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Article

Expression of T Lymphocyte p56^lck, a Zinc-Finger Signal Transduction Protein, Is Elevated By Dietary Zinc Deficiency and Diet Restriction in Mice

Department of Foods and Nutrition, University of Manitoba, Winnipeg, MB R3T 2N2

	ABSTRACT

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Compromised immune function is common to Zn deficiency, protein and energy malnutrition; however, the causative mechanisms at the molecular level have not been elucidated. The T lymphocyte signal transduction pathway contains several Zn-finger proteins, and it is possible that the in vivo functioning of these proteins could be affected by dietary deficiency of Zn and amino acids. Thus, the objective was to investigate the effects, on expression of the T lymphocyte signal transduction proteins p56^lck, phospholipase C1 (PLC1) and protein kinase C (PKC), of dietary Zn deficiency (ZnDF, < 1 mg Zn/kg diet) and protein-energy malnutrition syndromes [2% protein deficiency (LP), combined Zn and 2% protein deficiency (ZnDF+LP), and diet restriction (DR, body weight equal to ZnDF)] compared with control (C) mice. Indices of nutritional status and splenocyte counts were also determined. Based on serum albumin and liver lipid concentrations, the ZnDF+LP and LP groups had protein-type malnutrition, whereas the ZnDF and DR groups had energy-type malnutrition. For Western immunoblotting of the signal transduction proteins, mouse splenic T lymphocytes were isolated by immunocolumns. The expression of T lymphocyte p56^lck was significantly elevated in the ZnDF+LP, ZnDF and DR groups compared to the C group. In contrast, the expression of PLC1 and PKC was unaffected. There was a significant negative correlation between T lymphocyte p56^lck expression and serum Zn (r= -0.65, P = 0.0007) or femur Zn (r = -0.73, P = 0.0001) concentrations. We propose that elevated T lymphocyte p56^lck may contribute to altered thymoctye maturation, apoptosis and lymphopenia in Zn deficiency and protein-energy malnutrition syndromes.

KEY WORDS: • zinc • malnutrition • p56^lck • T lymphocytes • mice

	INTRODUCTION

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Immune dysfunction is common to zinc deficiency and protein and energy malnutrition syndromes. In experimental models of dietary Zn deficiency and in congenital errors of Zn metabolism (e.g., acrodermatitis enteropathica in humans, A46 mutation of Danish Fresian cattle), impaired immune function is characterized by atrophy of lymphoid tissue, reductions in lymphocyte numbers, decreased cell-mediated immunity (e.g., T lymphocyte proliferation) and increased susceptibility to infection (reviewed by Keen and Gershwin 1990 ). A similar pattern of impaired immune function has been reported in various types of protein and/or energy malnutrition syndromes (reviewed by Woodward 1998 ). However, the overlap of anorexia-induced malnutrition in Zn deficiency and mildly reduced Zn status in malnutrition syndromes makes it difficult to discern the effects of Zn per se from malnutrition. Furthermore, the type and magnitude of malnutrition was defined by biochemical analysis in very few experimental animal studies (Woodward 1998 ).

Although impaired immune function in Zn deficiency was characterized at the tissue and cellular levels, the molecular or biochemical function(s) of Zn for explaining this Zn deficiency pathology have not been established. The Zn-finger hypothesis for the molecular function of Zn proposes that Zn-finger proteins involved in signal transduction and gene transcription provide a mechanism for coordinating changes in plasma Zn status with the gene expression and cell proliferation required for immune function. The T lymphocyte signal transduction pathway contains several Zn-finger proteins in which the finger-like loop is formed by Zn binding to a combination of four cysteine sulfhydryl groups and/or histidine imidazole nitrogens (Berg 1990 ). Zn is believed to play an important role in maintaining the structural integrity and functionality of Zn-finger proteins based on in vitro chelation and point mutation experiments (Berg 1990 ).

In vitro manipulation of Zn can modulate the activity of several T lymphocyte signal transduction proteins, including 56^lck, phospholipase C1 (PLC1)⁵ and protein kinase C (PKC) (Csermely et al. 1988 ,Ottolenghi 1965 , Pernelle et al. 1991 ). p56^lck is a lymphoid-specific protein tyrosine kinase that is principally expressed in T lymphocytes (Weil and Viellette 1996 ). Association of p56^lck with the cytoplasmic tail of various cell surface receptors, as well as associations of p56^lck with intracellular targets of phosphorylation, suggest that this tyrosine kinase plays a central role in coordinating early signal transduction events (Anderson et al. 1994 ). Sequencing and point mutation experiments indicate that the association of p56^lck with the co-receptors CD4 or CD8 involves binding of a common cysteine motif in the cytoplasmic tails of CD4 and CD8 with a cysteine motif in the amino terminal sequence of p56^lck (Glaichenhaus et al. 1991 , Turner et al. 1990 ). The interaction of cysteine motifs in p56^lck and CD4 or CD8 does not involve disulfide linkage as demonstrated by co-precipitation experiments under reducing conditions (Turner et al. 1990 ). Rather, a role for Zn to stabilize the interaction was proposed because micromolar concentrations of Zn (in the absence of other divalent cations) elicits substantial tyrosine phosphorylation of p56^lckcompared to other divalent cations (Pernelle et al. 1991 ). Furthermore, it was demonstrated that Zn stimulates phosphorylation of p56^lck in a dose-dependent manner (Pernelle et al. 1991 ) and that addition of a Zn chelator disrupts the association of p56^lck with the Zn-finger proteins CD4 or CD8 (Turner et al. 1990 ). Similarly, activities of PLC and PKC are inactivated by chelator treatment and restored by in vitro Zn (Csermely et al. 1988 , Ottolenghi 1965 ). It appears that the lipid binding regulatory domain of PKC contains two cysteine-rich Zn-finger motifs (Quest et al. 1992 ), whereas the crystal structure of PLC indicates the presence of three Zn²⁺ in the active site (Hough et al. 1989 ). Thus, based on the results of in vitro experiments using cell extracts or transfected cells, Zn is critical for several steps in the T lymphocyte signal transduction pathway. However, the effects of Zn nutrition on the in vivo functioning of these T lymphocyte signal transduction proteins has not been investigated.

The objective of the present experiment was to investigate the effects, on the expression of the T lymphocyte signal transduction proteins, p56^lck, PLC1 and PKC, of dietary Zn deficiency (ZnDF) and protein-energy malnutrition syndromes [2% protein deficiency (LP), combined Zn and 2% protein deficiency (ZnDF+LP), and diet restriction (DR)] compared to control (C) mice. The effects of the dietary treatments on indices of nutritional status and on splenocyte counts were also determined. A young adult murine model of Zn deficiency and protein-energy malnutrition syndromes was used to minimize complications associated with rapid growth and immune system development in the post weaning stage. The DR group provided information on the effects of diet restriction, and it served as a control to interpret whether changes in the ZnDF group were due to Zn per se or the body weight changes associated with the anorexia of Zn deficiency. The LP group provided information on the effects of protein deficiency, caused in part by a dietary deficiency of sulfur amino acids. Comparison of Zn deficiency and protein deficiency, separately and in combination, provided information on the relative effects of Zn and amino acid deficiency on the expression of Zn-dependent proteins.

	MATERIALS AND METHODS

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Animals and diets.

Two-month-old female C57BL/6 mice were purchased from Charles River Laboratories (St.Constant, PQ) and fed a nutritionally complete nonpurified diet. At 4 mo of age (considered immunologically mature), mice were randomly assigned to one of the 5 dietary treatment groups for 4 wk: zinc-deficient (ZnDF, <1 mg Zn/kg and 12% protein), protein-deficient (LP, 2% protein and 30 mg Zn/kg), combined zinc- and protein-deficient (ZnDF+LP, <1 mg Zn/kg and 2% protein), diet restricted (DR, 30 mg Zn/kg and 12% protein), or control (C, 30 mg Zn/kg and 12% protein). The mice were given free access to the diets except for the DR group, which was fed the control diet in amounts necessary to maintain the same body weight as the ZnDF group, i.e., a "paired-weight" control group. The diet formulations, based on the AIN-93M diet (Reeves et al. 1993 ), are provided in Table 1 . Zinc content of the diets was verified by atomic absorption analysis. The mice were housed individually, in stainless steel hanging cages with mesh bottoms and were provided distilled water in plastic bottles with stainless steel sipper tubes. The mice were maintained in an environment of controlled temperature (21–23°C), humidity (55%) and light cycle (14 h light/10 h dark). Body weights were determined weekly, with the exception of daily weighing of the DR group. Animal care was provided in accordance with a protocol approved by the Local Animal Care Committee (University of Manitoba).

Mice were weighed and killed by CO₂ asphyxiation and cervical dislocation. Trunk blood was collected and placed on ice until centrifuged to obtain serum. Spleens were removed aseptically, weighed and processed immediately. Femurs with their accompanying musculature and livers were removed, frozen in liquid nitrogen and stored at -80°C.

Western immunoblotting.

    T lymphocyte lysate preparation. All reagents (molecular biology grade) were purchased from Sigma Chemical Company (St. Louis, MO) with exception of acid solutions, solvents and standard laboratory materials (VWR Canlab, Mississauga, ON) unless otherwise indicated. Spleen cell suspensions were prepared under aseptic conditions in a Nuaire biological tissue culture hood (Plymouth, MN). Sterile, bent needles were used to scrape spleen cells into sterile phosphate-buffered saline (PBS), pH 7.3, supplemented with 2% fetal calf serum (FCS)(Gibco, Grand Island, NY). After centrifugation of the total spleen cell preparation for 5 min at 300 x g, the cells were resuspended and gently inverted for 2 min in 2 mL Tris-buffered ammonium chloride (working solution: 90 mL of 0.16 mol NH₄Cl/L; 10 mL of 0.17 mol Tris/L, pH 7.65 adjusted to pH 7.2) to lyse erythrocytes. The lysis solution was diluted by adding 3 mL of PBS/2% FCS, and the cell suspension was centrifuged. This procedure was repeated once more to ensure that erythrocytes were no longer evident in the pellet. Cells were washed twice in PBS/2% FCS before determining the total nucleated spleen cell count using an AO Bright-Line Hemacytometer (American Optical Corporation, Buffalo, NY). Before loading onto the counting chamber, 10 µL cell suspension was diluted with 990 µL of 0.87 mol/L acetic acid to lyse any residual erythrocytes.

T lymphocytes were isolated from the mononuclear cell suspension using mouse T immunocolumns with polyclonal goat anti-mouse IgG (H+L) (Biotex Laboratories, Edmonton, AB) according to the protocol provided by the manufacturer. By a process of negative selection, B lymphocytes bind to the antibody on the column, resulting in an enrichment of T lymphocytes in the column eluant. There will also be non-specific binding of some macrophages and monocytes to the column. However, T cell enrichment is the important factor for analysis of p56^lck, a lymphoid-specific protein tyrosine kinase that is principally expressed in T lymphocytes (Weil and Viellette 1996 ). Briefly, columns were washed with 20 mL of PBS/2% FCS at a rate of 6–8 drops/min. The column reagent was reconstituted with 1.5 mL PBS/2% FCS, and allowed to incubate on the column for a minimum of 1 h. Excess column reagent was washed off with 15 mL PBS/2% FCS. Column setup and activation was completed 1 h prior to animal terminations. Cells (1 x 10⁸), adjusted to a concentration of 5 x 10¹⁰ cells/L, were allowed to flow through the column at a rate of 6–8 drops/min, and 20 mL of the eluant was collected. Cells were centrifuged at 300 x g for 10 min, resuspended in 0.8 mL PBS/2% FCS and counted as described above. T lymphocyte viability (>95%) was verified by trypan blue exclusion, and lymphocyte purity (>95% T lymphocytes) was verified by flow cytometric analysis of cells stained with fluorescein anti-mouse T3 complex CD3 (clone 145–2C11, Cedarlane Laboratories, Hornby, ON) and gated for the lymphocyte population using forward and light side scatter. T lymphocytes were repelleted in preparation for cellular lysis.

The T lymphocyte pellet was lysed by resuspension in 120 µL ice-cold RIPA buffer [1% Nonidet P-40 (BDH Laboratory Supplies, Toronto, ON); 6.4 mmol deoxycholate/L; 150 mmol NaCl/L; 50 mmol Tris/L, pH 7.5] containing protease inhibitors (1 mmol sodium orthovanadate/L, 1 mmol EGTA/L, 5 g aprotinin/L, 12.5 mg leupeptin/L, 1 mmol phenylmethylsulfonylfluoride/L), for 30 min (Kanner et al. 1990 ). The lysates were passed through a 27 gauge needle and centrifuged for 10 min at 15600 x g (Eppendorf Centrifuge #5414) to pellet the nuclear fraction. The supernate containing the cytosolic fraction was moved to fresh tubes and frozen at -80°C until measuring the protein concentration using the BCA Protein Assay (Pierce, Rockford, IL). Both protein standards and samples were prepared using RIPA buffer as the diluent.

    Western immunoblotting. The antibodies used were anti-human lck kinase N terminal domain (NT) (rabbit antiserum, Upstate Biotechnology, Lake Placid, NY) for p56^lck, anti-bovine PLC1 (mixed monoclonal of clones B-2–5, B-6–4, B-20–3, D-7–3, E-9–4; Upstate Biotechnology), and anti-human PKC (clone 3; Transduction Laboratories, Lexington, KY). An antibody for the NT domain of p56^lck was chosen because it is a cysteine motif in the NT domain of p56^lck, which requires the presence of Zn for interaction with the cysteine motifs in the cytoplasmic tails of CD4 and CD8 (Turner et al. 1990 ). PLC1 and PKC are the isoforms that undergo immediate tyrosine phosphorylation and translocation, respectively, in response to stimulation of the T-cell antigen receptor (Szamel et al. 1997 , Weiss et al. 1991 ). Each of these antibodies is cross-reactive with mouse, and detection was verified using the respective positive controls provided by the companies. Apparatus and reagents were purchased from Bio-Rad Canada (Mississauga, ON) unless otherwise indicated. Buffer formulations for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting were according to protocols provided by Bio-Rad and Boerhinger Mannheim (Laval, PQ, Canada). Samples (5 µg protein for p56^lck or 10 µg protein for PLC1 and PKC) were solubilized by boiling for 4 min with an equal volume of two times SDS sample buffer. Proteins in samples and the protein molecular weight ladder were subjected to SDS-PAGE, using a 10% (p56^lck, PKC) or 8% (PLC1) SDS separating gels and a 5% stacking gel. Electrophoresis was completed using 500 mL electrode buffer pH 8.3 (3 g Tris base/L, 14.4 g glycine/L, 1 g SDS/L) at 170 V (Power Pac 200) for 1 h using the Mini-Protean II electrophoresis cell. Separated proteins were transferred electrophoretically (Mini Trans-Blot Electrophoretic Transfer Cell) to a nitrocellulose membrane (0.45 µm) using transfer buffer pH 8.3 (3.03 g Tris/L, 14.4 g glycine/L, 200 mL methanol/L) and the transfer cell at 100 V for 1 h. The membrane was equilibrated in Tris-buffered saline (TBS) pH 7.5 (6.05 g Tris/L, 8.76 g NaCl/L).

The reagents used for immunodetection were purchased as a kit (BM Chemiluminescence Western Blotting Kit for Mouse/Rabbit, Boehringer Mannheim). To reduce nonspecific binding, the membrane was incubated with a 1% blocking solution for 1 h at room temperature or overnight at 4°C. The antibodies were diluted in 0.5% blocking reagent (1:1000 for anti-human lck kinase NT, 1:1250 for anti-bovine PLC1, 1:250 for anti-human PKC) and membranes were probed either overnight at 4°C (p56^lck) or for 1 h at room temperature (PLC1 and PKC). The membranes were washed twice with TBS-T [1 mL Tween 20/L TBS (50 mmol Tris base/L, 150 mmol NaCl/L), adjusted to pH 7.5] for 5–10 min, then equilibrated twice with 0.5% blocking solution for 5–10 min. The antigen-antibody complex was identified with anti-mouse/rabbit IgG horseradish peroxidase employing a 1:2500 dilution in 0.5% blocking solution. Following two 15 min washes in TBS-T, this enzyme coupled secondary antibody was allowed to react for 1–3 min, with the luminescent substrate contained in the detection solution. The bands representing p56^lck, PLC1 or PKC were scanned by image analysis and assigned arbitrary units that represent the band area combined with grey intensity.

As an additional step, India Ink staining, was used to visualize the transfer efficiency of the separated proteins blotted onto the membrane and for band identification. Membranes were washed in Tween 20 solution (0.3% v/v Tween-20 in PBS) at 37°C three times for 30 min each on an orbital shaker, followed by two 30-min washes at room temperature. The membrane was allowed to stain in India Ink solution [0.1% India Ink (Osmiroid International, Hampshire, UK) in Tween 20 solution] on an orbital shaker for 3 h at room temperature. The membrane was washed in Tween 20 solution until grey bands appeared on a white background and then air dried.

Zinc analysis.

Femurs were scraped of all musculature and connective tissue using a scalpel blade. After obtaining wet and dry weights, femurs and diet samples were wet-ashed using nitric acid as previously described (Clegg et al. 1981 ). After appropriate dilution of digests or serum, Zn concentration was determined by atomic absorption spectroscopy using a Spectra AA-30 Spectrophotometer (Varian Canada, Georgetown, ON). Quality control was monitored using bovine liver standard reference material 1577b (U. S. Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD).

Serum albumin analysis.

Serum albumin concentration was determined using the Sigma Diagnostics Albumin Reagent (Sigma Chemical) containing bromcresol green. This method is based on the procedure of Doumas et al. (1971) .

Liver lipid analysis.

Liver lipid content was determined by the Folch et al. (1956) method. Each liver was thawed, weighed and homogenized in 22 mL of a 2:1 mixture of chloroform and methanol, respectively, for 30 s using a Polytron (Brinkmann Instruments, Rexdale, ON). The homogenate was passed through a #1 Whatman filter, and the volume of the eluate, collected in a 25 mL graduated cylinder, was recorded. Twenty percent of this volume was added as water and then shaken. The milky suspension was covered with a stopper and allowed to separate overnight. The volume of the lower chloroform layer containing lipid was recorded, and the upper methanol layer was removed. Ten mL of the chloroform layer was placed in a pre-weighed dried 25 mL glass vial, and the chloroform was evaporated in a heated water bath (OA-SYS heating system, Organomation Associates, Berlin, MA) with nitrogen air (0.7 kg/cm²) for 1 h. The vials containing lipid were allowed to cool in a dessicator, and then weighed to determine the lipid content.

Statistical analysis.

Plots of data sets exhibited normal distributions and did not exhibit evidence of a pattern of lack of homogeneity, except for the data from the image analysis of p56^lck, which was normalized by log transformation prior to analysis by ANOVA. Differences among dietary treatment groups were analyzed by one-way ANOVA using the general linear models procedure (SAS software release 6.04, SAS Institute, Cary, NC). Significant differences between means (n = 9 unless otherwise specified) were determined using Duncan's New Multiple Range test. Correlation analysis (zinc status versus expression of p56^lck) was performed using Spearman's correlation coefficient. The probability level at which the differences were considered significant was P < 0.05.

	RESULTS

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Assessment of zinc status and malnutrition type.

The effects of a zinc-deficient , protein-deficient, combined zinc- and protein-deficient, diet restricted and control dietary treatments on body weight and some biochemical variables in young adult mice are presented in Table 2 . The initial body weight of the mice was 23.1 + 1.0 g. After the 4 wk feeding trial, mice fed the deficient diets weighed significantly less (~15%) than the C group.

The type and magnitude of malnutrition was assessed by liver lipid and serum albumin concentrations because protein-type malnutrition is characterized by an accumulation of lipid in the liver and a reduction in visceral protein status. Liver lipid concentration was significantly higher (29–32%) in the LP and ZnDF+LP mice compared to the C group; however, DR and ZnDF groups were not significantly different from the C group. The accumulation of liver lipid in the low protein groups (LP and ZnDF+LP) was paralleled by significantly lower liver weights (27–30%) and lower liver/body weight ratios (12–18%) compared to the C group. The LP and ZnDF+LP groups had serum albumin concentrations that were not significantly different from the C group. The serum albumin concentrations of the ZnDF and DR groups were significantly higher (11 and 28%, respectively) than the C group.

Assessment of spleen variables.

All mice fed deficient diets had significantly lower spleen weights, spleen/body weight ratios and total splenocyte (mononuclear cell) counts than the C group (Table 3). The spleen weight of the DR group was significantly less than the spleen weights of the ZnDF and ZnDF+LP mice. The spleen weight of the LP group was significantly less than the ZnDF group. However, the spleen/body weight ratio for the DR and LP groups was significantly less than the spleen/body weight ratio for mice fed zinc-deficient diets (ZnDF and ZnDF+LP groups). Total splenocyte counts (per spleen) were significantly lower in the DR group compared to the ZnDF and ZnDF+LP groups. The LP group had a greater reduction in splenocyte counts than the combined ZnDF+LP group (46 vs. 28%, respectively) when compared to the C group. However, the splenocyte concentration (per mg spleen) was significantly lower in the ZnDF and DR groups compared to the ZnDF+LP and C groups. The T lymphocytes from each dietary treatment group yielded similar amounts of cytosolic protein in the lysates prepared for Western immunoblotting.

Western immunoblotting of T lymphocyte lysates was used to determine expression of p56^lck (Fig. 1a), PLC1(Fig. 2a)and PKC (Fig. 3a). The results were quantified by image analysis scanning and expressed as arbitrary units relative to the C group. The two zinc-deficient groups, ZnDF+LP and ZnDF, had a significantly greater expression of p56^lck compared to the LP and C groups (Fig. 1 b). Expression of p56^lck in the DR group was significantly lower than the ZnDF+LP group and significantly higher than the C group. The T lymphocyte expression of PLC1(Fig. 2 b) and PKC (Fig. 3 b) was not significantly different among the dietary treatment groups.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effects of dietary zinc deficiency, protein deficiency or diet restriction on expression of p56lck in murine splenic T lymphocytes. (a) Representative Western blot of p56lck expression in T lymphocyte lysates as detected by chemiluminescence. The dietary treatments were Zn-deficient and 2% protein (ZnDF+LP, Lane 1), Zn-deficient (ZnDF, Lane 2), diet restriction (DR, Lane 3), 2% protein (LP, Lane 4) and control (C, Lane 5). All lanes were loaded with an equivalent amount of lysate cytosolic protein. (b) Arbitrary units for p56lck expression determined by image analysis scanning. Columns represent means ± SEM for nontransformed values for n = 5, and different lower case letters indicate significant differences among means, P < 0.05. The units represent arbitrary units relative to the C group.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effects of dietary zinc deficiency, protein deficiency or diet restriction on expression of PLC1 in murine splenic T lymphocytes. (a) Representative Western blot of PLC1 expression in T lymphocyte lysates as detected by chemiluminescence. The dietary treatments were Zn-deficient and 2% protein (ZnDF+LP, Lane 1), 2% protein (LP, Lane 2), Zn-deficient (ZnDF, Lane 3), diet restriction (DR, Lane 4) or control (C, Lane 5). All lanes were loaded with an equivalent amount of lysate cytosolic protein. (b) Arbitrary units for PLC1 expression determined by image analysis scanning. Columns represent means ± SEM for n = 7. The units represent arbitrary units relative to the C group.

View larger version (32K): [in this window] [in a new window]   Figure 3. Effects of dietary zinc deficiency, protein deficiency or diet restriction on expression of PKC in murine splenic T lymphocytes. (a) Representative Western blot of PKC expression in T lymphocyte lysates as detected by chemiluminescence. The dietary treatments were Zn-deficient and 2% protein (ZnDF+LP, Lane 1), 2% protein (LP, Lane 2), Zn-deficient (ZnDF, Lane 3), diet restriction (DR, Lane 4) or control (C, Lane 5). All lanes were loaded with an equivalent amount of lysate cytosolic protein. (b) Arbitrary units for PKC expression determined by image analysis scanning. Columns represent means ± SEM for n = 7, except n= 6 for ZnDF+LP and LP groups. The units represent arbitrary units relative to the C group.

View larger version (19K): [in this window] [in a new window]   Figure 4. Serum zinc concentration (upper panel) or femur zinc concentration (lower panel) versus the arbitrary units for expression of p56lck in murine splenic T lymphocytes. Data points are values from individual mice fed Zn-deficient and 2% protein (ZnDF+LP, n = 4), Zn-deficient (ZnDF, n = 5), 2% protein (LP, n = 5), diet restricted (DR, n= 4) or control (C, n = 5) diet for 4 wk. The data are nontransformed values and represent arbitrary units relative to the C group. Analysis by Spearman's correlation coefficient (r) revealed significant negative correlations between p56lck expression and serum zinc concentration (r = -0.65, P = 0.0007) or femur zinc concentration (r = -0.73, P = 0.0001).

	DISCUSSION

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Of the three T lymphocyte signal transduction proteins that were analyzed, elevated protein expression was limited to p56^lck; expression of PLC1 and PKC were unchanged by the dietary treatments. In each case, protein expression was analyzed relative to total cytosolic protein in the T lymphocyte lysates, i.e., a constant amount of protein loaded per lane for SDS-PAGE. Furthermore, the amount of lysate cytosolic protein per 10⁶ T lymphocytes isolated on the immunocolumns was not significantly different among the dietary groups (Table 3) . Thus, the significantly higher expression of p56^lck because of zinc or diet restriction also represents elevated p56^lck expression on a per cell basis. Further studies are required to determine if the elevated p56^lck was due to increased synthesis or decreased degradation of p56^lck and if the functional activity of this protein tyrosine kinase was altered. p56^lck is the first protein in the signal transduction pathway to be phosphorylated upon stimulation of the T cell antigen receptor (Weil and Veillette 1996 ); subsequent steps involve phosphorylation and activation of PLC1, resulting in hydrolysis of phosphoinositol bisphosphate into diacylglycerol and inositol triphosphate, which in turn lead to activation of PKC and an increase in intracellular calcium (Weil and Veillette 1996 ). Co-immunoprecipitation experiments suggest that PLC1 is a target of p56^lck and that there is a direct association between these signal transduction molecules after stimulation of the T cell antigen receptor (Weber et al. 1992 ). However, in the present experiment, elevated p56^lck expression was not associated with any changes in PLC1 or PKC expression. One of the downstream outcomes of the signal transduction cascade is cell proliferation. In other studies with these same dietary treatment groups, we have examined concanavalin A-stimulated cell proliferation and have found that all deficient groups have a similar reduction in proliferation and a lower percentage of cells in S phase compared to cells from the control group (Bossuyt and Taylor 1998 ). In the present study, only the Zn-deficient and diet restricted groups had elevated p56^lck expression. This suggests that functions of p56^lck, other than activation of mature lymphocytes (Weil and Veillette 1996 ), may explain the relevance of our results.

p56^lck has a pivotal role in signal transduction for development of T cells in the thymus and for positive selection (Weil and Veillette 1996 ). In terms of T cell development, mice lacking a functional lck gene (Molina et al. 1992 ) or overexpressing a catalytically inactive form of p56^lck (Levin et al. 1993 ) exhibit thymic atrophy, early arrest of thymocyte maturation (preservation of double negative CD4^-CD8^- thymocytes and a dramatic decrease in double positive CD4⁺CD8⁺ thymocytes), and very few peripheral T lymphocytes. Thus, p56^lck activity is critical as thymocytes mature from the double negative to double positive stage to ultimately yield mature single positive CD4⁺ or CD8⁺ peripheral T lymphocytes. It has been reported that 6-wk-old mice fed a Zn-deficient diet for 3–8 wk have an increase in immature T cells in the spleen based on their ability to form autologous rosettes, a characteristic of a certain population of immature T cells present in the thymus (Nash et al. 1979 ). Chandra (1979) reported that children with protein-energy undernutrition had a higher proportion of "null" cells in peripheral blood and that this was correlated with elevated leukocyte terminal deoxynucleotidyl-transferase activity, a marker for immature T and B lymphocytes. Although the percentage CD4⁺ or CD8⁺ lymphocytes and the CD4⁺/CD8⁺ ratio has been investigated in rodent models of Zn deficiency and protein-energy malnutrition (King and Fraker 1991 , Lee and Woodward 1996 , Taylor et al. 1997 ), it would be possible to use multiple antibody labeling and flow cytometry analysis to determine if there is an increase in immature T cells based on surface markers. This approach was used to demonstrate significant depletions in the proportion of small nucleated cells, early B cells and immature B cells in the bone marrow of Zn-deficient mice (King et al. 1995 ); however, B cell development is not p56^lck-dependent. Using CD1a for identification of immature T lymphocytes or cortical thymocytes, Parent et al. (1994) have reported that severely malnourished children, regardless of the clinical form of protein-energy malnutrition, had a significantly higher proportion of immature T lymphocytes, which correlated with severe involution of the thymus. Interestingly, in vitro incubation of the immature T lymphocytes (CD1a) with thymulin, a Zn-dependent lymphocyte differentiating factor secreted by thymic epithelial cells (Dardenne et al. 1984 ), resulted in the appearance of new, mature lymphocytes (CD3⁺, CD4⁺ and CD8⁺). Based on flow cytometry it also appears that the deficient groups used in this study, particularly the ZnDF+LP mice, have fewer single positive CD4⁺ and CD8⁺ cells, and thus more double negative cells within the CD3⁺ population (Bossuyt and Taylor 1998 ). In the present experiment, it is not known if there was an increase in immature T cells in the lysates used for Western immunoblotting, as the T lymphocyte population was isolated by negative selection on T immunocolumns, a process that would not distinguish between double negative, double positive or single positive T cells. Further experiments are required to address the potential interaction of Zn and p56^lck in thymocyte maturation and whether this contributes to a higher proportion of immature T lymphocytes in Zn deficiency and protein-energy malnutrition.

Activity of p56^lck also contributes to T cell receptor-derived signals that regulate positive selection of thymocytes that are single positive for CD4⁺ or CD8⁺ (Hashimoto et al. 1996 ). Positive and negative selection events result in the elimination of dysfunctional or self-reactive T cells by apoptosis. Jurkat T cell lines stably transfected with a constitutively active form of p56^lck were hypersensitive to apoptosis induced by crosslinking with antibodies to the T cell receptor, whereas cell lines defective for p56^lck were resistant to apoptosis (di Somma et al. 1995 ). In the present experiment, it is possible that overexpression of p56^lck could lead to increased T lymphocyte apoptosis, and this would be consistent with the peripheral lymphopenia of Zn deficiency and malnutrition syndromes. A role for increased apoptosis in the primary lymphoid portion of the bone marrow during Zn deficiency was proposed by Fraker (1996) . However, it is questionable whether the peripheral lymphopenia observed in adult mice after 4 wk of dietary intervention can be attributed to effects within the primary lymphoid organs. The present investigation provides support for the thesis that increased p56^lck expression in T lymphocytes could lead to increased apoptosis of peripheral T lymphocytes and that this may be a mechanism underlying the peripheral lymphopenia that is characteristic of diverse forms of malnutrition.

Atrophy of the spleen and depressed lymphocyte counts are characteristic features of experimental zinc deficiency and protein-energy malnutrition syndromes (Fraker 1996 , Woodward 1998 ). However, the effects of these nutritional deficiencies on immunological parameters have not been investigated simultaneously. In the present study, dietary protein deficiency and diet restriction (LP and DR groups) produced greater splenic atrophy and larger reductions in total splenocyte counts than dietary Zn deficiency (ZnDF and ZnDF+LP groups). Furthermore, the LP group had a significantly lower spleen/body weight ratio and splenocyte counts than the combined ZnDF+LP group, suggesting that the macronutrient deficiency alone was more detrimental than the combined macronutrient-micronutrient deficiency. In other Zn deficiency studies utilizing a pair-fed control, the Zn-deficient mice had significantly lower spleen weights and spleen cell counts and a body weight that was 14–19% less than their pair-fed counterparts (Cook-Mills & Fraker 1993 , Luecke et al. 1978 ). Thus, it would appear that the effects of Zn and protein deficiencies and diet restriction on splenic atrophy and lymphopenia also need to be interpreted in the context of body weight changes. Furthermore, the effects of these deficiencies on lymphoid morphology need to be investigated. In the present study, LP and ZnDF+LP groups had significantly decreased total splenocyte counts; however, the concentration of splenocytes in the spleen (expressed as splenocytes/mg spleen) was not significantly different from the C group (Table 3) . The splenocyte concentration was more affected by ZnDF and DR treatments. It has been suggested that the response of the supporting cell system in lymphoid organs may be partially responsible for the atrophy observed in protein-energy malnutrition (Woodward, 1992 ), but the relative role of this mechanism in the spleen during dietary zinc deficiency, protein deficiency and diet restriction has yet to be determined.

Although mice in the LP and DR groups received a Zn-adequate diet (30 mg Zn/kg), femur Zn concentration (both LP and DR groups) and serum Zn concentration (LP group) were significantly lower than the C group (Table 2) . Depression of serum Zn concentration (30–40%) by feeding a diet deficient only in protein has been previously demonstrated in weanling mice fed a 0.6 or 1.7% protein diet for 14 d (Filteau and Woodward 1982 and 1984 ). Also, children with protein-energy malnutrition have low plasma zinc concentrations (Golden and Golden 1979 ). It has been suggested that physiological changes in the intestinal mucosa brought about by malnutrition may adversely affect zinc absorption as demonstrated by the significantly depressed response to an oral plasma Zn tolerance test by children with kwashiorkor (Atalay et al. 1989 ). It was also suggested that low serum albumin concentrations may be associated with decreased serum Zn concentrations because albumin is an important carrier protein for circulating Zn. Yet, this seems unlikely because serum Zn concentrations were similar in the ZnDF and ZnDF+LP groups, despite the significantly lower serum albumin concentration in the ZnDF+LP group (Table 2) . Furthermore, plasma albumin concentrations were not correlated with low plasma Zn concentrations in malnourished children with severe wasting (Golden and Golden 1979 ). Also, the present study demonstrates that the type of malnutrition was different in Zn deficiency alone (energy-type malnutrition) versus combined Zn and protein deficiency (protein-type malnutrition). Investigating Zn deficiency alone is important for basic mechanistic studies; however, the combined Zn and protein deficiency model has significant clinical relevance because low protein intakes are a major contributor to inadequate Zn intake in the human population (Sandstead 1995 ).

In summary, expression of p56^lck, but not PLC1 and PKC, was elevated in splenic T lymphocytes from mice with Zn deficiency, combined Zn and protein deficiency, and diet restriction. Also, there was a significant correlation between elevated p56^lck expression and reduced Zn status. These results suggest an important interaction between p56^lck and nutritional status that needs to be addressed in future studies investigating thymoctye maturation, apoptosis and lymphopenia in Zn deficiency and protein-energy malnutrition syndromes.

	ACKNOWLEDGMENTS

 
We thank Marilyn Latta, Dept. of Foods and Nutrition, for technical assistance and Sergio Mejia, Dept. of Geological Sciences, for assistance with the image analysis.

	FOOTNOTES

 
⁴ To whom correspondence should be addressed.

¹ Presented in part at the World Conference of the International Society for Molecular Nutrition and Therapy, August 2–4, 1997: Lepage, L. M., Andres, P. J. and Taylor, C. G. Effects of dietary zinc deficiency and protein-energy malnutrition on the T lymphocyte zinc-finger protein p56^lck in mice.

² Supported by International Life Sciences Institute (ILSI) Future Leader Award in Nutrition, Natural Sciences and Engineering Research Council (NSERC) operating grant (PIN 186434), University of Manitoba Research Development grant, and University of Manitoba Research Grant Program to CGT.

³ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

⁵ Abbreviations used: C, control group; DR, diet restricted group; FCS, fetal calf serum; LP, protein-deficient group; NT, anti-human lck kinase N terminal domain; PBS, phosphate-buffered saline; PKC, protein kinase C; PLC1, phospholipase C1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; ZnDF, zinc-deficient group; ZnDF+LP, combined zinc- and protein-deficient group.

Manuscript received June 29, 1998. Initial review completed August 3, 1998. Revision accepted November 19, 1998.

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