Effect of metabolic control on the in vitro proliferation of peripheral blood mononuclear cells in type 1 and type 2 diabetic patients

ABSTRACT CONTEXT AND OBJECTIVE: Diabetes mellitus is a clinical syndrome that frequently leads to the development of chronic complications and high susceptibility to infections. It is probably due to defective immunological defense, which may be related to metabolic control of the disease. The aim of this study was to evaluate the effect of metabolic control on immune-cell behavior in type 1 and type 2 diabetic patients. For this, the in vitro proliferation of peripheral blood mononuclear cells (PBMC) was analyzed in patients with inadequate and adequate metabolic control. DESIGN AND SETTING: Experimental/laboratory study at a university hospital. METHODS: Eleven type 1 and thirteen type 2 diabetic patients were studied, together with 21 healthy individuals divided in two groups (11/10), who were matched by sex and age with those diabetic patients. PBMC cultures stimulated with concanavalin-A (Con-A) were used to measure 3H-thymidine incorporation after 72 hours of cell culturing. For patients with inadequate metabolic control, culturing was performed on the first day of patient hospitalization and again after intensive treatment to achieve adequate control. RESULTS: The proliferation index for Con-A-stimulated cultures from type 1 diabetic patients was significantly greater than that for cultures from healthy individuals and type 2 diabetic patients, independent of metabolic control. A negative correlation between the proliferation cell index and body mass index and serum C-reactive protein levels was also observed. CONCLUSION: The increase in the proliferation capacity of type 1 diabetic T lymphocytes was probably not caused by hyperglycemia and/or insulinopenia related to inadequate metabolic control.


INTRODUCTION
Diabetes mellitus is a chronic clinical syndrome caused by insulin defi ciency (defects of insulin secretion and/or action) that causes a set of metabolic abnormalities involving the metabolism of glucose, lipids, protein and other substances.It frequently presents chronic microvascular and macrovascular complications such as retinopathy, nephropathy, coronariopathy, peripheral and cerebral vascular disease, and peripheral neuropathy.][3] Besides vascular and neurological complications, a high susceptibility to infection has been described in diabetes mellitus patients, [4][5][6][7][8][9][10] which may be caused by several defects of the immunological defense mechanism.
2][13][14][15][16] The most dramatic defect that occurs in diabetes mellitus is related to abnormalities of T cell function. 17,18The reason for these alterations in the immune cell behavior of diabetic patients is still undefi ned, with few studies of lymphocyte proliferation and none regarding the infl uence of metabolic control in diabetic patients.

OBJECTIVE
Considering the importance of the immune system in the development of diabetic complications such as higher susceptibility to infections in diabetic patients, we proposed to study the in vitro proliferation of peripheral blood mononuclear cells (PBMCs) from type 1 and type 2 diabetic patients, and to correlate this with inadequate and adequate metabolic control, as measured by the glycemic para me-ters associated with body mass index (BMI), and with the peripheral infl ammatory response assessed by C-reactive protein levels.

METHODS
Twenty-four diabetic patients were selected at the outpatient clinics of the university hospital of Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (Table 1).We also studied 21 healthy individuals who were matched by sex, age and BMI with these type 1 and type 2 diabetic patients.
The patients presented inadequate metabolic control (defi ned as fasting glucose greater than 200 mg/dl and glycated hemoglobin greater than 11%), but did not present any infectious disease and were not using any drugs that might have interfered with the results.The patients were hospitalized for 2-3 weeks to obtain adequate metabolic control.This was achieved by using an intensive protocol with capillary blood glucose measurements at 7, 11, 17 and 23 hours, and administration of regular insulin injections until a reduction of at least 100 mg/dl in mean daily glycemic level was reached.In addition to the neurological examination, the presence of microvascular complications was evaluated by ophthalmological examination and urine protein measurement, and the presence of macrovascular complications by clinical and electrocardiographic evaluation.
The protocol was approved by the Ethics Committee of Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (HC-FMRPUSP) and, before giving their consent to participate, the volunteers were carefully informed about the nature, purpose and possible risks of the study.
Blood samples were obtained on the fi rst and last day of hospitalization.PBMCs were isolated by gradient density using Ficoll-Hypaque ® . 19PBMCs at the concentration of CONTEXT AND OBJECTIVE: Diabetes mellitus is a clinical syndrome that frequently leads to the development of chronic complications and high susceptibility to infections.It is probably due to defective immunological defense, which may be related to metabolic control of the disease.The aim of this study was to evaluate the effect of metabolic control on immune-cell behavior in type 1 and type 2 diabetic patients.For this, the in vitro proliferation of peripheral blood mononuclear cells (PBMC) was analyzed in patients with inadequate and adequate metabolic control.

DESIGN AND SETTING:
Experimental/laboratory study at a university hospital.

METHODS:
Eleven type 1 and thirteen type 2 diabetic patients were studied, together with 21 healthy individuals divided in two groups (11/10), who were matched by sex and age with those diabetic patients.PBMC cultures stimulated with concanavalin-A (Con-A) were used to measure 3 H-thymidine incorporation after 72 hours of cell culturing.For patients with inadequate metabolic control, culturing was performed on the fi rst day of patient hospitalization and again after intensive treatment to achieve adequate control.

RESULTS:
The proliferation index for Con-A-stimulated cultures from type 1 diabetic patients was signifi cantly greater than that for cultures from healthy individuals and type 2 diabetic patients, independent of metabolic control.A negative correlation between the proliferation cell index and body mass index and serum C-reactive protein levels was also observed.

CONCLUSION:
The increase in the proliferation capacity of type 1 diabetic T lymphocytes was probably not caused by hyperglycemia and/or insulinopenia related to inadequate metabolic control.

RESULTS
The three groups studied (type 1 and type 2 diabetic patients and healthy individuals) were statistically similar with regard to age, sex and BMI (Table 1).The patients did not present chronic macrovascular, microvascular or neurological complications.The metabolic control evaluated by daily mean glycemic profi les during the hospitalization period showed a signifi cant decrease with treatment, between the fi rst and last day of hospitalization for type 1 and type 2 diabetic patients (p = 0.0002 and p = 0.0001, respectively, Figure 1).
The proliferation index was higher in the type 1 diabetes group, independent of glycemic control [M = 100.1 (inadequate) and M = 96.8(adequate)], than in the type 2 group [M = 44.4(inadequate), p < 0.0001; and M = 43.4(adequate), p = 0.01] (Figure 2).Type 1 diabetic patients also showed higher proliferation index than for the healthy individuals (M = 62.5, p = 0.01) who were matched for sex and age (Figure 2).
The proliferation index showed a negative correlation (r = -0.5, p = 0.01) with the BMI of the diabetic patients with both adequate and inadequate metabolic control.A negative correlation (r = -0.7,p = 0.002) was also observed with the C-reactive protein levels of inadequately controlled diabetic patients.

DISCUSSION
Lymphoproliferation refl ects the capacity of a cell to produce clones in vitro in the presence or absence of polyclonal activators.In the present study, concanavalin-A (Con-A) was used as a polyclonal T cell activator.The use of soluble polyclonal activators is based on the knowledge that they present superfi cial epitopes that are recognized by human cell surface receptors, regardless of the presence of illness.
The present study evaluated the lymphoproliferative response and its correlation with glycemic control in diabetic patients, for the fi rst time.Type 1 diabetic lymphocytes showed a higher in vitro proliferation rate, independent of glycemic control, which could be explained by some metabolic defects of these diabetic patients.Some authors have suggested that hyperglycemia has a negative infl uence on immune-competent cells.According to Reinhold et al., 20 the amount of thymidine incorporation in PBMC cultures from normal individuals is inversely proportional to the glucose concentration of the culture.This hypothesis is supported by the evidence of improvement in the immunecell response of diabetic patients following Table 1.Clinical characteristics of type 1 and type 2 diabetic patients and healthy individuals who were matched with these type 1 (Control 1) and type 2 (Control 2) patients.Data are presented as mean ± standard deviation (SD)  2.5 x 10 6 cells/ml were cultivated in triplicate in the presence or absence of concanavalin-A (Con-A).After 72 hours of culturing at 37° C in a humid environment with approximately 5% CO 2 , 0.5 microcuries of 3 H-thymidine were added to each well.The cells were maintained in the same condition as described above for an additional period of 16 hours.They were then collected using an automated PHD cell harvester (Cambridge Technology Inc., Cambridge, UK) and radioactivity was measured over a period of 10 minutes using a scintillation spectrometer (Beckman Coulter Inc., Fullerton, California, USA).The lymphoproliferation results were evaluated in accordance with the cell incorporation of 3 H-thymidine.These results are presented as the proliferation index, obtained as the ratio between the mean counts per minute (CPM) from the triplicates of the stimulated culture (Con-A) and non-stimulated culture (culture medium-RPMI).A high-sensitivity assay for C-reactive protein measurement in blood samples was carried out on day 1 using a turbidimetric immunoassay (Dimension  , Dade Behring Inc. Newark, USA).
The results are presented as median (M), mean (X ) and standard deviation (SD).The GraphPad Prism program (San Diego, California, USA) was used for the statistical analysis.The Kruskal-Wallis test was used to analyze differences between the three groups (type 1 and type 2 patients, and controls), and the Wilcoxon test was used to compare the diabetic groups before and after metabolic control.The Spearman test was used to calculate all correlations.p values < 0.05 were considered to be signifi cant.metabolic control. 21On the other hand, the insulinopenic state may be the reason for these immunological defects. 22urrent studies 23,24 suggest that insulin has an anti-infl ammatory effect, even in situations of acute compensation for hyperglycemic metabolic disturbances.In our study, the glycemic control did not determine differences in the proliferation of type 1 diabetic lymphocytes.Hyperglycemia was completely normalized and adequate control was obtained using insulin therapy, making it diffi cult to conclude whether hyperglycemia and plasma insulin levels are modulators of the lymphocyte response.
Furthermore, the increase in proliferation index for type 1 diabetic patients was signifi cant when compared with type 2 diabetic patients and normal individuals.These results suggest that the increased proliferation index in type 1 diabetic patients is intrinsic to this disease and may be due to chronic activation of the immunological system, thus enhancing the stimulated PBMC response.This chronic lymphocyte activation may be determined by superantigen infections 25,26 and/or be related to immunological alterations determined by the type 1 autoimmune process.According to McCormack et al., 27 chronic exposure to superantigens initially causes a reduction in the number of T cells, probably due to superantigen stimulation that determines proliferation followed by an increased cell death rate. 28,29In fact, Gonzalo et al. 30 showed DNA fragmentation characteristic of apoptosis in T cells after 72 hours of culturing in the presence of superantigens.
However, although we did not observe any infl uence of metabolic control on cell proliferation, we noticed a negative correlation with BMI and also observed that the highest C-reactive protein levels correlated with the lowest lymphoproliferation values in diabetic patients with inadequate metabolic control.These are important results if we consider that C-reactive protein is an acute infl ammatory reactive protein that is usually related to degenerative processes induced by pro-infl ammatory cytokines like interleucin-6 (IL-6), tumor necrosis factor-α (TNF-α) and others.
It may be proposed that, under conditions of inadequate metabolic control, the production of this protein and other pro-infl ammatory factors can regulate the immunological system to favor susceptibility to infection.

ConclusioN
The results obtained in this study show that type 1 diabetic patients have an increased in vitro cloning capacity that probably is not due to hyperglycemia and/or insulinopenia.

Figure 1 .
Figure 1.Daily mean glycemic levels of type 1 and type 2 diabetic patients on the fi rst (D1) and last (D2) day of hospitalization.

Figure 2 .
Figure 2.Proliferation index in Con-A (10 µg/ml)-stimulated peripheral blood mononuclear cells cultures from type 1 and type 2 diabetic patients with inadequate (NA) and adequate (A) metabolic control, and from healthy individuals who were matched with these type 1 (C1) and type 2 (C2) diabetic patients.The horizontal lines represent the medians for each group.