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R. Ortiz, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, Avenida San Rafael Atlixco 186 CP 09340, México DF, México. E-mail: firstname.lastname@example.org
Malnutrition is distributed widely throughout the world and is a particular problem in developing countries. Laboratory animals have been very useful in studying the effects of varying levels of malnutrition because non-nutritional factors that affect humans may be controlled. The objective of the present study was to determine the effects of moderate and severe malnutrition on lymphocyte proportions and activation markers of T cells in experimentally malnourished rats during lactation by flow cytometry. Lower absolute (total) and relative (%) numbers of CD3+ and CD4+ lymphocyte subpopulations were observed in moderately (second degree) and severely (third degree) malnourished rats compared with well-nourished rats (P < 0·05). Both groups of malnourished rats showed a significant decrease in the percentage of CD71+ cells at 24 h post-activation with phytohaemagglutinin (PHA). After 24 h activation of spleen cells with PHA, a lower percentage of CD25+ cells was observed in malnourished than well-nourished rats (P < 0·05). In conclusion, the results of this study indicated an altered expression of CD71 and CD25 during activation of T lymphocytes in malnourished rats and may partially explain increased susceptibility to infection associated with malnutrition. Moreover, these results demonstrated that moderate malnutrition affects the response of T lymphocytes as much as severe malnutrition.
Malnutrition occurs as a consequence of inadequate food intake and/or low-protein diets and is distributed widely throughout the world, but particularly in developing countries. It is associated generally with poverty. Individuals of low socio-economic class have little or no access to health services and education and are more vulnerable to malnutrition than those from middle and high socio-economic classes . Approximately 12 million children younger than age 5 years die every year in developing countries, and half of those deaths are related to preventable disease and malnutrition . The effects of malnutrition may be particularly devastating during childhood because of rapid growth rate and increased nutrient requirements .
Laboratory animals have been very useful in studying the effects of malnutrition at different levels, because non-nutritional factors such as infections and drugs that affect human studies may be controlled. In addition, studies may be performed both in vivo and in vitro. Several studies on the effects of malnutrition with experimental animal models have been conducted during the lactation period, which is critical in the growth and development of breastfed pups or infants . The effects of malnutrition during nursing are more severe than those observed in adults [4,6]. Malnutrition has been classified as mild, moderate or severe, according to the degree of weight deficit or altered weight : height ratio [4,7]. Classification by weight deficit is useful in experimental malnutrition animal models.
It has been shown previously that malnutrition reduced in vitro and in vivo bone marrow cell proliferation and increased thymocyte apoptosis significantly in rats [8–10].
The relationship between malnutrition and immunosuppressive effect has been documented in experimental and human malnutrition. Several mechanisms by which malnutrition affects immunity and susceptibility to infection have been described in the literature [11,12]. Several studies have highlighted the fact that even children with mild to moderate malnutrition, not just those with more severe forms, have an increased risk of death [2,13].
Several authors have shown changes in peripheral lymphocyte subsets, but the results of these studies were controversial. In malnourished rodents, a decrease in the CD4+ and CD8+ T cell subsets was found [14,15]. However, other authors have observed that the proportion of CD4+ lymphocytes was not altered, but quiescent CD4+ lymphocytes tended to increase . In other studies, the response of lymphocytes from malnourished children to mitogens was decreased [17–19]. It is important to study the changes in cellular parameters of the immune response in animal models of malnutrition, as it is possible to control for non-nutritional confounding factors [4,10].
T lymphocytes are required for an adaptive immune response. These responses are characterized by the capacity to recognize and remember pathogen-specific antigens. When a foreign antigen is encountered lymphocytes become activated, undergo clonal proliferation and acquire functions that enable the activated cells to eliminate it . Lymphocytes use a complex array of signal transduction molecules to regulate proliferation, differentiation and effector functions. The first step in antigen receptor signal transduction is the activation of cytosolic tyrosine kinases which phosphorylate an array of adaptor molecules, initiating a cascade of signalling pathways .
The expression of cell surface activation antigens is useful in evaluating lymphocyte response to antigens or mitogens. CD25 [interleukin (IL)-2 receptor α-chain] is expressed after T cell activation. CD71 (transferrin receptor) surface expression represents the state of metabolic activity in the cell and must be increased in order to internalize iron required for T cell DNA synthesis and proliferation [22,23]. The effects of malnutrition [19,24] and certain drugs [25–27] on CD25 expression have been investigated previously, but the results are controversial.
The aims of the present study were twofold: (i) to determine the effects of moderate and severe malnutrition during lactation on splenic lymphocyte numbers in the pups; and (ii) to investigate the effects of malnutrition on T cell activation markers (CD25 and CD71), assessed by flow cytometry.
Materials and methods
Experimental malnutrition during lactation
Wistar rats from a closed breeding colony from the Division of Biological and Health Sciences of the Universidad Autónoma Metropolitana-Iztapalapa (UAM-I), México City were used. Rats were kept under 12-h controlled light–dark cycles at 22–25°C with 45% relative humidity. Dams had undergone a second delivery and were fed a balanced diet ad libitum for rodents (2018S; Harlan Teklad, Madison, WI, USA). They were bred in polysulphone cages (Harlan Teklad). Experiments were performed according to the guidelines for the use of experimental animals of the Universidad Autónoma Metropolitana (UAM-I), which are in accordance with those approved by the National Institutes of Health (Bethesda, MD, USA) and the Official Mexican Guidelines (Norma Oficial Mexicana NOM-062-ZOO-1999).
One-day-old Wistar rats from different litters were assigned randomly to either the experimental or control groups. In the control group pups were fed from nursing dams, each suckling six to eight pups. In the experimental or malnourished group, each nursing dam fed 16 pups. Pups were assigned to another dam 1 day after birth and the dams accepted the young without demur. The same proportion of male to female pups per litter was assigned to each group. Pups were weighed every 2 or 3 days and we estimated mean body weight in each litter from the first day until weaning (day 21). Well-nourished rats were selected from 10 different control litters and malnourished rats were selected from 10 different experimental litters. In the experimental group, the degree of malnutrition was established according to the classification used for children described by Gómez et al. . The number was categorized as mild or first degree when the weight deficit reached 10–25%; moderate or second degree (MN2nd) when the weight deficit reached 25–40%; and severe or third degree (MN3rd) when the weight deficit was greater than 40% of that of age-matched control rats (WN). These rats also had other physical signs of malnutrition, such as sparse hair, bone fragility and low activity levels.
The antibodies utilized were fluorescein isothiocyanate (FITC)–anti-CD3, phycoerythrin (PE)–anti-CD25, allophycocyanin (APC)–anti-natural killer (NK)R-P1A (Caltag Laboratories, Burlingame, CA, USA), Cy-Chrome (CyC)–anti-CD45RA, PE-anti-CD8, CyC-anti-CD4, and PE–anti-CD71 (Pharmingen-BD, San José, CA, USA).
Spleen cells were obtained by sieving the tissue through a nylon screen  and cells were suspended in Iscove's medium. Cell viability was determined with Trypan blue (Microlab, México City, México) and more than 95% of the cells were viable.
Staining lymphocyte subpopulations
Specific staining of the respective cell surface molecules was performed with (i) anti-rat CD3-FITC, anti-rat CD45RA CyC and anti-rat NK APC or (ii) anti-rat CD8 PE and anti-rat CD4 CyC. One million cells were incubated for 30 min with the recommended amount of antibodies in the dark at room temperature. As controls, FITC- and PE-labelled non-specific mouse immunoglobulin G1 antibodies were used to establish background fluorescence. The cells were washed with 1% bovine serum albumin (BSA; Sigma Chemical Co., St Louis, MO, USA) and prepared in phosphate-buffered saline (PBS; Microlab). Finally, cells were fixed in 1% paraformaldehyde (Sigma Chemical Co., St Louis, MO, USA) prior to analysis.
Cell culture and staining of activation cell surface antigens
Lymphocytes were isolated from spleen using a density gradient (Linfograd; Microlab). Spleen cells were cultured (5 × 106 cells/ml) for 24 h at 37°C and 5% CO2 in Iscove's medium without glutamine (Gibco, Carlsbad, CA, USA) and with or without phytohaemagglutinin (PHA 25 µg/ml; Sigma Chemical Co.). Following culture, 1 million cells were incubated with (i) anti-rat CD3 FITC, CD4 CyC and anti-rat CD71 PE or (ii) anti-rat CD3 FITC, CD4 CyC and anti-rat CD25 PE for 30 min at room temperature. The cells were washed with 1% BSA in PBS and fixed with 1% paraformaldehyde prior to analysis.
Flow cytometry analysis
After fixation, four-colour flow cytometry was performed using a fluorescence activated cell sorter (FACSCalibur) flow cytometer (BD, San José, CA, USA). A minimum of 10 000 events were acquired and analysed with CellQuest software (BD). Figure 1 shows how gates were set on the forward-scatter–side-scatter (SSC) to acquire all lymphocytes FITC-SSC distribution for gate on the basis of anti-CD3 labelling (gate on CD3). These gates were used to analyse CD71 or CD25 expression.
Results are expressed as mean ± standard error. All groups were compared by the non-parametric Mann–Whitney U-test. Level of significance was set at values of P < 0·05.
Experimental malnutrition during lactation
Table 1 shows the mean and standard deviation of body weight, spleen weight and number of cells per spleen for all groups of rats. At weaning age, the average weight deficit between WN and MN2nd was 33·7% and the average weight deficit between WN and MN3rd was 50·7%. The body and spleen weight and the number of spleen cells were significantly lower (P < 0·05) in both groups of malnourished rats compared with WN rats. However, the mean numbers of cells/100 mg of spleen were not statistically significant among the three groups.
Table 1. Body weight, spleen weight and spleen cell number (×106) of well-nourished and malnourished rats.
Values are means ± standard deviation. Means or values followed by different superscript letters are statistically significant. †Statistical differences from well-nourished (WN) group, P < 0·05;‡statistical differences from moderately (MN2nd) and severely (MN3rd) malnourished rats, P < 0·01. WN, n = 12; MN2nd, n = 10; MN3rd, n = 10.
Table 2 summarizes the data of isolated splenic lymphocyte subpopulations (T, B and NK) in WN and MN rats. Significantly lower percentages of CD3+ lymphocytes were observed in malnourished than in well-nourished rats (P < 0·05). In the same manner, total lymphocytes decreased significantly (P < 0·05); conversely, non-lymphoid cells present in the same region were increased (P < 0·05).
Table 2. Splenic lymphocytes from WN, MN2nd, and MN3rd rats.
(% ± s.e.)
Cells that appear in region of analysis. Data are based upon flow cytometric analysis of 10 000 events and are means ± standard errors (SE).
Statistical differences from WN, P < 0·05: CD3+ moderately (MN2nd) and severely (MN3rd); total lymphocytes (MN3rd) malnourished rats; non-lymphoid cells (MN3rd).
The CD3+ CD4+ and CD3+ CD8+ subpopulation are shown in Table 3. The percentages of CD4+ lymphocyte subset were significantly lower (P < 0·05) in both groups of malnourished rats than in WN rats. However, there was no statistically significant difference between the MN groups. Although the percentage of CD8+ cells tended to be elevated in malnourished rats compared with well-nourished animals, the increase was not statistically significant partly because of large standard deviations. The CD4– CD8– subpopulation increased in MN3rd rats in comparison with WN rats.
Table 3. T (CD3+) splenic lymphocyte subpopulations of WN, MN2nd and MN3rd rats.
(% ± s.e.)
Significantly different from WN rats, P < 0·05: in CD4+ moderately (MN2nd); in CD4+ and in CD4- CD8- severely (MN3rd) malnourished rats.
Figure 2 shows the percentage of T lymphocytes (CD3+) from cultured spleen cells. A small but significant difference was found between PHA-stimulated WN CD3+ lymphocytes and unstimulated lymphocytes. No significant difference was observed in PHA-stimulated and unstimulated lymphocytes between WN and MN groups. When spleen cells were incubated with PHA, the percentage of CD3+ cells tended to be higher in severely than in moderately malnourished rats. However, the difference between both groups was not statistically significant.
Effect of culture and malnutrition on expression of CD71+ cells
The percentages of activated CD3+ CD71+ cells are presented in Fig. 3a. No difference was observed between the groups for freshly isolated CD3+ CD71+ splenocytes. Malnutrition affected negatively the increase of CD71+ expression in CD3+ CD71+ spleenocytes cultured for 24 h with or without mitogen (Fig. 3a). Following cell activation, the mean percentage of CD3+ cells was higher in well-nourished than in malnourished rats (P < 0·05).
Effect of culture and malnutrition on expression of CD25+ on cells
CD3+ CD25+ splenocyte percentages are shown in Fig. 3b. No significant difference in CD25 expression was observed for freshly isolated cells, with 2·1% in WN rats, 2·0% for MN2nd, and 2·0% for MN3rd. Similar percentages were obtained for splenocytes cultured for 24 h without mitogen. After in vitro activation, the increase in CD3+ CD25+ cells was lower in MN rats. No significant difference was observed between the MN2nd and MN3rd group, probably because of large variations within treatment groups. There was a positive correlation between spleen weight and the percentages of spleen cells expressing CD71 (R2 = 0·97) receptors (Table 1 and Fig. 3).
In this study, we evaluated the ability of splenic T lymphocytes to become activated in moderately and severely malnourished rats in order to identify possible malnutrition-degree-related effects. The effects of malnutrition were assessed during lactation. Several studies have demonstrated the importance of this period, where the effects are more severe than those observed in adults and malnutrition in early life produces adverse consequences in adulthood . We used a well-established method for the induction of malnutrition in rats during the lactation period, in which malnourished rats mimicked the syndrome of malnutrition in children . Flo et al.  showed that malnutrition during the suckling period decreased the percentage of CD4+ and CD8+ cells in the gut-associated lymphoid tissues. It was reported that the decrease in K+ conductance by alteration of K(V) channels was associated with inhibition of activation of T lymphocytes in severely malnourished animals .
Previous studies in adult animals have reported that the number of splenic lymphocytes decreased during caloric restriction . Similar results were obtained for animals on a low and free protein and micronutrients-deficient diet (zinc copper, vitamins, etc.) [32–34]. In contrast to specific deficiencies, in the present study we evaluated the reduced intake of all nutrients in the lactation period. It has been pointed out that the analogous murine model of multiple nutrient deficiencies is similar to human malnutrition in developing regions of the world .
The results of the present study showed a decrease in the percentage of T lymphocytes (CD3+ cells) in the spleens of moderately and severely malnourished rats. Because statistical analysis showed that the percentage of lymphocyte subsets was reduced significantly in both experimental groups, our results suggest that severe and moderate malnutrition are equally deleterious. The decrease in the percentage of T lymphocytes observed in malnourished rats is probably related to reduced proliferation in bone marrow cells [8,9], and/or a defect in the thymus, and/or an increase in apoptosis .
Phytohaemagglutinin is a lectin composed of five tetramers derived from the combination of two subunits, namely erythroagglutinin PHA and leucoagglutinin PHA, which are associated non-covalently . It is used frequently as a mitogen in studies of lymphocyte activation and function. It recognizes the CD3 receptor and can influence the initiation and regulation of lymphocyte activation and proliferation . Furthermore, Kontny  showed that CD4+ and CD8+ are stimulated similarly by PHA. Lectin-stimulated T lymphocytes have been observed using CD71+ and CD25+ labelling [39–41].
Our data also showed a significantly reduced percentage of CD3+ CD71+ lymphocytes in moderately and severely malnourished rats after 24 h of culture. Cell surface transferrin receptor (CD71) is required for iron uptake through receptor-mediated endocytosis of diferric transferrin. This function is essential for cell proliferation, because iron must be available for the synthesis of haem- and iron-containing proteins . In addition, CD71 appears to play a co-stimulatory role in T cell activation  and in the formation of the immunological synapse . Low expression of CD71+ in T cells in malnourished subjects may contribute to the poor immunological response observed in malnutrition.
Expression of the α subunit of the IL-2 receptor (CD25) is induced in activated T lymphocytes, leading to an increased sensitivity to IL-2. IL-2 drives cellular proliferation and also has an important role in protecting immune cells from cell death by inducing anti-apoptotic mechanisms . The percentage of CD25+ T lymphocytes was decreased after 24 h of culture in both malnourished groups. These data may explain the epidemiological studies that showed increased risk of death of children with moderate malnutrition because of impaired immunity [2,13].
The lower proportion of CD71+ and CD25+ cells in splenic T lymphocyte populations in both MN groups compared with the WN group indicates a decreased response to mitogen. Two previous studie have demonstrated a decrease in splenic T lymphocyte proliferative response to mitogens in diet restriction in mouse  and in in vivo responses to pulmonary tuberculosis in guinea pigs [46,47]. Studies of lymphocytes from malnourished children showed a defective expression of activation markers. Nájera et al.  observed impaired expression of CD69 in activated lymphocytes. In addition, Rodríguez et al.  demonstrated that lymphocytes had reduced expression of activation markers − both CD69, an early activation marker and CD25, which is expressed later in response to stimuli. Moreover, the capacity of CD4+ and CD8+ cells to produce cytokines in response to stimuli was impaired. The generation of protective T cell responses to infectious agents is a complex process whereby cytokines and co-stimulatory molecules provide signals that direct the development of adaptive immunity . If malnutrition alters this complex process, the organism is vulnerable to infections. The results obtained in this and other studies [18,19] suggest that lymphocytes from malnourished subjects are unable to become activated fully and have defects in cytokine production.
Mild and moderate malnutrition also affects immunological status. In 1981, McMurray et al. provided evidence that there was a reduction in cell-mediated and humoral immune function in lymphocytes from children with mild and moderate malnutrition . Rodríguez et al.  observed lower ability of T lymphocytes to become activated in moderate and severe malnourished children, but did not observe a correlation between the degree of malnutrition. The results obtained in this study agree with this observation. It is important to study the effect of mild and moderate malnutrition because the prevalence of second degree malnutrition is higher than severe malnutrition [51,52].
An interesting finding from this study is that moderate or second degree malnutrition alters the activation of cultured splenic lymphocytes and should be investigated further. Preliminary results (data not shown) suggest that both CD4+ and CD8+ lymphocytes are affected. Further studies will also be necessary to confirm the alteration of CD71 and CD25 expression on both subsets of activated T lymphocytes and to evaluate the proliferative ability of activated cells.
In conclusion, the results of the current study suggest that moderate malnutrition is as bad as severe malnutrition with regard to lymphocyte function because it also reduced significantly the expression of lymphocyte activation markers. The data may explain, in part, the increased susceptibility to infection associated with malnutrition.
We thank M. V. Z Rocío González for the animal facilities and Dr Mario Altamirano for his suggestions. This work was supported in part by grants from FOMES (México), grant 98-35-28, and SEP (México), grant PIFI 2003-35-19.