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Institute of Nutrition and Food Technology, University of Chile, and University Iberoamericana, Santiago, Chile
Address all correspondence and requests for reprints to: Dr. Liana Schlesinger, Institute of Nutrition and Food Technology, Casilla, 138-11 Santiago, Chile. E-mail: . lschlesi{at}uec.inta.uchile.cl
Abstract
Circulating leptin levels, proinflammatory and T helper cells type 1 (Th1), Th2 cytokine production, and lymphoproliferative response were measured in 15 infants with primary moderate protein calorie malnutrition on admission and after recovery of 10% of weight. Sixteen healthy, well nourished infants of comparable age served as controls. A significant deficit in the z-score of weight for age, weight for height, body mass index, and circulating leptin and growth factors were detected in malnourished infants on admission compared with controls (P < 0.05). These deficits were associated with a significant suppression of the lymphoproliferative response, Th1, and proinflammatory cytokine production (P < 0.05). After a 10% weight gain, a significant increase in circulating leptin levels was produced in parallel with a significant increase in Th1 activity, as revealed by an enhancement in interferon-
and a suppression in IL-4 production. Concomitantly, the lymphoproliferative response became similar to that detected in control infants. Furthermore, a significant increase in IL-1 and TNF
production compared with that at admission was shown. These findings suggest an association between the increase in leptin and the immunological recovery observed following refeeding of malnourished infants.
ACUTE NUTRITIONAL deprivation affects immune function; in addition, it rapidly reduces circulating leptin in mice and humans (1, 2, 3). Furthermore, impaired cell-mediated immunity has been described in ob/ob and db/db mice, which are known to be defective in leptin or its receptor, respectively (4, 5, 6). Lord et al. (6) reported that the in vitro addition of leptin to mixed lymphocyte cultures increases the proliferative response of T lymphocytes and regulates the T helper type 1 (Th1)/Th2 balance. Moreover, Howard et al. (7) in a recent publication demonstrated that the regimen of recombinant leptin administration during acute starvation was able to completely reverse the effect of starvation on thymic involution and protected against the starvation-induced changes in both thymocyte number and subpopulations proportions. Also, these researchers showed that peripheral administration of recombinant leptin to ob/ob mice reduced thymocyte apoptosis and substantially increased both thymic cellularity and the CD4+ CD8+/CD4- CD8- ratio (7).
Recently, decreased levels of leptin have been demonstrated in severe malnourished infants by Soliman and co-workers (8); however, no link between this decrease and in vivo immune responses has been established to date, despite the fact that a great body of evidence indicates that chronic nutritional deprivation suppresses cellular immune function (9, 10, 11, 12, 13).
The purpose of this study was to investigate whether the decrease in leptin levels is associated with alterations of cell-mediated immunity and with proinflammatory and Th1/Th2 balance in human protein calorie malnutrition (PCM) of moderate severity. Furthermore, to confirm these findings, cell-mediated immunity, proinflammatory cytokines, and the Th1 and Th2 profile were also determined when a significant increase in the circulating leptin level was observed in malnourished infants.
Subjects and Methods
Subjects
In 15 infants with primary moderately intense PCM (seven males and eight females) leptin, growth factor levels, proinflammatory, Th1 and Th2 cytokine production, and lymphoproliferative responses were determined on admission to a Close Nutritional Recovery Center of the Chilean Nutrition Foundation, and after a 10% weight gain. All infants had a birth weight over 2500 g and were free from infections at the time of study (C-reactive protein, <10 mg/liter). Sixteen healthy, well nourished infants (seven males and nine females) of comparable age served as the control group. Informed consent was obtained from the mothers after the aims of the project had been fully explained to them. This study was approved by the ethics in human research committee of the Institute of Nutrition and Food Technology, University of Chile (Santiago, Chile).
Nutritional status
Anthropometric measurements, weight, length, body mass index (BMI) (kilograms per square meter), and skinfold thickness were determined by a registered nurse on admission and after a 10% weight gain. Nude weight was obtained before the first morning feeding with an infant scale (Detecto, Brooklyn, NY) with a precision of 5 g. Body length to the nearest 0.1 cm was determined in the recumbent position using a portable infantometer. The means of three measurements for weight and length were evaluated comparing them to the National Center for Health Statistics standard. Weight for age (W/A) and weight for height (W/H) z scores were calculated with the PCTL9Z anthropometric subroutine (Center for Health Promotion and Education, National Center for Disease Control and Prevention, Atlanta, GA). The percentage of body fat was calculated by skinfold measurements using a Lange caliper (Cambridge, MD, and Washington DC) (14).
Endocrinological measurements
A venous blood sample was obtained after an overnight, 6-h fast, and serum was separated and kept frozen at -20 C until analysis of leptin and growth factors was performed. Serum leptin was measured by RIA (Linco Research, Inc., St. Charles, MO) as previously described and validated (15). The intra- and interassay coefficients of variation were 3.4 and 3.6 ng/ml, respectively.
IGF-I and IGF-binding protein-3 (IGFBP-3) were determined by ELISA. The IGF-I intra- and interassay coefficients of variation were 4.5% and 7% respectively. The IGFBP-3 intra- and interassay coefficients of variation were 4.2% and 6.8%.
Cytokine measurements
Induction of IL-1 TNF and IL-6.
Peripheral blood mononuclear cells (PBMC) were isolated by Hypaque gradient (Sigma, St. Louis, MO) and washed twice in PBS, and 2.5 x 106 cells/ml were resuspended in RPMI 1640 culture medium supplemented with gentamicin, glutamine, 0.5% heat-inactivated AB plasma, and 1 µg/ml indomethacin. Two hundred microliters of cell suspension were dispensed in 96-well flat-bottom microtiter plates in the absence (spontaneous cytokine release) or the presence of endotoxin [lipopolysaccharide (LPS), 1 µg/ml]. Cultures were incubated for 18 h at 37 C in 5% CO2 and 95% air. Supernatants were collected and kept at -70 C until assays for released cytokines were performed.
Induction of Th1 and Th2 cytokines. The same methodology for cell isolation and culture conditions was applied for induction of these types of cytokines; their production per well was stimulated with 5 µg PHA, 5 ng phorbol acetate myristate, and 0.2 µg ionomycin, and the incubation period was 48 h.
Cytokine determinations
All cytokine determinations were performed using specific, commercial, ELISAs (R\|[amp ]\|D Systems, Inc., Minneapolis, MN). The assay sensitivity was 3.6 pg/ml.
Lymphoproliferative assay
Isolated PBMC were cultured in the presence of PHA and concanavalin A (Con A) for 3 and 5 d, respectively. Unstimulated cells served as controls. Eighteen hours before harvesting, the cells were incubated with 18.5 GBq tritiated thymidine (specific activity, 247.9 GBq/mol) and harvested in an automatic cell harvester (16). Results were expressed as counts per minute.
Statistical analysis
Parametric results were analyzed using Student’s paired and unpaired tests. Nonparametric data were logarithmically transformed to yield normal distributions before parametric analyses were performed. P value less than 0.05 was accepted as statistically significant.
Results
Anthropometric determinations
Table 1
shows a significant deficit in W/A, W/H, BMI, and percentage of sc fat in the malnourished infants on admission compared with well nourished controls (P < 0.05). A significant increase in the W/A z-score and BMI was induced after 30 d of refeeding, the time interval necessary for 10% weight gain.
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Serum leptin levels and IGF-I were significantly lower in the PCM children on admission (Fig. 1). After a 10% weight gain, PCM infants experienced significant increases in serum leptin and IGF-I levels, reaching the values observed in the control group. IGFBP-3 had a similar behavior, with mean ± SD values on admission and after 10% weight gain of 2703 ± 333.3 and 2959 ± 343.6 ng/ml (P < 0.05, respectively; mean ± SD of control group, 3213 ± 146.7 ng/ml; P < 0.05 vs. PCM admission values).
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). A similar situation was observed when the correlation between leptin and IGF-I levels was evaluated in PCM infants (Fig. 3); a significant correlation became apparent after 10% weight recovery in PCM children (Fig. 3).
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TNF production by LPS-stimulated PBMC of malnourished infants was similar to that in the control group on admission, increasing significantly after the 10% weight gain and reaching higher levels than in controls (P < 0.05, by t test; Fig. 4). IL-1 production by LPS stimulated PBMC from PCM infants tended to be low on admission compared with that in well nourished controls (0.05 > P < 0.1, by t test; Fig. 4). When 10% weight had been achieved, IL-1 production was comparable to that in the control group, with a tendency to become significant compared with the production on admission (0.05 > P < 0.1, calculated by paired t test). Malnourished infants released comparable quantities of IL-6 as well nourished controls on admission and after the 10% weight gain. No differences in spontaneous production of IL-1, TNF, and IL-6 from PBMC were detected between the groups in any period of time.
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Interferon- (IFN) production by mitogen-stimulated PBMC from PCM infants was similar to that in the control group on admission. However, a significant increase (P < 0.05, by t test and paired t test) was observed after the 10% weight gain compared with well nourished infants and with PCM on admission, respectively (Fig. 5). On the other hand, a significantly higher production of IL-4 was detected in PCM subjects compared with well nourished controls on admission (P < 0.05, by t test). After recovery of 10% of initial weight, a significant decrease in the production of this cytokine was shown (P < 0.05, by paired t test). No differences in spontaneous production of IFN and IL-4 by PBMC were detected between the groups in any time period.
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Malnourished infants showed a significant decrease in the lymphoproliferative response to PHA and Con A on admission compared with controls (P < 0.05). After gaining 10% of initial weight, this response increased to the levels observed in the well nourished group (Table 2).
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A significant reduction in leptin levels and a trend toward a decrease in proinflammatory and Th1 cytokine production was observed in PCM infants on admission. However, when the infants recovered 10% of their initial weight, circulating hormone levels became normal, associated with a significant increase in cytokine production.
It is well known that circulating leptin concentrations closely correlate with body fat mass and that higher levels of this hormone are observed in obese subjects (15, 17); a decline of leptin levels occurs during acute starvation and weight loss, with return to normal values after refeeding (18). In addition, leptin levels are low in many forms of malnutrition, including intrauterine growth retardation and untreated anorexia nervosa (19, 20). To date few data on leptin levels in protein calorie malnutrition have been reported. Our data show that PCM infants had low leptin serum levels; these results are in agreement with the data recently published by Soliman et al. (8). Interestingly, the recovery of only 10% of weight in our children, which took place approximately 30 d after refeeding, produced a significant increase in the levels of this hormone. The rise in leptin did not parallel the increase in body fat mass, a situation that was also observed in individuals subjected to acute fasting or energy restriction, in which the reduction of leptin levels was not concomitant to the diminution of fat body mass (19). These observations suggest that the leptin concentration is regulated by factors other than fat mass (21). Some researchers postulate that leptin behaves like a signal for energy stores, speculating that the rise of this hormone could represent an adaptive phenomenon necessary for growth development (19). Experimental evidence shows that low leptin levels could be a predictor of body weight gain based on the data of Jacquet et al. (19), who demonstrated that children born with intrauterine growth retardation had higher leptin levels at 1 yr of age compared with normal controls. These researchers (19) suggested that the higher leptin levels in their children could represent an adaptive response that stimulated catch-up growth. An alternative explanation could be that high serum leptin concentrations could reflect a defect in adipose tissue function that impairs the sensitivity of the regulatory system of leptin synthesis and secretion (19). The increasing risk of obesity during adult life of children born with intrauterine growth retardation supports this hypothesis (22).
Recent evidence indicates that cytokines play an important role in the nutrition-infection complex (23). On one hand, an impairment of cytokine production has been reported in malnutrition, and furthermore, infections induce cytokine release, which is known to interfere with metabolic activity and cause appetite loss that alter nutritional status (13, 23, 24). We observed a trend toward reduced PBMC IL-1 production in PCM infants on admission, which agrees with the data reported in malnourished children and anorexia nervosa (12, 25, 26), and explains why these children have an impaired capacity to mount a febrile reaction as a response to infectious agents. TNF production by PBMC from PCM infants on admission did not differ from that in normal controls, a finding in disagreement with the results reported by Keenan et al. (25) in adults with anorexia nervosa. On the other hand, IL-6 production in malnourished infants was similar to that in normal controls, results that differ from those of Doherty et al. (27), who reported a diminished production of this cytokine in severe PCM infants, a fact that could explain the difference in our cases with moderate PCM. Surprisingly, when 10% of weight had been gained, a significant increase in IL-1 and TNF production, concomitant with the increase in circulating leptin levels, was observed in the PCM patients. Furthermore, Faggione et al. (28) reported that the decrease in leptin levels during fasting contributes to the increased susceptibility to LPS toxicity. These researchers showed that LPS induced a 5-fold greater increase in serum TNF in fasted mice compared with fed animals (28). The TNF increase was blunted by exogenous leptin replacement, suggesting that leptin could be protective by both inhibiting TNF induction by LPS and by reducing TNF toxicity. These researchers concluded that leptin exerts a regulatory role on the immune response during the nutritional recovery period (28).
No information correlating leptin levels and cytokine production in human malnutrition is available at present in the current literature. The studies published evaluating the effect of leptin on immune function have been performed only in experimental animals and in in vitro experiments (6, 29). It has been reported that leptin-deficient ob/ob mice cannot clear and kill circulating Escherichia coli as efficiently as normal mice, suggesting that these genetically obese mice have an impaired immune system (29). The possibility that leptin-dependent signals regulate immune function is also supported by the observation that Zucker fa/fa rats, an obese and diabetic strain with deficient leptin receptors, produced suboptimal quantities of proinflammatory cytokines when challenged with LPS (29). Furthermore, studies of macrophages from ob/ob mice (which lack leptin) and db/db mice (which have dysfunctional leptin receptors) show an impaired ability to phagocytize and kill Candida (29, 30). Recombinant leptin restored the phagocytic function of cells from ob/ob mice, but not that of cells from db/db mice, indicating that leptin may regulate macrophage phagocytosis via mechanisms that require direct interactions with its receptor (29). The evidence that leptin can also increase phagocytic activity in macrophages isolated from lean, nondiabetic mice supports these findings and suggests that the phagocytic defect in ob/ob and db/db mice is a direct result of leptin deficiency rather than secondary to obesity-related diabetes (31). These researchers also have demonstrated that leptin is necessary for animals to mount an optimal cytokine response when challenged with endotoxin, a fact supporting our findings and suggesting that this hormone could stimulate proinflammatory cytokine production indirectly by a posttransductional mechanism (29).
Impaired cell-mediated immunity, demonstrated in our study by the decrease in the mitogen-stimulated lymphoproliferative response, is a feature of malnutrition that predisposes to infectious episodes; however, the mechanisms responsible for this alteration remain unknown. When the PCM infants recovered 10% of their weight, leptin levels and lymphoproliferative responses returned to normal, becoming similar to those of well nourished healthy controls. These results are consistent with the findings of Lord et al. (6), who demonstrated that the in vitro addition of leptin to mixed lymphocyte cultures enhanced their proliferative response, the increment being much higher when isolated CD4 cells were employed as responders, suggesting that T lymphocytes are the principal target for leptin action.
Until now, no information on Th1 and Th2 activity has been reported in malnutrition. Our study reveals an imbalance between lymphocyte subpopulations in PCM, demonstrated by the trend toward a decrease in IFN, and the increase in IL-4 production, concomitant with the presence of low leptin levels. When a 10% recovery of weight was achieved, a significant enhancement of IFN production associated with suppression of IL-4 was shown in parallel with the increase in circulating leptin levels. These findings are similar to those reported by Lord et al. (6), who observed that in vitro leptin addition to PBMC stimulated IL-2 and IFN production, indicating that leptin may favor a Th1 response, whereas its absence may promote Th2 activity.
It is possible to speculate that leptin could exert immunological modulation by acting directly on T cell receptors or indirectly, through the hypothalamic-pituitary-adrenal axis. Some time ago we demonstrated that central noradrenergic hyperactivity in malnourished rats could be one of the mechanisms responsible for immunosuppression in malnutrition (32). Leptin may also modulate central noradrenergic activity, and therefore, through this pathway it may regulate immune function (33).
A significant decrease in IGF-I and IGFBP-3 levels was observed in PCM infants on admission, in agreement with previous results (34). With the recovery of 10% of weight, these growth factors increased to levels similar to those in well nourished controls.
Many studies have shown that in rats central leptin infusion stimulates basal secretion of GH, indicating that leptin could act as a neuromodulator of the growth axis (35). We did not measure GH levels in our patients, but we assume that this may be increased. We also postulate that the increased leptin levels could induce an increase in GH sensitivity that contributes to stimulate IGF-I production.
Thus, leptin could play an important role in growth factor regulation as well as in modulation of the immune response. However, immunoregulation may not be attributed only to leptin, because growth factors function as immunomodulators (36, 37). It has been reported that hypophysectomy induces an immunodeficiency in rats that is reversed by administration of GH and PRL (37). Furthermore, GH and IGF-I receptors have been described on the surface of lymphocytes (37). However, in patients with Laron syndrome, a genetic disease characterized by low levels of IGF-I and IGFBP-3, similar to those observed in PCM, no immunological deficits have been reported (37). It is therefore possible that growth factors do not play a fundamental role in immune regulation in PCM.
In conclusion, our findings suggest an association between the increase in leptin and the immunological recovery observed after refeeding of malnourished infants. However, a therapeutic trial is needed as a proof that leptin could enhance Th1 activity in malnutrition.
Acknowledgments
We thank Dr. Oscar Brunser, M.D., and Dr. Illani Atwater, Ph.D., for critical reading of the manuscript.
Footnotes
This work was supported by a grant from University Iberoamericana, Santiago, Chile.
Abbreviations: BMI, Body mass index; Con A, concanavalin A; IFN, interferon-; IGFBP, IGF-binding protein; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cells; PCM, protein calorie malnutrition; Th1 and Th2, T helper cells type 1 and type 2; W/A, weight for age; W/H, weight for height.
Received July 20, 2001.
Accepted March 19, 2002.
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