Introduction

Natural killer (NK) cells were characterized over 30 y ago as lymphoid cells that mediate the spontaneous killing of certain virus-infected and tumor cells in a major histocompatibility complex (MHC)-unrestricted manner. NK cells are a key component of innate immunity. In the peripheral blood, they account for 5–20% of lymphocytes. In addition to their ability to promote rapid lysis of target cells, NK cells have been recently appreciated as an important source of cytokines. In turn, they are activated by numerous cytokines, notably interleukin (IL)-2, IL-12 and IL-15 and type I interferons.^{1, 2, 3} Several hormones and neurotransmitters exert modulatory effects on NK cell activity.^{4, 5, 6} Peptides and steroid hormones of the hypothalamic–pituitary–adrenal (HPA) axis have received special attention, due to their key role in stress reactions and in a number of psychoneuroendocrinological disorders.^{7, 8, 9, 10, 11, 12, 13}

It is held that glucocorticoids at physiological concentrations are potent inhibitors of spontaneous and cytokine-inducible NK cell activity. Interestingly enough, this activity has been shown to be modulated also by nutrients.^{14, 15, 16, 17} Therefore, it represents a valuable model to investigate neuroendocrine-immune interactions in clinical conditions characterized by abnormalities of the eating behavior. Indeed, we have recently documented an altered responsiveness in vitro to positive and negative modulators of circulating NK cells of patients with anorexia nervosa.¹⁸

Obesity is a condition defined by excess body weight, body mass index (BMI30 kg/m² according to the clinical World Health Organization classification)¹⁹ and fat mass (more than 20% in men and 25% in women), with systemic involvement and with overall increased morbidity and mortality.^{20, 21} Obesity has been long associated with psychoneuroendocrinological disturbances, notably subtle hyperfunction of the HPA axis leading to clinically relevant albeit inapparent hypercortisolism.²² Moreover, obesity has been associated with changes in both the innate and the acquired components of the immune system. Obese subjects show increased susceptibility to infections.^{23, 24} Obesity per se and high-fat diet have also emerged as independent risk factors for cancer.²⁵ On the other hand, obesity has been recently viewed as a condition of chronic systemic subclinical inflammation, involving the activation of pro-inflammatory cytokines.^{26, 27}

In overweight (BMI>25 but <30 kg/m²) and obese (BMI30 kg/m²) subjects, the absolute and relative numbers of phenotypically identifiable NK cells have been found in the normal range.^{24, 28, 29, 30} On the other hand, inconsistent and even divergent results have been obtained in studies that have focused on the killing activity.^{24, 29, 31, 32} Since NK cell activity is a dynamic and highly flexible process which depends on the balance between activating and inhibitory signals, it is important to evaluate the responsiveness to positive and negative modulators, respectively. To our knowledge, studies so far published do not address this issue, but are limited to the assessment of basal (spontaneous) lytic activity on well-established target cells in vitro. The aim of the present case–control study was to investigate NK cell activity measurable in peripheral blood mononuclear (PBM) cell preparations drawn from 21 obese subjects and age- and sex-matched nonobese controls. We evaluated both spontaneous cytotoxicity and changes after exposure to physiological modulators in the opposite directions, that is, an activating cytokine (IL-2) and an inhibitory hormone (cortisol). We also searched for correlations between NK cell activity and anthropometric indices of obesity, dietary components and the most common metabolic parameters.

Methods

Study population

A total of 21 subjects affected by uncomplicated²¹ obesity (male to female: 6/15, age (means.e.m.) 52.82.6 y, BMI 36.00.7 kg/m²) were enrolled. All cases were outpatients in a weight-stable phase in the previous 6 months; none of them was taking any medication or was under dietary restriction; post-menopausal women were not under hormone replacement therapy. Syndromic forms of obesity, thyroid dysfunction, hyperandrogenism and relevant renal, hepatic, cardiovascular diseases were excluded by accurate anamnestic recall (including familial weight distribution, associated symptoms and age of onset of overweight), clinical examination and laboratory tests (hemochromocytometric assay; serum protein electrophoresis; erythrocytes sedimentation rate; prothrombin and partial thromboplastin times; serum: C-reactive protein, sodium, potassium, calcium, phosphorus, iron, transferrin, ferritin, folic acid, vitamin B12, urea nitrogen, creatinine, bilirubin, transaminases, alkaline phosphatase, -glutamil-transpeptidase, glucose, albumin and pre-albumin, total and HDL cholesterol, tryacylglycerol, uric acid, immunoglobulins, thyroid-stimulating hormone; urine: cell count, creatinine, protein). Consumption of ethanol and smoking were not allowed in the last 72 h before blood was drawn.

Anthropometric indices were measured by the same operator: body weight and height were measured without clothes and shoes; body superficies was calculated according to anthropometric tables, BMI was calculated as weight (in kg) divided by square of height (in m²). Waist circumference was measured with the subject standing, using a 1 cm-wide plastic tape, as the minimum value between the iliac crest and the lateral costal margin, whereas hip circumference was determined as the maximum value over the buttocks.³³ Fat mass was measured by bioelectrical impedance analysis (BIA) (Tanita Body Fat Analizer TBF 511).²⁰ Dietary analysis was performed by a trained operator by personal interview according to the guidelines of the Istituto Nazionale di Nutrizione (INN—National Institute of Nutrition). Routine laboratory analyses were performed by standard techniques. LDL concentration was calculated according to Friedewald's formula (serum tryacylglycerol concentrations were always less than 4.51 mmol/l). In a representative subgroup of patients (male to female: 5/13, age 52.23.1 y; BMI: 36.00.9 kg/m²), serum leptin concentrations were measured by radioimmunoassay (Mediagnost, Reutlingen, Germany).

In total, 21 healthy, nonobese age- and sex-matched subjects (male to female: 6/15, age 55.52.8 y; BMI 24.30.4 kg/m²) were recruited from the general population as controls. None of them had a history of overweight or showed any abnormality on physical examination; routine blood and urine laboratory tests proved to be normal.

The local Ethical Committee approved the study and all subjects gave their written informed consent.

NK cell activity

1. Materials: Cortisol (Sigma, Milan, Italy) was initially dissolved in 95% ethanol, diluted in RPMI 1640 (HyClone Europe Ltd., Cramlington, UK) to 1 mmol/l solution and stored at 4°C up to 1 month. Before experiments, cortisol was promptly diluted in complete medium to a final concentration of 1 mol/l.

Recombinant human IL-2 (Eurocetus, Emeryville, USA) was reconstituted to 10⁶ IU/ml with bovine serum albumin 1% (w/v) in phosphate-buffered saline (PBS), and stored at -20°C. Before experiments, IL-2 was promptly diluted in complete medium to the final concentration of 100 IU/ml.

2. Cell cultures: RPMI 1640 medium enriched with 1% (w/v) glutamine, 50 g/ml gentamicin and 10% (v/v) heat-inactivated fetal bovine serum (Sigma) was the complete medium (CM) used for both K562 and PBM cells culture. The human myeloblastoma cell line K562 was used as a sensitive target for measurements of NK cytotoxicity, and was maintained in suspension culture flasks at 37°C in a humidified atmosphere of 95% air/5% CO₂ incubator. All targets used had a >90% viability measured by trypan blue dye exclusion procedure.

3. Separation and incubation of PBM cells: PBM cells were obtained from heparinized venous blood samples drawn between 0800 and 0900 h. PBM cells were separated by Ficoll–Hypaque density gradient centrifugation at 400 g for 25 min at room temperature.³⁴ The resultant preparations contained more than 98% mononuclear cells; 75–80% of them were lymphocytes identified by May–Grünwald–Giemsa staining. After separation, PBM cells were washed three times, counted and resuspended to a density of 7 10⁶ cells/ml in CM; they were then incubated for 20 h with CM alone or modulators (cortisol: 1 mol/l; IL-2: 100 IU/ml). After incubation, PBM cells were washed twice, assessed for viability by the trypan blue dye exclusion method and then assayed for cytotoxicity. The effect on cell viability of any tested compound was negligible.

4. Cytotoxicity assay: NK activity was measured by a 4-h enzyme-release cytotoxicity assay. Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity released in the supernatant by lysed target cells according to the method of Korzeniewski and Callewaert.³⁵ Results obtained with LDH-release method have been shown to correlate with those of ⁵¹Cr release.^{35, 36, 37} We have previously confirmed such correlation, and consequently used this non-radioactive method extensively in our work. K562 (target) cells were mixed with PBM (effector) cells to give different effector-to-target cell ratios (25 : 1, 12.5 : 1, 6.25 : 1, 3.125 : 1). LDH activity was measured by colorimetric procedure, performed by an automatic PC-aided microtiter plate reader (UV-Max™ provided with Soft-Max™ software, Molecular Devices Corporation, Menlo Park, USA). Data on the NK activity of PBM cells incubated with or without modifiers were expressed as lytic units (LU)/10⁷ cells according to Pross et al.³⁸

Statistical analysis

Statistical analysis of data was performed with Statistica 5.0 software package (Statsoft Inc., Tulsa, OK, USA). All results are presented as means.e.m. Parametric tests were used in those cases in which sample data verified the assumptions of normal distribution (assessed by Shapiro–Wilk's test) and homogeneity of variance (assessed by Levene's test). Only when these assumptions were violated were nonparametric tests employed. Accordingly, comparison between obese and nonobese subjects was performed by unpaired Student's t-test or Mann–Whitney U-test, as appropriate. Correlations between immune, anthropometric and nutritional variables were evaluated by Spearman's rank correlation test. Given the exploratory nature of this study, the statistical analysis was not adjusted for multiple comparisons.³⁹ A level of significance of 0.05 was chosen for all tests.

Results

Demographic data, anthropometric indices, dietary composition and metabolic parameters of obese subjects are resumed in Table 1. Representative effector-to-target dilution curves are shown in Figure 1. Both in obese and in nonobese subjects, the levels of spontaneous NK cell activity showed a wide interindividual variability; neither gender differences nor correlation with age were found. The mean spontaneous NK cell activity was not significantly different in obese subjects vs controls (Figure 2).

Figure 1.

Representative E : T dilution curves for spontaneous, cortisol-, and IL-2-modulated NK cell activity in obese and control subjects. In order to measure the total release of LDH, target cells were disrupted by sonication and released enzyme activity was measured by colorimetric procedure. Lytic units have been calculated for each E : T dilution curve (see also Methods). F, female.

Full figure and legend (24K)

Figure 2.

Spontaneous, cortisol-, and IL-2-modulated NK cell activity in obese and control subjects. After incubation, PBM cells were assayed for cytotoxicity. Bars and whiskers represent the mean+s.e., respectively. ^*P<0.001, ^**P<0.0001 vs spontaneous activity by Wilcoxon matched pair test (see also Methods). sp, spontaneous; F, cortisol.

Full figure and legend (63K)

Table 1 - Demographic, anthropometric, nutritional and metabolic data in obese subjects.

Full table

As expected, IL-2 stimulated while cortisol inhibited NK cell activity in both populations (Figure 2). When comparing obese vs control subjects, IL-2-dependent percent increment was not significantly different, while cortisol-dependent percent decrement was significantly lower in obese subjects (-24.42.9 vs -38.63.3%, P=0.002). In subgroup analysis, decreased sensitivity was restricted to women (-20.03.1 vs -41.74.1%, P=0.0007) (Figure 3). On the other hand, patterns of response to IL-2 and cortisol were not different when looking at central vs peripheral phenotypes of obesity.

Figure 3.

IL-2- and cortisol-dependent percent variation of NK cell activity in obese and control subjects: individual values. Black circles, post-menopausal women; white circles, pre-menopausal women; white triangles, men. ^*P=0.0007, obese vs nonobese women, by Mann–Whitney U-test.

Full figure and legend (24K)

In obese subjects, a negative correlation was apparent between BMI and percent inhibition by cortisol. When considering gender differences, statistical significance, which resulted at the limit for the whole population (P=0.052), was attained at a high level for women (r=-0.63, P=0.012) and not for men (Figure 4). No other anthropometric variable showed significant relationships with NK cell responsiveness to challengers, as inferred by percent changes vs baseline values. Since a negative correlation between serum leptin concentration and lymphocyte glucocorticoid receptor (GR) mRNA has been reported,⁴⁰ serum leptin was measured in a representative subgroup (n=18). Serum levels were higher in females than in males (32.14.7 vs 14.93.6 ng/ml, ns). When the whole subgroup was considered, leptin serum levels correlated positively with BMI (r=-0.59, P=0.01) and fat mass (r=0.70, P=0.001), and negatively with percent inhibition by cortisol (r=-0.54, P=0.02, Figure 5).

Figure 4.

Correlation between cortisol-dependent inhibition and BMI in obese women (r=-0.63, n=15, P=0.01). Black circles, post-menopausal women; white circles, pre-menopausal women; white triangles, men.

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Figure 5.

Correlation between cortisol-dependent inhibition and serum leptin concentrations in obese subjects (r=-0.54, n=18, P=0.02). Black circles, post-menopausal women; white circles, pre-menopausal women; white triangles, men.

Full figure and legend (20K)

With regard to nutritional and metabolic variables, no significant relationships were found for the degree of cortisol-dependent inhibition, but significant relationships were found for the degree of IL-2-dependent stimulation. We found that percent increase after exposure to the cytokine was positively correlated with percent dietary carbohydrates (Figure 6) and serum LDL (Figure 7) and negatively correlated with percent dietary lipids (Figure 8).

Figure 6.

Correlation between IL-2-dependent stimulation and dietary carbohydrates in obese subjects (r=0.61, n=19, P=0.005). Black circles, post-menopausal women; white circles, pre-menopausal women; white triangles, men. Dietary analysis was not available for two subjects.

Full figure and legend (26K)

Figure 7.

Correlation between IL-2-dependent stimulation and serum LDL in obese subjects (r=0.55, n=21, P=0.009). Black circles, post-menopausal women; white circles, pre-menopausal women; white triangles, men.

Full figure and legend (23K)

Figure 8.

Correlation between IL-2-dependent stimulation and dietary lipids in obese subjects (r=-0.71, n=19, P=0.0006). Black circles, post-menopausal women; white circles, pre-menopausal women; white triangles, men. Dietary analysis was not available for two subjects.

Full figure and legend (23K)

Discussion

NK cells represent a key component of innate immunity and are involved in MHC-unrestricted surveillance against virus-infected and tumor cells. A vast literature supports the view that obesity and high fat diet are associated with an increased risk of infections and breast, endometrial and colorectal cancer, and with chronic systemic inflammation.^{23, 24, 25, 26, 27} Surprisingly enough, NK cell activity has been scarcely investigated in obese subjects in relation to gender, age and dietary components. Moriguchi et al³¹ found divergent patterns as a function of age and sex: in men (but not women) over 60 y, NK-cell dependent cytotoxicity was lower in obese than in normal subjects; on the contrary, in both men and women under 60 y NK-cell dependent cytotoxicity was higher in obese than in normal subjects. Nieman et al³² reported a significant negative correlation between body fat and NK cell activity in elderly women, but found no difference between obese and nonobese adult subjects.²⁴

These studies have focused on the spontaneous NK cell activity measurable in vitro after exposure of established target cells to PBM cell preparations. In this respect, we did not notice significant differences between obese subjects and controls. Cases enrolled in this study were carefully selected and did not show any sign of associated morbidity or abnormal laboratory parameters; their baseline levels of NK cell activity were all within the established ranges for the normal population of our region. The main purpose of the study, however, was to assess the responsiveness of their particular immune function to endogenous modifiers. As NK cells mediate spontaneous killing of certain cells, it is important that this process is under tight regulatory control.^{1, 2, 3, 41, 42} We have previously documented that in certain psychoneuroendocrinological disorders NK cell responses to challengers are altered yet in the absence of apparent abnormalities in the spontaneous activity.^{12, 13, 18} Accordingly, one important finding in our study is that obese women have a decreased sensitivity of NK cells to glucocorticoid inhibition, as inferred by in vitro exposure to physiological concentrations of cortisol. With obvious caution due to the limited number of cases, such decrease is comparable in central and peripheral obesity, and is independent of the menopausal status. Moreover, it is directly related to the degree of overweight, as expressed by BMI. As far as obese men are concerned, negative findings in our study could be accounted for by low number of cases and/or gender difference. Decreased GC sensitivity could be explained by the recent works of Vettor et al⁴³ and Güven et al,⁴⁴ who consistently found that in PBM cells drawn from uncomplicated obese subjects the number of GR as measured by radioligand-binding assay was reduced with respect to healthy nonobese controls. These two studies did not address the issue of gender differences in GR expression, but is worth noticing that women were more than 2/3 of the enrolled subjects in the study by Vettor et al, and the work by Güven et al, was limited to women. Indeed, the concentration of receptor is widely accepted as a key factor that dictates the magnitude of GC effects both in vitro and in vivo.⁴⁵ Decreased GR number is unlikely to be due to downregulation by increased concentrations of serum cortisol, since most Authors have found normal GR number in PBM from Cushing's disease patients.^{46, 47} Moreover, Güven did not report any correlation between serum cortisol concentration and GR number,⁴⁴ while Vettor et al paradoxically found a positive relation between serum and 24-h urinary cortisol and GR number.⁴³ Øgard et al⁴⁰ reported a negative correlation between serum leptin and lymphocyte GR mRNA in normal weight male subjects, and we found that serum leptin was inversely correlated with GC sensitivity. Though the mechanisms subserving such correlations still remain obscure, it is tempting to speculate that decreased GR number and sensitivity could be accounted for by leptin. In any event, less inhibition by cortisol of an important component of innate immunity could contribute to the maintenance of systemic subclinical inflammation, which has been widely reported in obese subjects.^{26, 27}

Another interesting finding in our obese subjects concerns the dissociation of the putative effects of nutrients upon glucocorticoid-dependent inhibition and IL-2-dependent stimulation of NK cell activity, respectively. While no significant relationship was apparent for the former, highly significant correlations were found for the latter. Percent increments above baseline levels of cytotoxicity were clearly different as a function of percent carbohydrate or fat in the diet. Moreover, they were positively correlated with serum LDL.

Our data are consistent with those of previous studies aimed to investigate the effects of dietary components on immune functions, including NK cell activity.^{14, 15, 16, 17} It is worth noticing that these studies demonstrated modulatory actions of sustained exposure to high-fat or high-carbohydrate diets. Indeed, in our study, blood samples were drawn after an overnight fast, and nutritional data reflect habitual diet composition of our obese subjects. Taken together with previous results, our data are consistent with chronic rather than acute effects of diet composition on NK function. In particular, the negative modulatory effect of a high-fat diet on the activation of immunocytes by cytokines has recently received attention and different types of fatty acids have been credited with specific action. Polyunsaturated fatty acids (PUFA), and especially those of the (n-3) family, have been found to inhibit both basal and cytokine-stimulated cell activity in animal models;^{14, 17} there are also observations in humans pointing to a negative relationship between circulating concentrations of PUFAs and levels of NK cytotoxicity.^{16, 17} We did not perform an analysis of the fatty acid composition in the habitual diet of our subjects, and the issue remains to be investigated as far as NK cell activity in obesity is concerned.

With regard to serum LDL concentration, their role is supported by the presence of receptors on phenotypically identifiable (CD56+) NK cells.^{48, 49} A number of studies have been published on the regulatory effect of LDL on NK cell activity. Conclusions were not consistently supporting a positive modulatory role, as it could be hypothesized from our positive correlation, yet available data plausibly indicate a positive modulation for the native form and a negative one for oxidized or acetylated forms.^{48, 50} Notwithstanding limitations in assessing more precisely the dietary components, our results are the first to suggest that nutritional factors modulate much more effectively the NK cell responsiveness to activating cytokines than that to inhibitory hormones.

In conclusion, despite the limited number of subjects in this study, we show that obesity affects the sensitivity of NK cells to glucocorticoid inhibition in women. Moreover, our findings are consistent with previous observations on the influence of nutritional factors and specific dietary components on the activity of immune cells. In future studies, it will be important not only to confirm and expand the present findings but also to focus on NK cell regulatory mechanisms that may depend on the metabolic activity of the adipose tissue. Additional research is needed to examine interactions between dietary components, abnormal patterns of circulating cholesterol and innate immunosurveillance in relation to cancer risk in obesity.

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Acknowledgements

This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica, Cassa di Risparmio di Cuneo, Cassa di Risparmio di Saluzzo and Fondazione Rossini.