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BRIEF REPORT |
Dipartimento di Pediatria (R.I., A.S., A.G., F.C., M.I.S., A.F., A.V.), Università Federico II, 80131 Naples, Italy; Istituto di Endocrinologia e Oncologia Sperimentale (S.F., D.A., G.M.), Consiglio Nazionale delle Ricerche, 80125 Naples, Italy; and Laboratorio di Immunologia A.O. Monaldi (F.P.), 80131 Naples, Italy
Address all correspondence and requests for reprints to: Dr. Raffaele Iorio, Department of Pediatrics, University of Naples "Federico II," Via Sergio Pansini n. 5, 80131 Naples, Italy. E-mail: riorio{at}unina.it.
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Abstract |
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Objective: Thus, the objective of this study was to evaluate immune phenotype and leptin serum levels in obese children with and without obesity-related liver disease.
Design: The study was performed in two groups of consecutive obese children: the first formed by children with obesity-related liver disease, diagnosed in the presence of chronic hypertransaminasemia, liver steatosis at ultrasound, and absence of known etiologies; the second composed of children with isolated obesity. In all patients serum leptin, immunoglobulins, peripheral T, B, and natural killer (NK) cells were evaluated.
Results: Twenty-three children in the first group and 16 children in the second were considered eligible. Serum leptin was increased in both groups but without any significant difference. No significant correlation was found between leptin and aminotransferases, lipid serum levels, and all tested lymphocyte subpopulations. Patients with obesity-related liver disease showed significantly higher peripheral NK and B cell counts and IgA levels than children with isolated obesity. Furthermore, no correlation was found between severity of liver disease and lymphocyte subpopulations.
Conclusion: In our study, leptin did not correlate with hepatic steatosis, aminotransferases, and serum lipids. Children with obesity-related liver disease showed significantly higher peripheral NK and B cells and IgA levels. Additional studies are required to define the pathogenetic role of these immunological findings.
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Introduction |
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Potential steps involved in the pathogenesis of NAFLD include abnormalities of lipid metabolism, production of reactive oxygen species, increased hepatic lipid peroxidation, activated Stellate cells, and abnormal patterns of cytokine production (2). According to the multihit theory, the first hit involves accumulation of fat in the hepatic parenchyma, probably due to insulin resistance commonly observed in patients with NAFLD (2). Also, recently leptin has been involved in the pathogenesis of NAFLD (3). Leptin, whose main role is the regulation of metabolic, endocrine, and behavioral responses to starvation, might play a key role in the relationship between nutritional status and immune response (4).
The objective of this study was to evaluate the role of leptin and of immunologic parameters such as serum immunoglobulins, T and B lymphocyte subpopulation (CD3+, CD4+, CD8+, CD19+), and natural killer (NK) (CD3–/CD16+/CD56+) in the peripheral blood of obese children with or without liver disease.
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Subjects and Methods |
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The study was performed in two groups of consecutive obese children: the first formed by children with obesity-related liver disease, the second by children with isolated obesity. Patients were defined obese if body mass index (BMI) was at the 95th percentile or greater for age (5). Obesity-related liver disease was diagnosed in presence of chronic (>6 months’ duration) hypertransaminasemia (alanine aminotransferase, 
1.5 times above normal values) and echographic signs of liver steatosis, in absence of known causes of liver disease. Obesity was defined isolated in absence of genetic, metabolic, and/or endocrine syndromes.
In all patients liver function tests, cholesterol, triglycerides, serum iron levels, ferritin, complete blood count, erythrocyte sedimentation rate, and C-reactive protein were tested. Insulin resistance was calculated from fasting plasma insulin and glucose using the homeostasis model assessment (HOMA), a computer-based model of glucose/insulin interactions (6).
All patients underwent an ultrasonographic study of liver and biliary tract. Liver steatosis was graded as mild, moderate, and severe (7).
In children with signs of liver disease, the following causes were ruled out: infections, autoimmune hepatitis, drug-induced liver disease, celiac disease, biliary tract disease, genetic and metabolic diseases such as 
1-antitrypsin deficiency, Wilson disease, and hemochromatosis. Endocrinological disorders such as thyroid diseases, hypopituitarism, and adrenocortical insufficiency were also considered. Leptin, Ig serum levels, T and B lymphocyte subpopulations (CD3+, CD4+, CD8+, CD19+), and NK (CD3–/CD16+/CD56+) were tested in all children. All patients’ parents or guardians were informed about the aim and procedures of the study and gave their consent. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the local ethical committee.
Measurements
Leptin concentrations were determined with human leptin ELISA kits (Alexis Corp., Laüfelfingen, Switzerland) within 2 months of sampling. Leptin concentrations were calculated from standard curves generated for each assay using recombinant human leptin. The minimum detection limit of the assay was 0.2 ng/ml. Samples were measured in duplicate at 450-nm wavelength, using an ELISA plate reader (Bio-Rad Laboratories, Inc., Hercules, CA) (8).
Immunophenotypic analysis of peripheral blood cells was performed with an EPICS XL flow cytometer (Beckman Coulter, Marseille, France) using the XL System II software (Beckman Coulter). Triple combinations of different antihuman monoclonal antibodies (mAbs) such as the fluorescein isothiocyanate (FITC)- and phosphatidylethanolamine (PE)-anti-CD3, PE- and prohormone convertase (PC)-5-anti-CD4, PC5-anti-CD8, PE-anti-CD16, PC5-anti-CD19, PE-anti-CD25, FITC-anti-CD45, and PE-anti-CD56 were purchased from Coulter Immunotech (Marseille, France). All blood samples, obtained at 0830 h after an overnight fast, were analyzed within 3–4 h of sampling. Immunofluorescence staining was performed using the above specific mAbs according to standard procedures. Briefly, 100 µl of whole blood was added to the appropriate mAb mixes. After incubation in the dark for 30 min at room temperature, samples were washed twice with PBS and 10% fetal calf serum. Erythrocytes were lysed through incubation with the lysing solution (Becton Dickinson) for 10 min at room temperature. The different subsets were assessed by three-color analysis using an acquisition gate on lymphocytes by using fetal calf serum and saline sodium citrate parameters as well as the FITC-anti-CD45 labeling (9). Ig serum levels were quantified by nephelometry.
Statistical analysis
Statistical differences in quantitative data were determined using the Student t test and the Mann-Whitney U test, when applicable; the 
2 test and the Fisher’s exact test were used for qualitative data, as appropriate. P < 0.05 was considered significant.
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Results |
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. All patients were symptom free. No patient had signs of acute infectious disease at the time of the study. Furthermore, none of the children of group 1 showed signs of either chronic liver disease, such as spider nevi and palmar erythema, or portal hypertension or ascites. On clinical examination, 10 children (six males; median age 9 yr, range 6.1–12.5) with obesity-related liver disease showed hepatomegaly of normal consistency, confirmed at ultrasound scanning. None of the patients showed laboratory signs of either cholestasis or liver failure. Inflammatory parameters, iron status, and fasting blood glucose levels were in the normal range in both groups. Two children with obesity-related liver disease and none with isolated obesity had a HOMA value of 2.5 (Table 1).
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). No significant correlation was found between leptin levels and aminotransferases, triglycerides, cholesterol serum levels, and lymphocyte subpopulations.
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). In group 1, no correlation was found between severity of liver disease (in terms of hypertransaminasemia and steatosis) and levels of peripheral lymphocyte subpopulations. The other cells populations did not change significantly between the two groups. Serum IgG and IgM levels were normal and did not differ between the two groups, whereas serum IgA levels was significantly higher in group 1 (mean value 1.48 ± 0.50 g/liter) than group 2 (1.07 ± 0.34, P = 0.02). |
Discussion |
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, IL-4, and TNF-mediated system (12, 13, 14). So far, analysis of T and B lymphocyte subpopulations in the peripheral blood has been performed exclusively in adults with chronic hepatitis C; in these patients a correlation between NK cell blood value with histological activity and liver fibrosis was found (15). Although in the present study no information about phenotyping of intrahepatic lymphocytes was available, it can be hypothesized that NK count in the peripheral blood may reflect the number of intrahepatic lymphocytes, as previously reported (16).
Liver biopsy was not performed in our patients because in children the role of biopsy for diagnosis of NAFLD has yet to be established. Furthermore, histological diagnosis of NAFLD does not affect the treatment of obesity-related liver disease in children. In the studied patients, diagnosis was based on concomitance of chronic hypertransaminasemia and liver steatosis at ultrasound in absence of other causes of liver disease. Our findings are the first, to our knowledge, to show an increase in NK in the peripheral blood of children with obesity-related liver disease. Due to the cytotoxic activity of these lymphocytes, it is possible to hypothesize a role for these cells in the pathogenesis of the liver damage and increase of transaminases.
Chitturi et al. (17) reported a correlation between serum leptin and hepatic steatosis and inflammation, anthropometric indices, serum lipids, glucose, and transaminases in adult patients with nonalcoholic steatohepatitis. They also hypothesized that leptin may play a crucial role in the pathogenesis of NAFLD. In contrast with these data, in the current study, no direct pathogenetic role for leptin was found. In fact, although leptin was elevated in both groups of the studied children, leptin levels did not correlate with presence of liver disease. A pleiotropic effect of leptin on lymphoid organ homeostasis and T lymphocyte functions, in particular proinflammatory T helper immune response (18), was demonstrated. A preliminary study also indicated that leptin can play a role in innate immunity, affecting NK cell function (19). It has been observed that human NK cells expressed leptin receptor and that human recombinant leptin affects human NK cell function, inducing their cytotoxicity with a dose-dependent mechanism (19). Nevertheless, in the present study, a relationship between leptin serum levels and peripheral NK cells has not been found.
Another interesting evidence is the increase of peripheral B cells in our children with obesity-related liver disease. This increase could in part explain the slight but significant increase of IgA circulating levels found in these patients. Also previous studies described the presence of IgA deposition in histologic sections of NAFLD patients and an increase of serum IgA levels in about 25% of patients (20).
In contrast with previous data, the studied obese children did not show laboratory evidence of insulin resistance. This result could be related to the size of our sample.
The role of immunological findings of our study in the pathogenesis of obesity-related liver disease must be established. Of note, children represent an ideal model for the study of natural history of obesity-related liver disease for the absence of common confounding factors of liver disease such as alcohol consumption and drugs.
In summary, our results shows an increase in NK, B cells, and IgA in patients with obesity-related liver disease. This immunological finding did not correlate with serum leptin levels. It is therefore likely an immune-mediated pathogenesis of obesity-related liver disease and that fat deposition can alter the generation and eventually function of immune cells. Whether this can be pathogenic for an autoaggression of liver still needs to be demonstrated. Clarification of these mechanisms could help in the settlement of novel therapeutic approaches.
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Footnotes |
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First Published Online October 25, 2005
Abbreviations: BMI, Body mass index; FITC, fluorescein isothiocyanate; HOMA, homeostasis model assessment; mAb, monoclonal antibody; NAFLD, nonalcoholic fatty liver disease; NK, natural killer; PC, prohormone convertase; PE, phosphatidylethanolamine.
Received May 12, 2005.
Accepted October 19, 2005.
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References |
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plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology 111:462–471[CrossRef][Medline] during alcohol consumption. Gastroenterology 126:1387–1399[CrossRef][Medline]This article has been cited by other articles:
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  H. Nave, G. Mueller, B. Siegmund, R. Jacobs, T. Stroh, U. Schueler, M. Hopfe, P. Behrendt, T. Buchenauer, R. Pabst, et al. Resistance of Janus Kinase-2 Dependent Leptin Signaling in Natural Killer (NK) Cells: A Novel Mechanism of NK Cell Dysfunction in Diet-Induced Obesity Endocrinology, July 1, 2008; 149(7): 3370 - 3378. [Abstract] [Full Text] [PDF]
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 V. Nobili, M. Manco, P. Ciampalini, V. Diciommo, R. Devito, F. Piemonte, D. Comparcola, R. Guidi, and M. Marcellini Leptin, free leptin index, insulin resistance and liver fibrosis in children with non-alcoholic fatty liver disease. Eur. J. Endocrinol., November 1, 2006; 155(5): 735 - 743. [Abstract] [Full Text] [PDF]
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