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T-helper lymphopenia and decreased mitogenic response in cafeteria diet-induced obese rats
Nutrition Research 22 (2002) 497—506 www.elsevier.com/locate/nutres
T-helper lymphopenia and decreased mitogenic response in cafeteria diet-induced obese rats O. Lamas, J.A. Martinez, A. Marti* Department of Physiology and Nutrition, University of Navarra, 31080-Pamplona, Spain Received 8 June 2001; received in revised form 11 January 2002; accepted 13 January 2002
Abstract Obese individuals are more susceptible to infection than lean subjects. However, the underlying causes are not fully understood. To investigate whether obesity status influences immune response, we examined the immune function of diet-induced obese rats after 5 weeks of cafeteria feeding. Spleen lymphocyte subsets, blastogenic response to mitogens, phagocytosis, oxidative burst activity and the production of several cytokines by splenocytes were determined. Flow cytometric analysis revealed that spleen T helper cells were significantly reduced in obese rats without changes in T cytotoxic cells. Proliferative responses of splenocytes to LPS (lipopolysaccharide) and PHA (phytohaemagglutinin) from obese rats were significantly lower compared to their lean mates. Although no differences were found in phagocytic activity, a lower production of reactive oxygen metabolites (ROS) was noted in diet-induced obese animals as compared to lean rats. Finally, IL-2 production after mitogen addition was lower in cafeteria fed rats than in those receiving the control diet whereas IL-10 production tended to be higher in obese rats compared to lean animals. These results suggest that diet-induced obesity is accompanied by a reduction in the size of the T-helper pool, an impairment of splenocyte and monocyte responsiveness, decreased IL-2 and slightly increased IL-10 release by spleen, which may be related to potential health problems. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Lymphocyte subsets; Lymphoproliferation; Oxidative burst; Interleukin-2
1. Introduction Besides the well-known complications of obesity, including coronary heart disease [1], hypertension [2] and diabetes mellitus [3], obese subjects have been reported to have a higher
* Corresponding author. Tel.: 34-948-425600; fax: 34-948-425649. E-mail address: [email protected] (A. Marti). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 3 6 2 - 7
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incidence of infection and of several types of cancer [4 – 6]. Furthermore, obesity has also been associated with a poor antibody response to hepatitis B vaccination [7,8]. The mechanisms responsible for the increased risk of infection and poor antibody response among obese subjects are unknown, but may be linked to the negative effect that their metabolic milieu produces on immunity [9]. Although most data provide indirect evidence of impairment of immunocompetence among the obese, only a small number of studies have compared immune function in obese and nonobese individuals, and those have included a limited range of immune measurements [10 –17]. Nieman et al found decreased T- and B-cell proliferation in obese people and changes in lymphocyte subsets have been found [14]. Moreover, monocyte and granulocyte phagocytosis and oxidative burst were higher in obese subjects compared with nonobese [15]. In regard to animal studies, genetically obese rodents such as obese mice (ob/ob) [18] and obese diabetic mice (db/db) [19] as well as Zucker fatty rats (fa/fa) [20] also presented T-lymphopenia and diminished B cells. Lymphocyte responsiveness to different mitogens as well as the capacity to produce oxidative burst by monocytes and granulocytes was lower in obese compared with lean rodents [21]. Furthermore, the splenocytes cytokine production (IL-2, TNF-␣, IFN-␥, IL-10. . .) was diminished in obese animals compared with controls [22–25]. The majority of these immunity studies have used genetically obese animals with mutations in the leptin or leptin receptor gene. However, few cases of these mutations have been detected in obese people [26,27]. Therefore, we have adopted a “cafeteria” rodent model [28,29], in order to test the hypothesis that diet-induced obesity impairs lymphocyte and monocyte function by comparing several measurements of the immune response in dietinduced obese and lean animals.
2. Materials and methods 2.1. Animals and treatment Five week old male Wistar rats, obtained from the Applied Pharmacobiology Center (CIFA-Spain) were selected for this study. The animals were housed in cages under controlled conditions of light (12/12 h light/dark) and temperature (22⫾2°C). All experimental procedures were performed according to National and Institutional Guidelines for Animal Care and Use at the University of Navarra. Eighteen rats were assigned into two dietary groups for 5 weeks. The control group (n⫽9) was fed with standard laboratory pelleted diet and free access to water, while the second group (Cafeteria diet, n⫽9) was fed a fat-rich hypercaloric diet containing pate, chips, chocolate, bacon, biscuits and chow in a proportion of 2:1:1:1:1:1 as previously published [28,29]. Food was offered in excess and food intake and body weight were measured daily. After the 5 week experimental feeding period, animals were euthanized and spleen, thymus, interscapular BAT, abdominal, retroperitoneal, mesenteric and epididymal WAT, spleen and thymus were immediately removed, weighed and frozen. Serum glucose, proteins, choles-
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terol, glycerol, triglycerides and free fatty acids were analyzed using an Autoanalyzer (Cobas Roche Diagnostic, Basel, Switzerland) by routine procedures. Plasma insulin was assayed by RIA using 125I-labeled insulin (Diagnostic Products Corporation, Los Angeles, CA, USA) with a human insulin standard. Total plasma PAI-1 was determined by an ELISA kit purchased from Molecular Innovations (Royal Oak, MI, USA). 2.2. Measurement of lymphocyte subsets and “in vitro” blastogenic response of splenocytes to mitogens The spleens were aseptically removed, gently squashed between glass slides and resuspended in RPMI 1640 (Gibco BRL, Life Technologies, Barcelona, Spain) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Complete medium) as previously described [30,31]. Cell suspensions were centrifuged and washed twice in RPMI 1640. Erythrocytes were lysed with 155 mmol/l NH4Cl, 10 mmol/l KHCO3 and 0.1 mmol/l EDTA. The viability of mononuclear cells was above 95% as assayed by trypan blue dye exclusion. Viable splenocytes (1⫻105/ml) were incubated with mouse anti-rat PE-conjugated CD4⫹ (Clon W32, Labgen, Barcelona, Spain) and anti-rat FITC-conjugated CD8⫹ (Clon MRC OX-8, Labgen) for 20 minutes on ice in the dark. Samples were then centrifuged for 10 minutes at 3000 ⫻ g and 4°C. The supernatant was discarded and the cells were resuspended in 500 l of PBS. Cells stained with monoclonal antibodies were analyzed by flow cytometry with a FACScan Cell Sorter (Becton Dickinson, Mountain View, CA, USA). For each experiment, 5,000 cells with the forward and 90° light scattering characteristics of mononuclear cells were analyzed. The relative proportion of T-cell subsets were obtained as a percentage of the total cells and the count of each subset was calculated. For in vitro blastogenic response experiments, viable cells (1⫻105/ml) were placed in a tissue culture plate with or without concanavalin A (Con A; Sigma Chemical Co, St Louis, MI, USA) and phytohaemagglutinin-P (PHA; Sigma Chemical Co) at 5g/ml and 50g/ml, respectively as described elsewhere [30, 31]. Lipopolysaccharide (LPS; Sigma Chemical Co) was added to B-lymphocyte cultures at 100 g/ml and preliminary experiments confirmed that these concentrations of lectins were mitogenic for both T and B-lymphocytes from both obese and lean rats. The plates were cultured at 37°C in a 5% CO2-humidified air incubator (Haereus, Bilbao, Spain) for 72h, after which [3H]-thymidine was added at a concentration of 1Ci/well. Twenty-four hours later, cells were harvested and [3H]-thymidine incorporation (Amersham, Barcelona, Spain) was counted in a Beckman LS 5801 liquid scintillation counter (Beckman Instruments, Fullerton, CA, USA). Data are expressed as stimulation index (SI) calculated according to the formula: (cpm culture stimulated with mitogen/cpm culture non-stimulated). 2.3. Phagocytic and oxidative burst activity of monocytes To determine the phagocytic activity of monocytes a kit purchased from Orpegen Pharma (Heidelberg, Germany) was used according to the supplier’s instructions. Briefly, 100l of
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heparinized whole blood was incubated for 10 minutes on ice. Then, 20l of precooled E. coli was added per sample and test samples were incubated for 10 minutes at 37°C in a water bath. The phagocytosis was stopped by adding 100l of ice-cold quenching solution and mixing the samples. Samples were washed twice with 3 ml of washing solution and centrifuged for 5 minutes at 250 ⫻g and 4°C. Erythrocytes were then lysed and samples were fixed with 2 ml of prewarmed lysing solution for 20 minutes at room temperature. The samples were washed twice and 200l of DNA staining solution were added and incubated in a dark room for 10 minutes at 4°C. Finally, cells were analyzed using a FACScan Cell Sorter (Becton Dickinson). For each experiment, 10,000 monocytes were analyzed and the percentage of cells having performed phagocytosis was analyzed. Oxidative burst activity was measured using a kit purchased from Orpegen Pharma. Briefly, 100l of heparinized whole blood was incubated on ice for 10 minutes. Then, 20l of E. coli per test was added to the sample and incubated in a water bath at 37°C for 10 minutes. 20l of substrate solution was then added to the samples and they were incubated in a water bath for another 10 minutes. Then, 2 ml of prewarmed lysing solution were added and incubated for 20 minutes at room temperature. Samples were washed twice with 3 ml of washing solution. Later, 200l of DNA staining solution were added. Samples were incubated for 10 minutes on ice in the dark. Finally, 10,000 monocytes for each experiment were analyzed by flow cytometry using a FACScan Cell Sorter (Becton Dickinson). The percentage of cells having produced reactive oxygen metabolites were analyzed. 2.4. Determination of cytokines in the culture supernatant Spleen cells at a concentration of 1⫻105 cells/ml were set in 96-well plates in RPMI 1640 (Gibco BRL, Life Technologies, Barcelona, Spain) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Complete medium) and in the presence or absence of Con A at 5g/ml or LPS at 100g/ml. After 48h of incubation, the supernatant fraction was collected and assayed for cytokines. To determine the concentration of IL-2, IL-10, TNF-␣ and IFN-␥, specific sandwich-ELISA kits purchased from Endogen (Woburn, MA, USA) were used. Briefly, 96-well ELISA plate was coated with specific anti-rat capture antibodies in a coating buffer and incubated at room temperature for 2h. Plates were then washed with washing solution and incubated with 200l of blocking buffer for 2h. Appropriately diluted samples or standards (final volume 100l) were added to the wells in duplicates and incubated for 2h. The wells were washed and secondary antibodies were added. After washing, 100l of detection reagent (horseradish peroxidase-conjugated Streptavidin) was added and incubated for 30 minutes. The wells were washed again and 100l of substrate [TMB (3,3', 5,5'-tetramethylbenzidine)] was added into the wells. The reaction was stopped by the addition of 50l of 1M sulfuric acid and the intensity of the yellow color was read at 450 nm in a microplate reader (Titertek Multiskan Plus MKII). Absorbance was corrected by subtracting background from the mean values of the samples and other standards. A standard curve was generated to quantify the concentration of specific cytokines in the samples.
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Table 1 Effects of the cafeteria diet on final body weight, weight gain, daily food intake and fat depotsa
Body Weight (g) Weight Gain (g) Food Intake (g) Abdominal WAT (%) Retroperitoneal WAT (%) Mesenteric WAT (%) Epididymal WAT (%) Interscapular BAT (%) Spleen (%) Thymus (%)
Control (standard diet)
Obese (cafeteria diet)
p
298.11 ⫾ 7.06 133.00 ⫾ 6.20 57.96 ⫾ 0.69 1.28 ⫾ 0.14 1.18 ⫾ 0.12 0.83 ⫾ 0.06 1.39 ⫾ 0.04 0.13 ⫾ 0.01 0.22 ⫾ 0.01 0.13 ⫾ 0.01
333.44 ⫾ 6.35 162.89 ⫾ 6.87 74.65 ⫾ 0.83 1.55 ⫾ 0.12 1.93 ⫾ 0.14 1.12 ⫾ 0.08 1.82 ⫾ 0.07 0.25 ⫾ 0.03 0.20 ⫾ 0.01 0.15 ⫾ 0.01
0.003 0.008 0.001 n.s. 0.001 0.04 0.002 0.006 n.s. n.s.
a WAT, white adipose tissue; BAT, brown adipose tissue. Organ weights (%) refer to final body weight. Food intake was recorded for 24 h per cage. n.s. non significative differences. Values are means ⫾ SEM, n ⫽ 9.
2.5. Statistical analysis Values are given as the means ⫾ SEM. The non-parametric Mann-Whitney U test was used to verify the difference between groups, with a minimum significance level of P⬍0.05.
3. Results Rats eating a cafeteria diet for 5 weeks had a significantly increased body weight (p⬍0.01) as well as body weight gain (p⬍0.01) when compared to control rats (Table 1). Furthermore, food intake (g) was higher in obese rats (p⬍0.005) compared to lean controls. Also, relative interscapular BAT (p⬍0.01), retroperitoneal (p⬍0.005), mesenteric (p⬍0.04), and epididymal WAT (p⬍0.005) were significantly increased in cafeteria fed rats as compared to animals fed on the standard diet (Table 1). Spleen and thymus relative weights remained unchanged (Table 1). Serum triglycerides (p⬍0.001), cholesterol (p⬍0.001), glycerol (p⬍0.001) and FFA (p⬍0.001) were significantly higher in obese animals than in controls, while serum glucose (Table 2) and proteins remain unchanged (data not shown). Plasma PAI-1 levels were significantly increased (p⬍0.001) in obese rats as compared to control rats (Table 2). Splenic T-helper cells were significantly decreased in obese rats compared to control mates (p⬍0.05), while no change in cytotoxic/suppressor T cells was observed (Table 2). Proliferative responses of splenocytes to various B and T-cells mitogens were lower in rats receiving the cafeteria diet than control rats, the [3H]-thymidine incorporation was significantly diminished upon stimulation with LPS (p⬍0.001) and PHA (p⬍0.001) in obese rats compared to controls. No differences were observed after Con A stimulation of splenocytes (Table 2). While cafeteria feeding (Fig. 1) did not modify phagocytosis activity, the activated monocyte oxidative burst was significantly (p⬍0.005) lower among obese rats compared to
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Table 2 Effects of the diet on serum levels of glucose, TG, cholesterol, glycerol, FFA and plasma levels of PAI-1a
Glucose (mmol/l) TG (mmol/l) Cholesterol (mmol/l) Glycerol (mmol/l) FFA (mol/l) PAI-1 (ng/ml)
Control (standard diet)
Obese (cafeteria diet)
p
4.82 ⫾ 0.37 1.4 ⫾ 0.2 1.20 ⫾ 0.08 0.13 ⫾ 0.01 370 ⫾ 30 5.27 ⫾ 1.00
5.86 ⫾ 0.63 2.2 ⫾ 0.2 1.53 ⫾ 0.08 0.21 ⫾ 0.02 770 ⫾ 70 15.62 ⫾ 2.06
n.s. 0.003 0.019 0.007 0.003 0.002
a TG, Triglycerides; FFA, free fatty acids; PAI-1, Plasminogen Activator Inhibitor-1. n.s. non significative differences. Values are means ⫾ SEM, n ⫽ 9.
control rats. The IL-2 production was lower in obese rats (p⬍0.05) compared to lean rats (Fig. 2a). No change were found in other cytokines (data not shown) such as IFN-␥ (type 1 T-helper cells) and TNF-␣ (macrophages). However, IL-10 (type 2 T-helper cells) tended to be higher (p⫽0.09) in obese rats compared to controls (Fig. 2b).
4. Discussion In order to further understand disturbances in immune function under dietary situations of obesity, we have characterized the immune response of control rats and diet-induced obese rats. To our knowledge this is the first time, that immune function is analyzed in a diet-induced (cafeteria) model of obesity, since most of the studies have been performed in genetically obese animals. It has been suggested [32] that diet-induced obese animals are a more comparable model for human obesity than genetically-obese animals. Obese rats show hyperlipidemia and hypertriglyceridemia (Table 2). Furthermore, in this same model, a situation of hiperleptinemia has been reported [29]. However, it might be pointed out that not
Fig. 1. Phagocytic capacity and oxidative burst production by spleen macrophages of control and obese rats stimulated wih opsonied E. coli bacterias. Data are expressed as mean ⫾ SEM, n ⫽ 10. Bars with different letters indicate statistically significant differences (p ⬍ 0.05).
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Fig. 2a. IL-2 production by isolated splenocytes with or without Con A (5 g/ml) for 48h from control and obese rats. Values are means ⫾ SEM. Different letters indicate statistically significant differences (p ⬍ 0.05). 2b. IL-10 production by isolated splenocytes with or without LPS (100 g/ml) for 48h from control and obese rats. Values are means ⫾ SEM. Different letters indicate statistically significant differences (p ⬍ 0.05).
only was fat intake different, but also the micronutrients and protein intake of the obese rats was different compared to control rats. Different nutrient intake (zinc, iron, etc. . . ) may have some implications [33]. In this study we have found that T helper lymphocytes, but not T cytotoxic/suppressor lymphocytes, were diminished in obese rats compared to lean rats. In this context, Tanaka et al [11] observed a progressive and significant T lymphopenia in all the lymphocyte subsets of genetically obese Zucker rats. In other animal models such as db/db mice, a T lymphopenia was also reported [10]. Interestingly, other authors [14,15] reported an elevation of helper lymphocytes without differences in T cytotoxic/suppressor lymphocytes in obese individuals. The lymphoproliferative response was reduced in obese rats compared to control after LPS or PHA stimulation. A lower mitogenic response of lymphocytes was also found in genetically obese animals, which was independent of the mitogen used [11–14]. However, in lymphoproliferation we could not find any change when we stimulated with Con A, perhaps the difference may be attributed to the animal model (genetic vs. dietary obesity). It is uncertain whether the reduced responsiveness of lymphocytes in obese rats is directly
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Table 3 Effects of diet-induced obesity on lymphocyte subsets and lymphoproliferative response of splenocytes to mitogensa
% T helper (CD4⫹) % T cytotoxic (CD8⫹) LPS (100 g/ml) PHA (50 g/ml) Con A (5 g/ml)
Control (standard diet)
Obese (cafeteria diet)
p
26.79 ⫾ 0.88 17.83 ⫾ 1.23 1.95 ⫾ 0.25 2.52 ⫾ 0.32 1.22 ⫾ 0.04
21.25 ⫾ 1.55 17.69 ⫾ 1.55 0.78 ⫾ 0.14 1.37 ⫾ 0.09 1.11 ⫾ 0.17
0.045 n.s. ⬍0.001 ⬍0.001 n.s.
a
For lymphocyte subsets data are expressed as % of total splenocytes in spleen. For lymphoproliferation data are expressed as Stimulation Index to LPS, PHA and Con A. n.s. nonsignificative differences. Values are means ⫾ SEM, n ⫽ 9.
attributable to lymphopenia or, at least in part, related to a reduced functional response of lymphocytes. Elevated serum free fatty acid concentrations have been found to inhibit T lymphocyte signaling [34] and serum FFA levels were higher in the diet-induced obese rats compared to the controls (Table 2). While these data could explain the impaired responsiveness of lymphocyte proliferation in obese rats, other investigators [16] reported a decreased mitogen response of splenic lymphocytes in obese Zucker rats associated with the decreased expression of glucose transporter 1 (GLUT-1). Although there was no change in phagocytosis by macrophages and monocytes between obese and lean rats, oxidative burst production was lower in obese rats suggesting that cafeteria fed rats had a diminished ability to kill bacteria as compared to control fed animals. These results are in agreement with those of Plotkin et al [21], who were not able to found in fa/fa rats no statistical differences in phagocytosis. Obese fa/fa rats display a significant decrease in their ability to kill phagocytosed yeast cells as compared to the corresponding controls. Furthermore, IL-2 production by cultured splenocytes after stimulation with Con A was lower in obese rats than control rats. It is commonly accepted that cytokine production is modulated by high-fat diet intake [22–25]. The intra-group variability of cytokine levels may explain that IFN-␥ and TNF-␣ production were not statistically significant. On the other hand, IL-10 production tended to be higher in obese rats compared to control rats. IL-10 is a major inhibitory cytokine of the immune function [35]. Therefore, these results could, at least in part, contribute to the impaired immune function observed in cafeteria fed animals. In summary, we have shown that diet-induced obese rats have a decreased immune response and specifically a lower CD4⫹ T cells subset, in lymphocyte proliferative response to LPS and PHA, a lower production of ROS by macrophages and monocytes as well as IL-2 levels by cultured splenocytes, which may explain the reduced immune function of obese individuals with a positive energy balance [36].
Acknowlegment The authors are grateful for the collaboration of Dr. J.J. Lasarte from the Internal Medicine Department. They also thank Dr. B. De Fanti for helpful suggestions for the discussion. This
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work was supported by grants from the Navarra Government and Linea Especial: Nutricio´ n y Obesidad, (University of Navarra). O. Lamas is a Predoctoral Fellow from AAUN (Pamplona, Spain) and DANONE. References [1] National Institutes of Health Consensus Development Panel on the Health implications of Obesity, Health implications of obesity. National Institutes of Health consensus development conference statement. Ann Intern Med 1985;103:1073–7. [2] Feinleib M. Epidemiology of obesity in relation to health hazards. Ann Intern Med 1985;103:1019 –24. [3] Kannel WB, Brand N, Skinner J, Dawber TR, McNamara PM. The relation of obesity to blood pressure and development of hypertension: the Framingham study. Ann Inter Med 1967;67:48 –59. [4] Marks HH. Influence of obesity on morbidity and mortality. Bull NY Acad Med 1960;36:296 –312. [5] Meares EM. Factors that influence surgical wound infections. Urology 1975;6:535– 46. [6] Garfinkel L. Over weight and cancer. Ann Inter Med 1985;103:1034 – 6. [7] Weber DJ, Rutala WA, Samsa GP, Santimaw JE, Lemon SM. Obesity as a predictor of poor antibody response to hepatitis B plasma vaccine. JAMA 1985;254:3187–9. [8] Weber DJ, Rutala WA, Samsa GP, Bradshaw SE, Lemon SM. Impaired immunogenicity of hepatitis B vaccine in obese persons [letter]. N Engl J Med 1986;314:1393. [9] Stallone DD. The influence of obesity and its treatment on the immune system. Nutr Rev 1994;52:37–50. [10] Kimura M, Tanaka S, Isoda F, Sekigawa K, Yamakawa T, Sekihara H. T lymphopenia in obese diabetic (db/db) mice is non-selective, and thymus independent. Life Sciences 1998;14:1243–50. [11] Tanaka S, Isoda F, Yamakawa T, Ishihara M, Sekihara H. T lymphopenia in genetically obese rats. Clin Immun and Immunop 1998;86:219 –25. [12] Tanaka S, Isoda F, Kiuchi Y, Ikeda H, Mobbs CV, Yamakawa T. T lymphopenia in genetically obesediabetic wistar fatty rats: effects of body weight reduction on T cells. Metabolism 2000;49:1261– 6. [13] Tanaka S, Inoue S, Isoda F, Waseda M, Ishihara M, Yamakawa T, Sugiyama A, Takamura Y, Okuda K. Impaired immunity in obesity: suppressed but reversible lymphocyte responsiveness. Int J Obes 1993;17: 631– 6. [14] Nieman DC, Henson DA, Nehlsen-Cannarella SL, Ekkens M, Utter AC, Butterworth DE, Fagoaga OR. Influence of obesity on immune function. J Am Diet Assoc 1999;99:294 –9. [15] Nieman DC, Nehlsen-Cannarella SL, Henson DA, Butterworth DE, Fagoaga OR, Warren BJ, Rainwater MK. Immune response to obesity and moderate weight loss. Int J Obes 1996;29:353– 60. [16] Moriguchi S, Kato M, Sakai K, Yamamoto S, Shimizu E. Decreased mitogen response of splenic lymphocytes in obese Zucker rats is associated with the decreased expression of glucose transporter 1 (GLUT-1). Am J Clin Nutr 1998;67:1124 –9. [17] Marti A, Marcos A, Martı´nez JA. Obesity and immune function relationships. Obes Rev 2001;2:131– 40. [18] Zang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–32. [19] Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM. Abnormal splicing of the leptin receptor in diabetic mice. Nature 1996;379:632–5. [20] Chua SCJ, Chung WK, Wu-Peng XS, Zang Y, Liu SM, Tartaglia L, Leibel RL. Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 1996;271:994 – 6. [21] Plotkin BJ, Paulson D, Chelich A, Jurak D, Cole J, Kasimos J, Burdick JR, Casteel N. Immune responsiveness in a rat model for type II diabetes (Zucker rat, fa/fa): susceptibility to Candida albicans infection and leucocyte function. J Med Microbiol 1996;44:277– 83. [22] Mito N, Hosoda T, Kato C, Sato K. Change of cytokine balance in diet-induced obese mice. Metabolism 2000;49:1295–1300. [23] Jolly CA, Fernandes G. Diet modulates Th-1 and Th-2 cytokine production in the peripheral blood of lupus-prone mice. J Clin Immunol 1999;19:172– 8.
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T-helper lymphopenia and decreased mitogenic response in cafeteria diet-induced obese rats O. Lamas, J.A. Martinez, A. Marti* Department of Physiology and Nutrition, University of Navarra, 31080-Pamplona, Spain Received 8 June 2001; received in revised form 11 January 2002; accepted 13 January 2002
Abstract Obese individuals are more susceptible to infection than lean subjects. However, the underlying causes are not fully understood. To investigate whether obesity status influences immune response, we examined the immune function of diet-induced obese rats after 5 weeks of cafeteria feeding. Spleen lymphocyte subsets, blastogenic response to mitogens, phagocytosis, oxidative burst activity and the production of several cytokines by splenocytes were determined. Flow cytometric analysis revealed that spleen T helper cells were significantly reduced in obese rats without changes in T cytotoxic cells. Proliferative responses of splenocytes to LPS (lipopolysaccharide) and PHA (phytohaemagglutinin) from obese rats were significantly lower compared to their lean mates. Although no differences were found in phagocytic activity, a lower production of reactive oxygen metabolites (ROS) was noted in diet-induced obese animals as compared to lean rats. Finally, IL-2 production after mitogen addition was lower in cafeteria fed rats than in those receiving the control diet whereas IL-10 production tended to be higher in obese rats compared to lean animals. These results suggest that diet-induced obesity is accompanied by a reduction in the size of the T-helper pool, an impairment of splenocyte and monocyte responsiveness, decreased IL-2 and slightly increased IL-10 release by spleen, which may be related to potential health problems. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Lymphocyte subsets; Lymphoproliferation; Oxidative burst; Interleukin-2
1. Introduction Besides the well-known complications of obesity, including coronary heart disease [1], hypertension [2] and diabetes mellitus [3], obese subjects have been reported to have a higher
* Corresponding author. Tel.: 34-948-425600; fax: 34-948-425649. E-mail address: [email protected] (A. Marti). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 3 6 2 - 7
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incidence of infection and of several types of cancer [4 – 6]. Furthermore, obesity has also been associated with a poor antibody response to hepatitis B vaccination [7,8]. The mechanisms responsible for the increased risk of infection and poor antibody response among obese subjects are unknown, but may be linked to the negative effect that their metabolic milieu produces on immunity [9]. Although most data provide indirect evidence of impairment of immunocompetence among the obese, only a small number of studies have compared immune function in obese and nonobese individuals, and those have included a limited range of immune measurements [10 –17]. Nieman et al found decreased T- and B-cell proliferation in obese people and changes in lymphocyte subsets have been found [14]. Moreover, monocyte and granulocyte phagocytosis and oxidative burst were higher in obese subjects compared with nonobese [15]. In regard to animal studies, genetically obese rodents such as obese mice (ob/ob) [18] and obese diabetic mice (db/db) [19] as well as Zucker fatty rats (fa/fa) [20] also presented T-lymphopenia and diminished B cells. Lymphocyte responsiveness to different mitogens as well as the capacity to produce oxidative burst by monocytes and granulocytes was lower in obese compared with lean rodents [21]. Furthermore, the splenocytes cytokine production (IL-2, TNF-␣, IFN-␥, IL-10. . .) was diminished in obese animals compared with controls [22–25]. The majority of these immunity studies have used genetically obese animals with mutations in the leptin or leptin receptor gene. However, few cases of these mutations have been detected in obese people [26,27]. Therefore, we have adopted a “cafeteria” rodent model [28,29], in order to test the hypothesis that diet-induced obesity impairs lymphocyte and monocyte function by comparing several measurements of the immune response in dietinduced obese and lean animals.
2. Materials and methods 2.1. Animals and treatment Five week old male Wistar rats, obtained from the Applied Pharmacobiology Center (CIFA-Spain) were selected for this study. The animals were housed in cages under controlled conditions of light (12/12 h light/dark) and temperature (22⫾2°C). All experimental procedures were performed according to National and Institutional Guidelines for Animal Care and Use at the University of Navarra. Eighteen rats were assigned into two dietary groups for 5 weeks. The control group (n⫽9) was fed with standard laboratory pelleted diet and free access to water, while the second group (Cafeteria diet, n⫽9) was fed a fat-rich hypercaloric diet containing pate, chips, chocolate, bacon, biscuits and chow in a proportion of 2:1:1:1:1:1 as previously published [28,29]. Food was offered in excess and food intake and body weight were measured daily. After the 5 week experimental feeding period, animals were euthanized and spleen, thymus, interscapular BAT, abdominal, retroperitoneal, mesenteric and epididymal WAT, spleen and thymus were immediately removed, weighed and frozen. Serum glucose, proteins, choles-
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terol, glycerol, triglycerides and free fatty acids were analyzed using an Autoanalyzer (Cobas Roche Diagnostic, Basel, Switzerland) by routine procedures. Plasma insulin was assayed by RIA using 125I-labeled insulin (Diagnostic Products Corporation, Los Angeles, CA, USA) with a human insulin standard. Total plasma PAI-1 was determined by an ELISA kit purchased from Molecular Innovations (Royal Oak, MI, USA). 2.2. Measurement of lymphocyte subsets and “in vitro” blastogenic response of splenocytes to mitogens The spleens were aseptically removed, gently squashed between glass slides and resuspended in RPMI 1640 (Gibco BRL, Life Technologies, Barcelona, Spain) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Complete medium) as previously described [30,31]. Cell suspensions were centrifuged and washed twice in RPMI 1640. Erythrocytes were lysed with 155 mmol/l NH4Cl, 10 mmol/l KHCO3 and 0.1 mmol/l EDTA. The viability of mononuclear cells was above 95% as assayed by trypan blue dye exclusion. Viable splenocytes (1⫻105/ml) were incubated with mouse anti-rat PE-conjugated CD4⫹ (Clon W32, Labgen, Barcelona, Spain) and anti-rat FITC-conjugated CD8⫹ (Clon MRC OX-8, Labgen) for 20 minutes on ice in the dark. Samples were then centrifuged for 10 minutes at 3000 ⫻ g and 4°C. The supernatant was discarded and the cells were resuspended in 500 l of PBS. Cells stained with monoclonal antibodies were analyzed by flow cytometry with a FACScan Cell Sorter (Becton Dickinson, Mountain View, CA, USA). For each experiment, 5,000 cells with the forward and 90° light scattering characteristics of mononuclear cells were analyzed. The relative proportion of T-cell subsets were obtained as a percentage of the total cells and the count of each subset was calculated. For in vitro blastogenic response experiments, viable cells (1⫻105/ml) were placed in a tissue culture plate with or without concanavalin A (Con A; Sigma Chemical Co, St Louis, MI, USA) and phytohaemagglutinin-P (PHA; Sigma Chemical Co) at 5g/ml and 50g/ml, respectively as described elsewhere [30, 31]. Lipopolysaccharide (LPS; Sigma Chemical Co) was added to B-lymphocyte cultures at 100 g/ml and preliminary experiments confirmed that these concentrations of lectins were mitogenic for both T and B-lymphocytes from both obese and lean rats. The plates were cultured at 37°C in a 5% CO2-humidified air incubator (Haereus, Bilbao, Spain) for 72h, after which [3H]-thymidine was added at a concentration of 1Ci/well. Twenty-four hours later, cells were harvested and [3H]-thymidine incorporation (Amersham, Barcelona, Spain) was counted in a Beckman LS 5801 liquid scintillation counter (Beckman Instruments, Fullerton, CA, USA). Data are expressed as stimulation index (SI) calculated according to the formula: (cpm culture stimulated with mitogen/cpm culture non-stimulated). 2.3. Phagocytic and oxidative burst activity of monocytes To determine the phagocytic activity of monocytes a kit purchased from Orpegen Pharma (Heidelberg, Germany) was used according to the supplier’s instructions. Briefly, 100l of
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heparinized whole blood was incubated for 10 minutes on ice. Then, 20l of precooled E. coli was added per sample and test samples were incubated for 10 minutes at 37°C in a water bath. The phagocytosis was stopped by adding 100l of ice-cold quenching solution and mixing the samples. Samples were washed twice with 3 ml of washing solution and centrifuged for 5 minutes at 250 ⫻g and 4°C. Erythrocytes were then lysed and samples were fixed with 2 ml of prewarmed lysing solution for 20 minutes at room temperature. The samples were washed twice and 200l of DNA staining solution were added and incubated in a dark room for 10 minutes at 4°C. Finally, cells were analyzed using a FACScan Cell Sorter (Becton Dickinson). For each experiment, 10,000 monocytes were analyzed and the percentage of cells having performed phagocytosis was analyzed. Oxidative burst activity was measured using a kit purchased from Orpegen Pharma. Briefly, 100l of heparinized whole blood was incubated on ice for 10 minutes. Then, 20l of E. coli per test was added to the sample and incubated in a water bath at 37°C for 10 minutes. 20l of substrate solution was then added to the samples and they were incubated in a water bath for another 10 minutes. Then, 2 ml of prewarmed lysing solution were added and incubated for 20 minutes at room temperature. Samples were washed twice with 3 ml of washing solution. Later, 200l of DNA staining solution were added. Samples were incubated for 10 minutes on ice in the dark. Finally, 10,000 monocytes for each experiment were analyzed by flow cytometry using a FACScan Cell Sorter (Becton Dickinson). The percentage of cells having produced reactive oxygen metabolites were analyzed. 2.4. Determination of cytokines in the culture supernatant Spleen cells at a concentration of 1⫻105 cells/ml were set in 96-well plates in RPMI 1640 (Gibco BRL, Life Technologies, Barcelona, Spain) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Complete medium) and in the presence or absence of Con A at 5g/ml or LPS at 100g/ml. After 48h of incubation, the supernatant fraction was collected and assayed for cytokines. To determine the concentration of IL-2, IL-10, TNF-␣ and IFN-␥, specific sandwich-ELISA kits purchased from Endogen (Woburn, MA, USA) were used. Briefly, 96-well ELISA plate was coated with specific anti-rat capture antibodies in a coating buffer and incubated at room temperature for 2h. Plates were then washed with washing solution and incubated with 200l of blocking buffer for 2h. Appropriately diluted samples or standards (final volume 100l) were added to the wells in duplicates and incubated for 2h. The wells were washed and secondary antibodies were added. After washing, 100l of detection reagent (horseradish peroxidase-conjugated Streptavidin) was added and incubated for 30 minutes. The wells were washed again and 100l of substrate [TMB (3,3', 5,5'-tetramethylbenzidine)] was added into the wells. The reaction was stopped by the addition of 50l of 1M sulfuric acid and the intensity of the yellow color was read at 450 nm in a microplate reader (Titertek Multiskan Plus MKII). Absorbance was corrected by subtracting background from the mean values of the samples and other standards. A standard curve was generated to quantify the concentration of specific cytokines in the samples.
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Table 1 Effects of the cafeteria diet on final body weight, weight gain, daily food intake and fat depotsa
Body Weight (g) Weight Gain (g) Food Intake (g) Abdominal WAT (%) Retroperitoneal WAT (%) Mesenteric WAT (%) Epididymal WAT (%) Interscapular BAT (%) Spleen (%) Thymus (%)
Control (standard diet)
Obese (cafeteria diet)
p
298.11 ⫾ 7.06 133.00 ⫾ 6.20 57.96 ⫾ 0.69 1.28 ⫾ 0.14 1.18 ⫾ 0.12 0.83 ⫾ 0.06 1.39 ⫾ 0.04 0.13 ⫾ 0.01 0.22 ⫾ 0.01 0.13 ⫾ 0.01
333.44 ⫾ 6.35 162.89 ⫾ 6.87 74.65 ⫾ 0.83 1.55 ⫾ 0.12 1.93 ⫾ 0.14 1.12 ⫾ 0.08 1.82 ⫾ 0.07 0.25 ⫾ 0.03 0.20 ⫾ 0.01 0.15 ⫾ 0.01
0.003 0.008 0.001 n.s. 0.001 0.04 0.002 0.006 n.s. n.s.
a WAT, white adipose tissue; BAT, brown adipose tissue. Organ weights (%) refer to final body weight. Food intake was recorded for 24 h per cage. n.s. non significative differences. Values are means ⫾ SEM, n ⫽ 9.
2.5. Statistical analysis Values are given as the means ⫾ SEM. The non-parametric Mann-Whitney U test was used to verify the difference between groups, with a minimum significance level of P⬍0.05.
3. Results Rats eating a cafeteria diet for 5 weeks had a significantly increased body weight (p⬍0.01) as well as body weight gain (p⬍0.01) when compared to control rats (Table 1). Furthermore, food intake (g) was higher in obese rats (p⬍0.005) compared to lean controls. Also, relative interscapular BAT (p⬍0.01), retroperitoneal (p⬍0.005), mesenteric (p⬍0.04), and epididymal WAT (p⬍0.005) were significantly increased in cafeteria fed rats as compared to animals fed on the standard diet (Table 1). Spleen and thymus relative weights remained unchanged (Table 1). Serum triglycerides (p⬍0.001), cholesterol (p⬍0.001), glycerol (p⬍0.001) and FFA (p⬍0.001) were significantly higher in obese animals than in controls, while serum glucose (Table 2) and proteins remain unchanged (data not shown). Plasma PAI-1 levels were significantly increased (p⬍0.001) in obese rats as compared to control rats (Table 2). Splenic T-helper cells were significantly decreased in obese rats compared to control mates (p⬍0.05), while no change in cytotoxic/suppressor T cells was observed (Table 2). Proliferative responses of splenocytes to various B and T-cells mitogens were lower in rats receiving the cafeteria diet than control rats, the [3H]-thymidine incorporation was significantly diminished upon stimulation with LPS (p⬍0.001) and PHA (p⬍0.001) in obese rats compared to controls. No differences were observed after Con A stimulation of splenocytes (Table 2). While cafeteria feeding (Fig. 1) did not modify phagocytosis activity, the activated monocyte oxidative burst was significantly (p⬍0.005) lower among obese rats compared to
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Table 2 Effects of the diet on serum levels of glucose, TG, cholesterol, glycerol, FFA and plasma levels of PAI-1a
Glucose (mmol/l) TG (mmol/l) Cholesterol (mmol/l) Glycerol (mmol/l) FFA (mol/l) PAI-1 (ng/ml)
Control (standard diet)
Obese (cafeteria diet)
p
4.82 ⫾ 0.37 1.4 ⫾ 0.2 1.20 ⫾ 0.08 0.13 ⫾ 0.01 370 ⫾ 30 5.27 ⫾ 1.00
5.86 ⫾ 0.63 2.2 ⫾ 0.2 1.53 ⫾ 0.08 0.21 ⫾ 0.02 770 ⫾ 70 15.62 ⫾ 2.06
n.s. 0.003 0.019 0.007 0.003 0.002
a TG, Triglycerides; FFA, free fatty acids; PAI-1, Plasminogen Activator Inhibitor-1. n.s. non significative differences. Values are means ⫾ SEM, n ⫽ 9.
control rats. The IL-2 production was lower in obese rats (p⬍0.05) compared to lean rats (Fig. 2a). No change were found in other cytokines (data not shown) such as IFN-␥ (type 1 T-helper cells) and TNF-␣ (macrophages). However, IL-10 (type 2 T-helper cells) tended to be higher (p⫽0.09) in obese rats compared to controls (Fig. 2b).
4. Discussion In order to further understand disturbances in immune function under dietary situations of obesity, we have characterized the immune response of control rats and diet-induced obese rats. To our knowledge this is the first time, that immune function is analyzed in a diet-induced (cafeteria) model of obesity, since most of the studies have been performed in genetically obese animals. It has been suggested [32] that diet-induced obese animals are a more comparable model for human obesity than genetically-obese animals. Obese rats show hyperlipidemia and hypertriglyceridemia (Table 2). Furthermore, in this same model, a situation of hiperleptinemia has been reported [29]. However, it might be pointed out that not
Fig. 1. Phagocytic capacity and oxidative burst production by spleen macrophages of control and obese rats stimulated wih opsonied E. coli bacterias. Data are expressed as mean ⫾ SEM, n ⫽ 10. Bars with different letters indicate statistically significant differences (p ⬍ 0.05).
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Fig. 2a. IL-2 production by isolated splenocytes with or without Con A (5 g/ml) for 48h from control and obese rats. Values are means ⫾ SEM. Different letters indicate statistically significant differences (p ⬍ 0.05). 2b. IL-10 production by isolated splenocytes with or without LPS (100 g/ml) for 48h from control and obese rats. Values are means ⫾ SEM. Different letters indicate statistically significant differences (p ⬍ 0.05).
only was fat intake different, but also the micronutrients and protein intake of the obese rats was different compared to control rats. Different nutrient intake (zinc, iron, etc. . . ) may have some implications [33]. In this study we have found that T helper lymphocytes, but not T cytotoxic/suppressor lymphocytes, were diminished in obese rats compared to lean rats. In this context, Tanaka et al [11] observed a progressive and significant T lymphopenia in all the lymphocyte subsets of genetically obese Zucker rats. In other animal models such as db/db mice, a T lymphopenia was also reported [10]. Interestingly, other authors [14,15] reported an elevation of helper lymphocytes without differences in T cytotoxic/suppressor lymphocytes in obese individuals. The lymphoproliferative response was reduced in obese rats compared to control after LPS or PHA stimulation. A lower mitogenic response of lymphocytes was also found in genetically obese animals, which was independent of the mitogen used [11–14]. However, in lymphoproliferation we could not find any change when we stimulated with Con A, perhaps the difference may be attributed to the animal model (genetic vs. dietary obesity). It is uncertain whether the reduced responsiveness of lymphocytes in obese rats is directly
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Table 3 Effects of diet-induced obesity on lymphocyte subsets and lymphoproliferative response of splenocytes to mitogensa
% T helper (CD4⫹) % T cytotoxic (CD8⫹) LPS (100 g/ml) PHA (50 g/ml) Con A (5 g/ml)
Control (standard diet)
Obese (cafeteria diet)
p
26.79 ⫾ 0.88 17.83 ⫾ 1.23 1.95 ⫾ 0.25 2.52 ⫾ 0.32 1.22 ⫾ 0.04
21.25 ⫾ 1.55 17.69 ⫾ 1.55 0.78 ⫾ 0.14 1.37 ⫾ 0.09 1.11 ⫾ 0.17
0.045 n.s. ⬍0.001 ⬍0.001 n.s.
a
For lymphocyte subsets data are expressed as % of total splenocytes in spleen. For lymphoproliferation data are expressed as Stimulation Index to LPS, PHA and Con A. n.s. nonsignificative differences. Values are means ⫾ SEM, n ⫽ 9.
attributable to lymphopenia or, at least in part, related to a reduced functional response of lymphocytes. Elevated serum free fatty acid concentrations have been found to inhibit T lymphocyte signaling [34] and serum FFA levels were higher in the diet-induced obese rats compared to the controls (Table 2). While these data could explain the impaired responsiveness of lymphocyte proliferation in obese rats, other investigators [16] reported a decreased mitogen response of splenic lymphocytes in obese Zucker rats associated with the decreased expression of glucose transporter 1 (GLUT-1). Although there was no change in phagocytosis by macrophages and monocytes between obese and lean rats, oxidative burst production was lower in obese rats suggesting that cafeteria fed rats had a diminished ability to kill bacteria as compared to control fed animals. These results are in agreement with those of Plotkin et al [21], who were not able to found in fa/fa rats no statistical differences in phagocytosis. Obese fa/fa rats display a significant decrease in their ability to kill phagocytosed yeast cells as compared to the corresponding controls. Furthermore, IL-2 production by cultured splenocytes after stimulation with Con A was lower in obese rats than control rats. It is commonly accepted that cytokine production is modulated by high-fat diet intake [22–25]. The intra-group variability of cytokine levels may explain that IFN-␥ and TNF-␣ production were not statistically significant. On the other hand, IL-10 production tended to be higher in obese rats compared to control rats. IL-10 is a major inhibitory cytokine of the immune function [35]. Therefore, these results could, at least in part, contribute to the impaired immune function observed in cafeteria fed animals. In summary, we have shown that diet-induced obese rats have a decreased immune response and specifically a lower CD4⫹ T cells subset, in lymphocyte proliferative response to LPS and PHA, a lower production of ROS by macrophages and monocytes as well as IL-2 levels by cultured splenocytes, which may explain the reduced immune function of obese individuals with a positive energy balance [36].
Acknowlegment The authors are grateful for the collaboration of Dr. J.J. Lasarte from the Internal Medicine Department. They also thank Dr. B. De Fanti for helpful suggestions for the discussion. This
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