 226(11): 1045 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Sun, A. R. Muthukumar, R. A. Lawrence, and G. Fernandes Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice Clin. Vaccine Immunol., September 1, 2001; 8(5): 1003 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Kristan and K. A. Hammond Parasite infection and caloric restriction induce physiological and morphological plasticity Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2001; 281(2): R502 - R510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Weindruch, K. P. Keenan, J. M. Carney, G. Fernandes, R. J. Feuers, R. A. Floyd, J. B. Halter, J. J. Ramsey, A. Richardson, G. S. Roth, et al. Caloric Restriction Mimetics: Metabolic Interventions J. Gerontol. A Biol. Sci. Med. Sci., March 1, 2001; 56(90001): 20 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Ing, Z. Su, M. E. Scott, and K. G. Koski Suppressed T helper 2 immunity and prolonged survival of a nematode parasite in protein-malnourished mice PNAS, June 20, 2000; 97(13): 7078 - 7083. [Abstract] [Full Text] [PDF]
|
|
