Diabetes & Metabolism 33 (2007) 439443

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Diabetes Metabolism

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Original article

Impaired cytokine production by peripheral blood mononuclear cells in type 1 diabetic patients
N.T. Fossa, M.C. Foss-Freitasb, M.A.N. Ferreiraa, R.N. Cardilia, C.M.C. Barbosab, M.C. Fossb,*
Division of Dermatology, Department of Medicine, Ribeiro Preto Medical School, So Paulo University, Av. Bandeirantes, 3900. Monte Alegre, 14049-900 Monte Alegre, Brazil b Division of Endocrinology, Department of Medicine, Ribeiro Preto Medical School, So Paulo University, Av. Bandeirantes, 3900. Monte Alegre, 14049-900 Monte Alegre, Brazil Received 19 October 2006; accepted 19 May 2007 Available online 07 November 2007
a

Abstract Aims. The objective of the present investigation was to study the production of IL-1, IL-6, IL-10, IFN and TNF in cultures of peripheral blood mononuclear cells (PBMC) taken from type 1 diabetic patients with inadequate metabolic control. Methods. Seventeen type 1 diabetic patients and a gender- and age-matched group of 17 healthy individuals were studied. PBMC cultures were stimulated with phytohemagglutinin (PHA; 20 g/ml) and lipopolysaccharide (LPS; 10 g/ml), and enzyme immunoassay (Elisa) was used to measure IL-1, IL-6, IL-10, IFN and TNF in the cell-culture supernatants. Results. IFN levels in PHA-stimulated cultures were lower in the type 1 diabetics than in the non-diabetic controls (P < 0.0001) while, in contrast, IL-10 levels were increased in the PHA-stimulated culture supernatants of the diabetics compared with the controls (P < 0.0001). In addition, supernatant levels of the cytokines IL-1, IL-6 and TNF released in the presence of LPS in the cell cultures from the diabetic patients were significantly lower than in the non-diabetic subjects (P < 0.0001, P < 0.0001 and P < 0.03, respectively). Conclusions. The impaired production of IL-1, IL-6, TNF and IFN, and the increased production of IL-10, in PBMC cultures from type 1 diabetics with inadequate metabolic control compared with healthy subjects may be an indication of a deficiency in mononuclear cell activation and, consequently, a deficient immune cellular adaptive response that, in turn, may be the cause of the increased incidence of infections in people with type 1 diabetes. 2007 Elsevier Masson SAS. All rights reserved. Rsum Altration de la production de cytokines par les cellules mononucles du sang priphrique des patients atteints de diabte type 1 Objet. Lobjectif de cette tude tait dvaluer la production de IL-1, IL-6, IL-10, IFN et TNF dans les cultures de cellules mononuclees obtenues du sang priphrique des patients porteurs de diabte type 1 non quilibrs. Mthode. Dix-sept patients diabtiques de type 1 et 17 tmoins, apparis selon le sexe et lge, ont t tudis. Des cellules mononucles du sang priphrique ont t stimules en culture par phytohmaglutinine (PHA ; 20 g/ml) et lipopolysaccharide (LPS ; 10 g/ml). La technique Elisa (essai dun immunosorbent li aux enzymes) a t utilise pour mesurer les taux dIL-1, IL-6, IL-10, IFN et TNF dans le surnageant des milieux de culture. Rsultats. Aprs la stimulation par PHA, les taux dIFN taient plus bas dans le groupe diabtique par comparaison au groupe tmoin non diabtique (P < 0,0001). En revanche, les taux dIL-10 taient plus levs chez les diabtiques que chez les tmoins (P < 0,0001). Par ailleurs, les taux des cytokines IL-1, IL-6 et TNF taient galement plus bas chez les diabtiques que chez les tmoins (P < 0,0001, P < 0,0001 et P < 0,03, respectivement). Conclusions. Les anomalies de la production dIL-1, IL-6, TNF et IFN ainsi que laugmentation de la production dIL-10 dans les cultures de cellules mononucles du sang priphrique des diabtiques de type 1 insuffisamment quilibrs pourraient indiquer un dficit dactivation des cellules mononucles, et par consquent un dficit cellulaire adaptatif immun qui pourrait constituer une des raisons de lincidence plus leve dinfections chez le patient diabtique. 2007 Elsevier Masson SAS. All rights reserved.

* Corresponding

author. E-mail address: mcfoss@fmrp.usp.br (N.T. Foss).

1262-3636/$ - see front matter 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.diabet.2007.10.001

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N.T. Foss et al. / Diabetes & Metabolism 33 (2007) 439443

Keywords: Lymphocytic transformation; IL-1, -6 and -10; IFN; TNF; Type 1 diabetes mellitus Mots cls : Transformation lymphocytaire ; Interleukine (IL)-1, -6 et -10 ; IFN ; TNF ; Diabte de type 1

1. Introduction The increased incidence of infectious diseases in diabetic patients has been reported in several studies [18], and is probably due to impairment of the immunological defense system of these patients. In previous studies, we found a higher prevalence of skin lesions (59%), especially dermatophytoses, in type 1 diabetes patients who are inadequately metabolically controlled [9]. Chronic hyperglycemia, causing an increase in glycated proteins, can stimulate the production of cytokines, which are involved in the activation of the immune system [10]. It appears that metabolic control is important not only in the pathogenesis and progression of the macro- or microvascular and neurological complications of diabetes [1113], but also in the high susceptibility of these patients to infectious diseases [9]. The most likely damage that occurs in diabetes is to the cellular immune response more specifically, T-cell function [1416]. Moreover, it has been demonstrated that polymorphonuclear cells from diabetic patients have impaired chemotaxis and phagocytosis capability [1720], defects that can be reversed by insulin therapy. In fact, recent studies have shown an anti-inflammatory effect of insulin [21]. Therefore, it may be important to determine immune-cell behavior in diabetic patients, focusing on mononuclear cells and especially the interaction between macrophages and T cells, to assess the mechanisms underlying the high susceptibility of diabetic patients to infections. On this basis, we have studied the production of interleukin (IL)-1, IL-6, IL-10, IFN and TNF in cultures of peripheral-blood mononuclear cells (PBMC) from type 1 diabetic patients with inadequate metabolic control, and compared them with cell cultures from healthy volunteers. 2. Subjects and methods Seventeen type 1 diabetic patients selected from the Outpatient Clinics at the University Hospital of the School of Medicine of Ribeiro Preto (USP), and 17 healthy, non-smoking volunteers were included in our study (Table 1). The patients presented with inadequate metabolic control (fasting plasma glucose and glycated haemoglobin levels higher than 200 mg/dl and 11%, respectively), had no infectious diseases and were not using immunosuppressive drugs. In addition to undergoing neurological examination, the patients were evaluated for the presence of microvascular complications using ophthalmological examination and urine-protein measurement. The presence of macrovascular complications was determined by clinical and electrocardiographic evaluation. Body mass index (BMI) was calculated as weight (kg)/height (m2). The Ethics Committee of our institution approved the study protocol, and patients gave their written informed consent to participate in the study.

A whole blood (12 ml) sample was taken from each study participant and placed in a sterile tube containing lithium heparin as anticoagulant (Vacutainer, BD) for the cell proliferation test and cytokine quantification. Additional blood samples were taken for the measurement of fasting plasma glucose and glycated haemoglobin (Labtest Diagnstica) [22]. The cell cultures were based on a previously described protocol [23], using a Ficoll-Hypaque density gradient for PBMC separation. The adherent cells (1.0 106) were exposed to lipopolysaccharide (LPS; 10 g/ml), and the supernatant collected after 24 h of culture for quantification of IL-1, IL-6 and TNF. The nonadherent cells, at a concentration of 2.5 105 cells/ml, were cultivated in triplicate and stimulated with 20 g/ml of phytohemagglutinin (PHA). After 72 hours of culture at 37 C, in a humid environment with approximately 5% CO2, the supernatant was collected for cytokine measurement (IL-10 and IFN), and 0.5 Ci of 3H-thymidine was added to each well for the cell-proliferation assay. The cells were maintained under the same conditions as described above for an additional 16 h. The cells were then collected using an automated cell harvester (Cambridge Technology Inc., Cambridge, UK) and radioactivity measured for a period of 10 min using a scintillation spectrometer (Beckman Coulter Inc., Fullerton CA, USA). Monoclonal antihuman IL-1, IL-6, IL-10 IFN and TNF antibody was used (Pharmingen International (Life Science Research), San Diego, CA, USA) as the capture antibody, and biotinylated antihuman antibodies for the five analyzed cytokines (Pharmingen) as the detecting antibody. Binding was detected with peroxidase-labeled streptavidin (DAKO, Glostrup, Denmark) and o-phenylenediamine-2HCl/substrate (OPD, Sigma, St Louis, MO, USA). The intra- and interassay variations were below 10%. Detection limits of cytokine Elisas were 31 pg/ml for IL-1, 78 pg/ml for IFN and 39 pg/ml for IL-10, IL-6 and TNF. The results are reported as median (M), mean (X) and standard error of the mean (SEM). The GraphPad Prism program (San Diego, CA, USA) was used for the statistical analysis. Comparisons between groups were made by the MannWhitney test and P < 0.05 was considered to be statistically significant. 3. Results The age and BMI of the diabetic group were similar to those of the non-diabetic group (Table 1). All of the diabetic patients were diagnosed during a ketoacidosis episode, and none had any chronic diabetic macro- or microvascular or neurological complications. Inadequate metabolic control among the diabetics was defined by high levels of glycated hemoglobin (11.7 2.0% SEM) and by fasting plasma-glucose concentration (249.3 mg/dl 67.8 SEM). The PBMC proliferation index of the PHA-stimulated cultures was higher in the non-diabetic controls (M = 97.5) than

N.T. Foss et al. / Diabetes & Metabolism 33 (2007) 439443 Table 1 Clinical and biochemical characteristics of type 1 diabetic patients and nondiabetic control subjects Type 1 diabetic patients (N = 17) 7/10 21.3 5.4 23.6 3.0 10.2 3.8 249 68 11.7 2.0 Control subjects (N = 17) 8/9 23.6 1.1 23.0 1.7 95 4 5.5 0.5

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Sex (m/f) Age (years) BMI (kg/m2) Diabetes duration (years) Fasting glucose (mg/dl) Glycated haemoglobin (%)

tants from type 1 diabetics (M = 3931 pg/ml) compared with non-diabetics (M = 1767 pg/ml; P < 0.03) (Fig. 2). The cytokines IL-1, IL-6 and TNF levels detected in the supernatants of LPS-stimulated cultures of PBMC taken from type 1 diabetics were significantly lower compared with the levels detected in non-diabetic subjects (IL-1: M = 1722 and 7840 pg/ml, P < 0.0001; IL-6: M = 444 and 1644 pg/ml, P < 0.0001; TNF: M = 670 and 1708 pg/ml, P < 0.03; in patients and controls, respectively) as shown in Fig. 3. 4. Discussion

in the type 1 diabetics (M = 42.4; P = 0.001) (Fig. 1). The supernatant levels of IFN in the PHA-stimulated cultures from type 1 diabetic patients were decreased (M = 1592 pg/ml) compared with levels in those from non-diabetic subjects (M = 5606 pg/ml; P = 0.0001) (Fig. 1). In contrast, IL-10 levels were increased in the PHA-stimulated culture superna-

Fig. 1. Proliferation index of PHA-stimulated cultures from type 1 diabetic patients and non-diabetic subjects. The horizontal lines represent the median of each group.

Cytokines are produced in response to microorganisms or other antigens and act as important immune modulators in host defense against aggressors [16,24]. They also regulate cellular function in other systems, and may play a role in the development of chronic complications of diabetes mellitus involving neurological and vascular pathological processes [10,25,26]. Previous studies have demonstrated that plasma levels of cytokines in type 1 diabetic patients can be different from those of non-diabetic patients [27,28]. It is also well known that plasma concentrations of cytokines are influenced by many factors and that several types of cells could produce cytokines. Thus, we used PBMC cultures to evaluate cells of the immune system, focusing on the susceptibility to infections seen in diabetic patients. In our study, the mononuclear cells in type 1 diabetics showed impaired in vitro proliferation, probably due to an immunological abnormality of the disease itself and to chronic activation of the immune system, leading to a diminished response of PBMC to the stimulus. This chronic lymphocytic activation may be triggered by superantigen infections [29,30] and/or be related to immunological alterations brought about by the type 1 autoimmune process. IFN is a cytokine produced by antigen-activated T cells that acts on the clones of T cells, stimulating cell proliferation using an autocrine mechanism to regulate the proliferation and differentiation of immune effector cells, including natural-killer

Fig. 2. IL-10 (pg/ml) and IFN (pg/ml) levels in PHA (20 g/ml)-stimulated PBMC-culture supernatants from type 1 diabetic patients (DM1) and non-diabetic controls (C). The horizontal lines represent the median of each group.

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Fig. 3. IL-1 (pg/ml), IL-6 (pg/ml) and TNF (pg/ml) levels in LPS (10 g/ml)- stimulated PBMC-culture supernatants of type 1 diabetic patients (DM1) and nondiabetic controls (C). The horizontal lines represent the median of each group.

(NK) cells [31]. Decreased levels of this cytokine in the PHAstimulated cultures from type 1 diabetics who are inadequately metabolically controlled reflect impaired activation of the adaptive immune system in such patients. In addition, we found lower levels of IL-1, IL-6 and TNF in the supernatants of LPS-stimulated PBMC cultures from the diabetic patients compared with those produced in non-diabetics, findings that confirm the hypothesis of an impaired adaptive immune response in type 1 diabetics. Finally, it must be borne in mind that hyperglycaemic memory confounds the relationship between cell function and blood glucose concentration: hyperglycaemia-induced changes in gene expression and biological reactions such as advanced glycation end-products in long-lived macromolecules may not be reversed when blood glucose returns to normal and, as these changes are progressively imprinted on cells [32], the result is impaired immune-cell activity. On the other hand, IL-10 levels were increased in the culture supernatant from the diabetics, reflecting enhancement of the Th2 subset of CD4 helper T cells, with stimulation of immunoglobulin(Ig) production by B cells and suppression of IFN-dependent macrophage function. These results suggest depressed macrophage activity, as determined by IL-1, IL-6 and TNF cytokine release in non-adherent PBMC cell cultures from diabetic patients compared with that from nondiabetic subjects. The higher production of IL-10 may be a regulatory response to IFN production. Despite the small number of patients in our study, the data show significant differences in cytokine levels between type 1 diabetic patients and healthy subjects. The impaired production of IFN and the inflammatory cytokines IL-1, IL-6 and TNF, and the increased production of the regulatory cytokine IL-10, in PBMC cell cultures of type 1 diabetic patients suggest a deficiency in mononuclear cell activation and, consequently, a deficient immune response, which may be one of the reasons that type 1 diabetic patients have an increased incidence of, in particular, extracellular infections.

Acknowledgements The authors are grateful to Maria Aparecida Nunes Ferreira and Sebastio L. Brando Filho for their technical assistance, and to E. Greene for reviewing the manuscript in English. References
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