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Capacity to Produce Cytokines During Weight Restoration in Patients With Anorexia Nervosa

From the Department of Neuropsychiatry, Osaka City University Medical School, Osaka, Japan.

Address reprint requests to: Toshihiko Nagata, MD, PhD, Department of Neuropsychiatry, Osaka City University Medical School, 1-4-3, Asahimachi, Abenoku, Osaka 545-8585, Japan. Email: toshi{at}med.osaka-cu.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
OBJECTIVE: Anorexic patients are surprisingly free of infectious complications despite their seriously undernourished state. To study this phenomenon, we longitudinally measured the capacity to produce cytokines in restricting-type anorexic patients.

METHODS: Lymphoproliferative responses with phytohemagglutinin (PHA) and the capacity of whole blood to produce cytokines, such as interleukin-1 (IL-1), IL-6, tumor necrosis factor- (TNF-), interferon- (IFN-), and granulocyte-colony stimulating factor (G-CSF), were longitudinally measured before and after weight gain, that is, at admission and at less than 60, 65, and 75% of standard body weight (SBW), in 17 patients with restricting-type anorexia nervosa and in 17 control subjects.

RESULTS: Cytokine production of IL-1, IL-6, and TNF- per monocyte in the anorexic patients recovered only with the start of refeeding, whereas IFN- production per lymphocyte was similar to that in control subjects and did not change during weight restoration. Only G-CSF production, even at 75% SBW, did not improve during weight restoration. Between the weight at admission and 65% SBW, the increase in the percentage of SBW and improvement of the total protein level were significantly correlated with improvement of the lymphocyte proliferative response with PHA.

CONCLUSIONS: The capacity to produce most cytokines recovered with the start of weight gain; however, recovery was not correlated with weight gain. The results suggest that the capacity to produce cytokines in these anorexic patients was dependent on something other than the absolute value of body weight, such as the start of refeeding, the neuroendocrine system, or the autonomic nervous system.

Key Words: anorexia nervosa • cytokine • lymphocyte • monocyte • nutrition

Abbreviations: HPA axis = hypothalamo-pituitary-adrenal axis; LPS =lipopolysaccharide; IL = interleukin; TNF- = tumor necrosisfactor-; PHA = phytohemagglutinin; G-CSF =granulocyte-colony stimulating factor; IFN- = interferon-; SBW = standard body weight; PBMC = peripheral bloodmononuclear cell; ELISA = enzyme-linked immunosorbent assay; ANOVA = analysis of variance; TGF-ß = transforming growthfactor-ß; DST = dexamethasone suppression test.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Anorexia nervosa is characterized by an individual’s refusal to maintain minimal healthy weight, intense fear of becoming obese, and distorted body image (1) and is associated with an increased mortality rate (2, 3). Nutritional deprivation, such as protein-calorie malnutrition, often causes immunodeficiency, leading to increased susceptibility to infection (4, 5). However, patients with anorexia nervosa have been anecdotally reported to be relatively free of such viral infections as colds and influenza (6, 7). These observations suggest that a compensatory mechanism is working to protect such patients from infection and that conditions in these patients may differ from those in other patients suffering from nutritional deprivation.

The major mechanism by which the brain influences the immune system is indirectly mediated by pituitary hormones. Glucocorticoids are well known to be highly immunosuppressive for T cells (8). Many researchers have reported activation of the HPA axis by cytokines (for a review, see (9)), and there is general agreement that underweight anorexic patients exhibit elevated cortisol levels (10).

An additional route of cytokine-to-brain communication, that is, an autonomic nervous system route rather than a humoral route, was recently proposed (11). The activation of subdiaphragmatic vagal afferents mediates a wide range of illness responses produced by intraperitoneal administration of LPS, IL-Iß, and TNF- in animal models (11). Lymphocytes (both T and B cells) and macrophages possess ß-adrenergic receptors, and both epinephrine and norepinephrine have been indicated to be able to modulate immune function (12). Autonomic dysfunction during weight reduction has been reported regardless of absolute numbers of body weight in normal, obese, and anorexic subjects (13, 14). Therefore, there is "bilateral" communication between the immune and neuroendocrine systems (15) and between the immune and autonomic nervous systems. A compensatory mechanism in anorexics might be evoked through either the neuroendocrine system or the autonomic nervous system.

Results of studies of cell-mediated immunity, such as lymphoproliferative responses and the lymphocyte subset in patients with anorexia nervosa, are conflicting. For example, lymphocyte proliferative responses in patients with anorexia nervosa have been reported to be reduced in most studies (16–20), whereas other investigators have reported similar (21–25) or elevated responses (26). These conflicting results are probably due to the heterogeneity of subjects. Most of the above-mentioned studies did not specify whether the patients underwent refeeding or whether they were bulimic or restricting-type anorexics. A longitudinal study in a homogenous population of only restricting-type anorexia nervosa patients may be helpful to better understand immunological functions in such patients. A whole-blood assay was recently used as a simple and reliable method to measure a patient’s capacity to produce cytokines (27). Using this method, we can measure the capacity to produce several cytokines in one culture with a small amount of blood.

In the present study, we longitudinally measured lymphoproliferative responses with PHA, as well as the capacity to produce several cytokines (IL-1, IL-1ß, IL-6, TNF-, G-CSF, and IFN-), in a homogenous sample of patients with restricting-type anorexia nervosa who had never binge-eaten, during weight restoration to examine the hypothesis that immune functions in anorexic patients are well preserved despite the severely malnourished state of the patients.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Subjects
The original subjects were 19 female anorexic patients. However, two patients were excluded because they withdrew from the study midway and their data were incomplete. All patients were inpatients at our department. They were interviewed by two psychiatrists (T.N. and N.K.), and all met the DSM-IV criteria (1) for anorexia nervosa, restricting type. Patients who had a history of binge eating were excluded from the study to keep the subject group homogenous. The immunological functions in restricting-type anorexia nervosa patients and those in binge eating/purging-type anorexia nervosa were found to be different (28). All of the anorexic patients in this study were underweight, at less than 60% of their SBW (29), and they had not started refeeding when they entered the study. The control group consisted of 17 normal-weight, age-matched, healthy women. These subjects had no history of physical or psychiatric disorders. Acute infectious diseases were ruled out by measuring the body temperature (<37°) and C-reactive protein level (<0.4 mg/dl). No subjects had liver or renal dysfunction or signs of inflammation, and none had other clinical or laboratory evidence of medical illness. None of the subjects had taken any medication for at least 2 months before entering the study. Informed consent to participate in the study was obtained from all 36 subjects.

Time of Measurements
Immune function of the patients with anorexia nervosa was assessed three times during weight restoration. The first was within 4 days after admission (at admission). At this time, the patients had not begun or had just started refeeding. This was confirmed by the daily food logs they kept while on the waiting list for admission. The second time was when the patients reached 65% of SBW. At that time, the patient had been refeeding for a short time and had just started to gain weight. We missed the data of two patients when they reached 65% SBW; thus, these two patients were excluded from the analysis. The third time was when the patient reached 75% of SBW. Treatment was provided in an open general psychiatric unit, mainly through a behavioral program with some cognitive treatment. None of the patients received any psychopharmacological medication throughout the inpatient treatment.

PHA-Stimulated Lymphoproliferation
After an overnight fast, blood samples were taken between 7 and 8 AM. Blood samples for the lymphoproliferative response assay and for whole-blood cultures were taken at the same time. To measure the mitogenic response of T lymphocytes to PHA, PBMCs were obtained by density-gradient centrifugation on Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) and suspended to a concentration of 5 x 105 cells/ml in RPMI 1640 (Difco Laboratories, Detroit, MI) culture medium containing 10 µl of fetal calf serum. The suspension was placed in 96-well microplates (200 µl per well) and incubated for 64 hours with PHA (1.5 µg/ml per well) at 37°C in water saturated with 5% C0₂. Then, [3H]-thymidine (0.25 µCi per well) was added to each well, and cells were collected using a cell harvester after an 8-hour incubation. Radioactivity incorporated by the cells was measured using a scintillation counter. Results are expressed both as tritiated thymidine incorporation (cpm) and as a stimulation index (ie, the ratio of radioactivity in maximally stimulated lymphocyte cultures compared with that in unstimulated control cultures).

Whole-Blood Assay
For the whole-blood assay, 0.5 ml of heparinized whole blood, from the same sample for the lymphoproliferation assay, was added to each 5-ml culture of RPMl 1640 with and without 10 µg/ml LPS (Difco) and incubated for 24 hours at 37°C. The culture was centrifuged (1000g, 10 minutes, 4°C), and the supernatant was kept at -70°C until assayed. The levels of IL-1, IL-1ß, IL-2, IL-6, TNF-, G-CSF, and IFN- in the serum and supernatant of the culture were determined using ELISA kits (Otsuka Pharmaceutical Co., Tokushima, Japan), which uses a horseradish peroxidase–labeled antibody as a tracer. Measurements were made according to the manufacturer’s instructions. All samples were examined in triplicate. The ranges of sensitivity of the cytokine assays were as follows: IL-1, 10 to 2500 pg/ml; IL-1ß, 20 to 5000 pg/ml; IL-2, 50 to 5000 pg/dl; IL-6, 20–5000 pg/ml; TNF-, 20 to 5000 pg/ml; G-CSF, 20 pg/ml to 10 ng/ml; and IFN-, 20 to 5000 pg/ml. Intraassay and interassay coefficients of variation were less than 4.9 and 7.5%, respectively.

Data Analysis
All statistical analyses were performed using SPSS software (30). Student’s t test with Levene’s test for equality of variances, repeated-measures ANOVA with simple contrasts, and Pearson correlation coefficients were used when appropriate. Cytokine production per absolute number of monocytes (for IL-1, IL-1ß, IL-6, TNF-, and G-CSF) or absolute number of lymphocytes (for IFN-) were also calculated because a lower capacity to produce a cytokine in the whole-blood culture might be due solely to a lower absolute number of monocytes or lymphocytes in the blood. Even in the supernatants of the whole-blood culture, cytokines were not detectable in some samples. In such cases, the lower limit of the sensitivity range was entered rather than zero or a missing value. This procedure controls Type I error, because we expected a significantly lower capacity to produce cytokines in the patients.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Demographic characteristics of the subjects are presented in Table 1 . There was no significant difference in age or height between patients and control subjects. The onset of anorexia nervosa occurred at a mean (±SD) age of 18.4 ± 3.9 years, and the duration of illness was 4.0 ± 3.7 years. The minimal body weight and minimal body mass index of patients were significantly lower than those of control subjects. All patients were amenorrheic, and the mean duration of amenorrhea was 3.4 years; even at end of this study, menstruation had not restarted in any of the patients.

The results regarding the capacity to produce cytokines with stimulation of LPS are presented in Table 3 . The serum levels and nonstimulated production of these cytokines could not be detected in most of the patients and control subjects. IL-2 was not detected even in the supernatants of whole-blood cultures. Thus, these results are not presented in the table.

There were no significant correlations between the cytokine production values and demographic and clinical variables such as body weight, SBW, total protein level, or total cholesterol level. Correlations between changes of immunological function and changes of clinical values during weight restoration are presented in Table 4 . Between admission and 65% SBW, the increases in proliferative responses were positively correlated with the increases of SBW and total protein. The decrease in the stimulation index also tended to be negatively correlated with the increase of SBW (p = .07). p < .05 by Pearson correlation coefficient.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

Data on blood cytokine levels in patients with anorexia nervosa are also conflicting. Pomeroy et al. (1994) (31) reported that the serum IL-6 and TGF-ß concentrations in patients with anorexia nervosa were elevated and that with weight restoration these high levels returned to levels comparable to those of normal-weight control subjects, but this was not confirmed in other studies (32, 33). In children with protein-energy malnutrition, the plasma levels of IL-6 and soluble receptors of TNF- were also found to be high (34). Thus, cachexia induced by TNF- has been hypothesized to have a role in the pathogenesis of anorexia nervosa (35), although blood TNF- levels were undetectable in anorexic patients (19, 31, 32, 36). We also did not detect elevated concentrations of IL-6 and TNF- in our patients; nonetheless, our patients were seriously malnourished and underweight. This discrepancy may be explained as follows. First, the condition of anorexia nervosa is somewhat different from that of malnourished patients in developing countries, because anorexic patients usually eat something, especially some protein. Second, differences in the methods used to measure cytokines (eg, ELISA (our study), radioimmunoassay (32), or bioassay (31)) may have led to the different results; however, the sensitivity range is not a factor because Pomeroy et al. (31) reported three-fold higher IL-6 blood levels, which would be easily detectable by ELISA or radioimmunoassay.

In contrast to a number of studies on the PHA lymphoproliferative response, lymphocyte subsets, and cytokine concentrations, few studies have measured the capacity to produce cytokines in patients with anorexia nervosa. In patients with malnutrition, impaired capacities to produce IL-1 (37–39), IL-6 (40), and TNF- (39, 40) have been noted, and after nutritional rehabilitation, the impaired capacity to produce IL-1 (39), IL-6 (40), and TNF- (39, 40) improved. Even 7 days of fasting also attenuated IL-2 production by PBMCs (41). Anorexic patients had significantly lower capacities to produce IL-2 (42), TNF- (19), and IFN- (20, 23), and the capacity to produce IFN- improved with weight gain and restoration of the menstruation cycle (20). Our findings are in line with those from patients with malnutrition and anorexia nervosa. The capacity of the anorexia nervosa patients in this study to produce these cytokines (IL-1, IL-6, and TNF- but not G-CSF) quickly recovered only with the start of weight gain. Even at the end of the study, the patients still had severe proteinemia and leukopenia, the number of lymphocyte and monocyte had not increased significantly, and the patients’ menstruation had not restarted. As a result, the capacity of one monocyte to produce IL-1, IL-6, and TNF- at 75% SBW was higher than the level of control subjects (although we did not statistically compare them because we did not repeatedly measure the levels in controls), suggesting some type of rebound hyperactivity to produce these cytokines during the refeeding period. In contrast, the capacity to produce IFN- per lymphocyte in the anorexia nervosa patients was similar to that of the control subjects even at admission, and this capacity did not change significantly during the weight restoration period.

In the present group of patients, only the capacity to produce G-CSF remained low. CSF is important for the maintenance of basal hematopoiesis (43). The number of white blood cells in the anorexia nervosa patients also did not change during this study. Thus, a low capacity to produce G-CSF might be responsible for the continuous leukopenia in the anorexic patients. We could follow their weight restoration only until they reached 75% of SBW because of the limitation of our treatment setting (this situation is common in Japan because of the medical system); therefore, this impaired capacity of G-CSF might recover when the body weight returns to normal or when menstruation restarts, although little is known about the relationship between gonadal hormones and immunological function (44). A recent study (45) suggested that calorie restriction in anorexia nervosa patients and obese dieters is associated with significant decreases in lymphocyte CD8⁺ counts. In addition, the low CD8⁺ count in anorexia nervosa patients persisted even after weight gain; thus, a factor other than weight change per se might be important for some immunological functions, such as G-CSF production or lymphocyte subpopulation.

How does the compensatory mechanism for the stimulation index and rebound hyperactivity to produce cytokines work in anorexic patients? A direct effect of the nutritional state cannot be neglected. The nutritional state is one of the very influential factors in immunological function (46). In our study, the increase in proliferative responses with PHA was correlated with the increase of SBW and total protein between before and after the start of refeeding (ie, between admission and 65% of SBW). These correlations suggested a close relationship between the nutritional condition and lymphoproliferative responses around the beginning of refeeding. However, there were no significant correlations between the increase in the capacity to produce cytokines and improvements of body weight and laboratory data. Thus, improvement of the capacity to produce cytokines during weight restoration cannot be explained only by recovery of the nutritional state.

There are many reports concerning activation of the HPA axis by IL-1, IL-6, and TNF- (9), and, conversely, glucocorticoids are well known as an immunosuppressive hormone (8). Both anorexia nervosa and protein-calorie malnutrition induce hypercortisolism, presumably as a consequence of an altered hypothalamic set point for pituitary-adrenal regulation (10, 47, 48). We also previously found that plasma cortisol levels after an oral DST were significantly correlated with the rate of weight gain (in 2 weeks) rather than the percentage of SBW or Hamilton Depression Rating Scale scores in six patients with anorexia nervosa during weight restoration (49). Thus, the effect of acute weight loss, rather than the degree of weight loss itself, may also be a factor in the activation of the HPA axis. Our present findings regarding patients’ capacity to produce cytokines can also be interpreted as the result of interactions among the immediate nutritional state, HPA axis, and immunological function, especially after the beginning of refeeding. First, the start of weight gain and improvement of the nutritional condition led to the normalization of hypercortisolism. Consequently, recovery of the capacity to produce cytokines occurred. However, DST and plasma cortisol measurements were not performed in this study; thus, the above explanation is only speculation.

An autonomic nervous system route was recently suggested to be an additional route for cytokine-to-brain communication (11). Autonomic dysfunction during weight reduction has been reported to exist regardless of the absolute number of body weight (13, 14). Therefore, a compensatory mechanism for maintaining a stable stimulation index and rebound hyperactivity to produce cytokines per monocyte in anorexics might be evoked via the autonomic nervous system; however, little is known about the relationship between immune function and the autonomic nervous system in patients with anorexia nervosa.

According to anecdotal reports, patients with anorexia nervosa are free of common viral infections when underweight (50). The nonreduced stimulation index in our study and decreased CD8 level (51) and elevated CD4/CD8 ratio (28, 45) may explain this phenomenon (52).

There are some limitations of this study. These compensatory mechanisms also work in weight loss in normal and obese people (45). However, there were no controls for weight reduction in obese or normal-weight control subjects in our study. In addition, another study (45) suggested that the compensatory mechanism might act even after weight gain. Thus, we cannot conclude that the weight change other than the absolute number of body weight is truly a factor in immunological function. Questions also remain about how great a percentage of body weight change, how large an absolute number of body weight change, and how rapid a body weight change are crucial for immunological function. In the control subjects, we did not repeatedly measure immunological function indicators of immediate nutritional state, such as transferrin, or indicators of the neuroendocrine system, such as DST.

Despite these limitations, our results indicate that the start of refeeding or the start of weight gain rather than the absolute number of body weight seems to be a factor in the recovery of cytokine production; however, the exact mechanism remains to be identified, and factors other than nutritional factors also remain to be studied. Additional longitudinal follow-up studies on immune function, the neuroendocrine system, autonomic nervous system, and psychological state of patients with anorexia nervosa are needed to clarify the relationships among these factors.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Masahiro Kokai, MD, at the Department of Neuropsychiatry, Hyogo College of Medicine, for valuable comments on the study design.

Received for publication April 28, 1998.

Revision received January 20, 1999.

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TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 

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