Protein-energy malnutrition (PEM)⁵ is associated with decreased antioxidant and immune defense. In experimental animal models of PEM, increased susceptibility to oxidative stress from xenobiotics and pulmonary oxygen toxicity has been linked to decreased liver and lung concentrations of glutathione (GSH) (Deneke et al. 1985, Jung 1985, Taylor et al. 1992). Children with PEM have compromised GSH status as indicated by significantly decreased blood GSH concentrations that return to control values after treatment and recovery from PEM (Golden and Ramdath 1987, Jackson 1986, Sive et al. 1993). Protein-energy malnutrition is also characterized by a high incidence of opportunistic infections and diarrhea. However, the effects of PEM on GSH status and functioning of the immune system have not been addressed in these studies. In human immunodeficiency virus (HIV)-infected patients with wasting malnutrition, decreased GSH concentrations have been reported in T-lymphocytes, mononuclear cells, plasma and lung epithelial lining fluid (Buhl et al. 1989, Eck et al. 1989). T-lymphocyte GSH concentrations are decreased even though only a small percentage of T-lymphocytes are actually infected with HIV (Staal et al. 1992). However, it is difficult to differentiate the effects of wasting malnutrition and those of HIV or other infections on GSH status and immune function.

The hypothesis that decreased GSH status in PEM may contribute to impaired immune defense is based on several lines of evidence (Bray and Taylor 1994). Spleen GSH concentration is decreased in a rat model of PEM (Bray and Taylor 1994), and a role for GSH in immune function has been demonstrated in a variety of in vitro systems investigating T-lymphocyte activation, proliferation and interleukin-2 (IL-2). For example, depletion of lymphocyte GSH in vitro by conjugation with 1-chloro-2,4-dinitrobenzene or by alkylation with N-ethylmaleimide inhibits T-cell receptor-induced mobilization of intracellular calcium and tyrosine phosphorylation of several proteins, including phospholipase C1 (Kanner et al. 1992, Kavanagh et al. 1993). Glutathione supplementation of culture medium modulates the synthesis and turnover of surface IL-2 receptors (Liang et al. 1992). When T-lymphocytes are sorted on the basis of GSH content and stimulated with mitogen, T-lymphocytes with low levels of GSH have a lower capacity to activate and proliferate in vitro (Kavanagh et al. 1990).

Given that there is considerable evidence that in vitro manipulation of intracellular GSH alters T-lymphocyte function, the goal of the present study was to investigate the effects of in vivo GSH depletion and repletion on tissue GSH concentrations and on T-lymphocyte functions. Synthesis of GSH (L--glutamyl-L-cysteinylglycine) is primarily dependent on availability of cyst(e)ine and methionine in the diet (Taylor et al. 1996). Thus, dietary sulfur amino acid deficiency (0.5% protein) can be used as an in vivo model of tissue GSH depletion (Taylor et al. 1992 and 1996) to investigate the relationship of GSH status (assessed in various tissues and splenocytes) to functional responses of T-lymphocytes. In the present study, immunologically mature young adult mice (3-4 mo) were fed a 0.5% protein diet for 4 or 6 wk until significant weight loss occurred; however, it cannot be assumed that the 0.5% protein dietary regimen in young adult mice will produce depressed thymus-dependent immunity and edematous PEM as previously reported in weanling mice fed a 0.5% protein diet for 21 d (Filteau and Woodward 1987, Woodward and Miller 1991). Thus, the objectives of the present investigation were 1) to assess GSH status in various tissues and splenocytes, and CD3-stimulated calcium mobilization and cell proliferation of splenocytes from mice fed 0.5% protein (LP group) or 15% protein (CP group) diets for 4 or 6 wk, and 2) to determine the effects of dietary repletion (3 d, 1 wk, 2 wk and 3 wk) with the 15% protein control diet on GSH concentration, CD3-stimulated calcium mobilization and cell proliferation of splenocytes from mice with PEM.

MATERIALS AND METHODS

Table 1. Ingredient compositions of the 0.5% (LP) and 15% (CP) protein diets1 [View Table]

Table 2. Body weight and organ weights of mice fed 0.5% (LP) or 15% (CP) protein diets for 4 or 6 wk1 [View Table]

Table 3. Spleen cell numbers and subpopulations of mice fed 0.5% (LP) or 15% (CP) protein diets for 4 or 6 wk1 [View Table]

RESULTS

Glutathione was measured in various organs (Table 4) and in gradient-isolated splenocytes (Fig. 1). Glutathione concentrations were significantly lower in liver, lung, heart and spleen of the LP group compared with the CP group at both 4 and 6 wk (Table 4). Splenocyte GSH concentration in the LP group was significantly lower by 32% at 4 wk and 64% at 6 wk compared with the CP group (Fig. 1). The close agreement in the reduction of spleen (27%) and splenocyte (32%) GSH concentration in the LP group at 4 wk (Table 4 and Fig. 1) provides evidence that the splenocyte isolation procedure does not significantly alter GSH concentration.

Table 4. Tissue glutathione (GSH) concentrations of mice fed 0.5% (LP) or 15% (CP) protein diets for 4 or 6 wk1 [View Table]

CD3-stimulated intracellular calcium mobilization was measured in CD4⁺ (T-helper) and CD8⁺ (T-suppressor) splenocytes (Table 5). In the CD4⁺ subset, CD3-stimulated calcium mobilization was not significantly different in LP and CP mice. In the CD8⁺ subset, however, less CD3-stimulated intracellular calcium mobilization was observed in splenocytes from the LP group compared with the CP group at both 4 and 6 wk.

Table 5. CD3-stimulated intracellular calcium mobilization in CD4+ or CD8+ splenocytes from mice fed 0.5% (LP) or 15% (CP) protein diets for 4 or 6 wk1,2 [View Table]

Proliferation assessed by the BrdU-Hoechst assay is shown in Figure 2. CD3 monoclonal antibody to the T cell receptor was used to stimulate T cell proliferation of splenocytes cultured in media with and without supplementation of 2-ME or IL-2 for 72 h. In the absence of 2-ME, T-lymphocyte proliferation was significantly lower in the LP group than in the CP group at both 4 and 6 wk. Thiol supplementation of culture media with 2-ME increased T cell proliferation in splenocytes from both LP and CP groups. The percentage of increase in T cell proliferation in the presence of 2-ME was greater in splenocytes from the LP group (188-225%) than in splenocytes from the CP group (53-75%), and this resulted in a higher percentage of proliferating cells in the LP group than the CP group under this condition. Addition of IL-2 to the culture media did not alter the proliferation rates of splenocytes from LP and CP mice in the absence or presence of 2-ME. Similar trends were observed when cell proliferation was assessed by [³H]thymidine incorporation (data not shown).

Table 6. Effect of dietary repletion (RP) on body weight, spleen weight and splenocyte number in mice previously fed the 0.5% protein diet (LP) for 4 wk compared with mice fed the 15% protein diet (CP) for 7 wk1 [View Table]

Splenocyte GSH concentration (Fig. 3) and functional variables, including CD3-stimulated Ca mobilization (Table 7) and proliferation (Fig. 4), were determined during the RP experiment. Glutathione concentration was significantly lower in splenocytes from LP mice than in splenocytes from CP mice (Fig. 3). Dietary RP for 1 wk or longer significantly increased splenocyte GSH concentration to levels equivalent to that of the CP group. Similar results were obtained when splenocyte GSH concentration was expressed per cell number (Fig. 3) or per milligram of protein (data not shown). CD3-stimulated intracellular Ca mobilization in CD4⁺ or CD8⁺ T-lymphocytes was not significantly altered by LP or RP (Table 7). The CD3-stimulated proliferation response of the splenocytes was assessed in vitro using the BrdU-Hoechst proliferation assay. Thiol supplementation of culture media with 2-ME produced a significantly higher percentage of proliferating cells regardless of dietary treatment (Fig. 4). The proliferation response after 2-ME supplementation was greater in splenocytes from LP mice (183%) than splenocytes from CP mice (61%). After 3 d RP, the proliferation response in the presence of 2-ME was similar (83%) to that for the splenocytes from the CP group (61%).

Table 7. Effect of dietary repletion (RP) on intracellular calcium mobilization in mice previously fed the 0.5% protein diet (LP) for 4 wk compared with mice fed the 15% protein diet (CP) for 7 wk1,2 [View Table]

DISCUSSION

The LP mice had reduced GSH status as indicated by lower GSH concentrations in liver, lung, heart, spleen and splenocytes (Table 4). The 0.5% protein dietary regimen has been used previously as a model for tissue GSH depletion in rats (Taylor et al. 1992). The present study demonstrates that depletion of tissue GSH in this form of PEM includes depletion of GSH in mouse spleen and splenocytes (Table 4, Fig. 2 and 4). Glutathione concentration was assayed in the entire splenic mononuclear population using the Tietze recycling assay (Baker et al. 1990). Decreased T-lymphocyte GSH has been reported in HIV patients with wasting malnutrition; however, only the relative intracellular GSH content of T-lymphocytes, not absolute GSH concentration, was obtained using a flow cytometric assay (Roederer et al. 1993).

T-lymphocyte proliferation in vitro and splenocyte cell number in vivo seem to be associated with GSH status. The LP mice had significantly lower splenocyte cell numbers (Tables 3 and 6), lower splenocyte GSH concentrations (Fig. 1 and 3) and lower CD3-stimulated T-lymphocyte proliferation in unsupplemented culture media (Fig. 2 and 4). Although the entire splenic mononuclear population was used in the proliferation assay, the CD3 monoclonal antibody would stimulate only T-lymphocytes. Decreased lymphocyte proliferation and/or increased apoptosis probably occurs in vivo in mice with PEM as indicated by the substantial reduction of spleen cell numbers in LP mice (Tables 3 and 6). Dietary protein repletion restored both splenocyte GSH concentration (Fig. 3) and receptor-mediated T-lymphocyte proliferation in unsupplemented culture media (Fig. 4) by 1 wk RP. Splenocyte number increased 1.6-fold after 3 d RP, but interestingly, the splenocyte number had recovered to only 70% of that for CP mice after 3 wk RP (Table 6). Significant reductions in nucleated spleen cells and primary acquired cell-mediated immunity (assessed by delayed hypersensitivity to sheep red blood cells) have been reported in a mouse model of preadolescent wasting PEM (Woodward et al. 1992). However, tissue or lymphocyte GSH status has not been previously reported in investigations of immunological function in animal models of PEM. The intracellular GSH concentration seems to be important for the proliferation of lymphocytes and other cell types in in vitro proliferation assays. When CD4⁺ T-lymphocytes from normal human donors were sorted by flow cytometry on the basis of GSH content, the proliferative response was lower in CD4⁺ T-lymphocytes containing lower levels of GSH (Kavanagh et al. 1990). Also, fibroblasts and endothelial cells have decreased intracellular GSH concentrations during quiescence compared with the proliferative growth phase (Mallery et al. 1993, Shaw and Chou 1986).

Thiol supplementation of culture media with 2-ME substantially increased the receptor-mediated proliferation response of T-lymphocytes regardless of dietary treatment (Fig. 2 and 4). However, T-lymphocytes from LP mice displayed a significantly greater enhancement of proliferation by in vitro thiol supplementation than did T-lymphocytes from RP or CP mice (Fig. 2 and 4). The greater proliferative response of splenocytes from LP mice than of splenocytes from CP mice in thiol-supplemented culture media (Fig. 2) may represent either greater intrinsic proliferative capacity of the supplemented LP cells, or alternatively may be due to the slightly greater proportion of CD4⁺ cells in the spleens of LP mice (Table 3). It has been demonstrated previously that 2-ME facilitates the transport of cyst(e)ine from culture media into cells as the mixed disulfide of 2-ME and cysteine (Ishii et al. 1981). Addition of 2-ME or cysteine to culture media increases transport of cysteine into resting or activated lymphocytes and retards time-dependent decreases in intracellular GSH during in vitro culture (Ishii et al. 1987, Zmuda and Friedenson 1983). Co-addition of 2-ME (50 µmol/L) and cysteine (500 µmol/L) acts synergistically to significantly increase intracellular GSH concentration and the proliferative response of concanavalin A-stimulated mouse splenocytes compared with adding either 2-ME or cysteine alone (Zmuda and Friedenson 1983). In agreement with our results that T-lymphocytes from LP mice display greater in vitro sensitivity to 2-ME during proliferation (Fig. 2 and 4), Noelle and Lawrence (1981) reported that 2-ME exhibits the greatest enhancement of concanavalin A-stimulated cell proliferation on lymphocytes with relatively lower concentrations of intracellular GSH.

Thiol supplementation of culture media may increase the proliferative response of lymphocytes by additional mechanisms. Many of the genes involved in cell proliferation are regulated by transcription factors, such as NFkB, that are sensitive to thiol redox status for DNA binding activity (Droge et al. 1991). It has also been reported that the synthesis and turnover of surface IL-2 receptors may be sensitive to thiol redox status (Liang et al. 1992). In the present study, however, the CD3-stimulated proliferation response of T-lymphocytes from LP or CP mice was not altered by addition of IL-2 to culture media in the presence or absence of 2-ME (Fig. 2). Thus, it seems that the amount of IL-2, the number of IL-2 receptors and binding of IL-2 to IL-2 receptors were not limiting factors for in vitro proliferation of T-lymphocytes from LP or CP mice in this study. The enhanced proliferation response of T-lymphocytes from LP mice in the presence of 2-ME suggests that accessory cells from LP mice were not a limiting factor for the proliferation response measured in this assay.

The early steps of the T-lymphocyte signal transduction pathway can be assessed by mobilization of calcium, an important second messenger. In this study, CD3-mediated Ca mobilization in CD4⁺ T-lymphocytes, but not CD8⁺ T-lymphocytes, was maintained in LP mice (Table 5). In other studies, depletion of lymphocyte GSH in vitro by conjugation with 1-chloro-2,4-dinitrobenzene or by alkylation of thiol groups with the permeant sulfhydryl-reactive nonpolar maleimide N-ethylmaleimide inhibited receptor-induced mobilization of intracellular calcium and tyrosine phosphorylation of several proteins, including phospholipase C1 and an associated 35-kDa protein (Kanner et al. 1992, Kavanagh et al. 1993). In the setting of weight loss (wasting malnutrition), decreased spleen cell numbers and decreased splenocyte GSH (especially after 6 wk of consumption of the LP diet), it is perhaps surprising that lymphocyte proliferation was not reduced to a greater extent than observed and that Ca mobilization in CD4⁺ T-lymphocytes was unaffected. We hypothesize that the in vivo survival of lymphocytes in PEM may be determined in part by selection of lymphocytes that maintain the functional capacity to respond to receptor stimulation, mobilize Ca and proliferate. We speculate that this selection process may be an adaptive response to minimize loss of immune cell function during wasting malnutrition and might be mediated by apoptotic death of those cells least capable of maintaining cellular redox status (Sato et al. 1995).