Differential effect of hyperglycaemia on the immune response in an experimental model of diabetes in BALB/cByJ and C57Bl/6J mice: participation of oxidative stress

Abstract

Diabetes is associated with an increased risk of death from infectious disease. Hyperglycaemia has been identified as the main factor contributing to the development of diseases associated with diabetes mellitus. However, experimental evidence indicates individual susceptibility to develop complications of diabetes. In this context, the aim of this work was to study the immune response in a streptozotocin-induced type 1 diabetes in two mouse strains: BALB/cByJ and C57Bl/6J. The participation of hyperglycaemia and oxidative stress was also analysed. Diabetic BALB/cByJ mice showed a decrease in both the in-vivo and in-vitro immune responses, whereas diabetic C57Bl/6J mice had higher blood glucose but exhibited no impairment of the immune response. The influence of hyperglycaemia over the immune response was evaluated by preincubation of lymphocytes from normal mice in a high glucose-containing medium. T and B cells from BALB/cByJ mice showed a decrease in cell viability and mitogen-stimulated proliferation and an increase in apoptosis induction. An increase in oxidative stress was implicated in this deleterious effect. These parameters were not affected in the T and B lymphocytes from C57Bl/6J mice. In conclusion, BALB/cByJ mice were sensitive to the deleterious effect of hyperglycaemia, while C57BL/6J were resistant. Although an extrapolation of these results to clinical conditions must be handled with caution, these results highlight the need to contemplate the genetic background to establish models to study the deleterious effect of diabetes in order to understand phenotypical variations that are of clinical importance in the treatment of patients.

Figure 1

Glycaemia values in BALB/c and C57 normal and diabetic mice. Average glycaemia values versus days after streptozotocin treatment in BALB/c and C57 mice. Data shown are the mean ± standard error of the mean of 12 animals in each group. Statistical significance was determined with two-way repeated-measures analysis of variance with a 4 × 12 design followed by Student–Newman–Keuls (SNK) post-test. **P < 0·01 with respect to control mice; ##P < 0·01 with respect to BALB/c diabetic mice.

Figure 2

Antibody production following sheep red blood cells (SRBC) and lipopolysaccharide (LPS) immunization in BALB/c and C57 control and diabetic mice. Antibody titres in controls and at 15 days, 1 month and 6 months after diabetes induction in BALB/c and C57 mice. (a) Anti-LPS immunoglobulin (Ig)M production. (b) Anti-SRBC IgM production. (c) Anti-SRBC IgG production. Data shown are the mean ± standard error of the mean of three animals in each group. Statistical significance was determined with the Kruskal–Wallis test followed by Conover post-test. *P < 0·05 with respect to control mice.

Figure 3

Mitogen-induced proliferative response in T and B cells from controls and at 15 days, 1, 3 and 6 months of diabetes in BALB/c and C57 mice. (a) T lymphocytes stimulated with 1 μg/ml of concanavalin A. (b) B lymphocytes stimulated with 25 μg/ml of lipopolysaccharide (LPS). Results shown are the mean ± standard error of the mean of three independent experiments performed in triplicate. Statistical significance was determined with two-way analysis of variance with a 4 × 4 design followed by Student–Newman–Keuls (SNK) post-test. *P < 0·05 with respect to cells from control mice.

Figure 4

Effect of high concentrations of glucose and mannitol on proliferation, viability and apoptosis of T and B normal lymphocytes. T lymphocytes (a,c) or B lymphocytes (b,d) from BALB/c and C57 mice were incubated for 24 h in RPMI-1640 either with or without (control) addition of glucose in the culture medium. Proliferation (a,b) and cell viability (c,d) were determined. (e) BALB/c lymphocyte apoptosis. Table: effect of high concentrations of mannitol on T and B cells from BALB/c mice. Results shown are the mean ± standard error of the mean of five independent experiments performed in triplicate for proliferation and viability and of three independent experiments performed in triplicate for apoptosis. Statistical significance was determined with two-way analysis of variance with a 2 × 4 design, followed by Student–Newman–Keuls (SNK) post-test for proliferation and viability and with one-way analysis of variance for three factors for apoptosis and for mannitol effect. *P < 0·05 and **P < 0·01 with respect to control values (standard medium alone).

Figure 5

Effect of high concentrations of glucose on oxidative stress. Reactive oxygen species (ROS) production and lipid peroxidation. Lymph node (a,c) and spleen (b,d) lymphocytes from BALB/c and C57 mice were incubated for 24 h in RPMI-1640 either with or without (control) addition of glucose in the culture medium. ROS (a,b) or malondialdehyde (MDA) production (as a measure of lipid peroxidation) (c,d) were determined. Data shown are the mean ± standard error of the mean of five independent experiments performed in duplicate. Statistical significance was determined with two-way analysis of variance with a 2 × 3 design followed by Student–Newman–Keuls (SNK) post-test. **P < 0·01with respect to control values.

Figure 6

N-acetylcysteine (NAC) action on the effect of high glucose on mitogen-induced proliferative response, viability and apoptosis in T and B cells from normal BALB/c mice. Lymph node (a,c,e) and spleen lymphocytes (b,d,f) from BALB/c mice were incubated for 24 h in RPMI-1640 either with or without (control) the addition of glucose or NAC in the culture medium. Proliferation (a,b), cell viability (c,d) and apoptosis (e,f) were determined. Results shown are the mean ± standard error of the mean of five independent experiments performed in triplicate for proliferation and viability and of three independent experiments performed in triplicate for apoptosis. Statistical significance was determined with two-way analysis of variance with a 2 × 3 design followed by simple effects analysis. **P < 0·01 with respect to control values.

Figure 7

Effect of high concentrations of glucose on total anti-oxidant capacity (TAC), total glutathione (GSH) levels and reduced glutathione (GSH) : oxidized glutathione (GSSG) ratio. Lymph node lymphocytes from BALB/c and C57 mice were incubated for 24 h in RPMI-1640 either with or without (control) addition of glucose in the culture medium. TAC (a), total GSH (b) and GSH : GSSG ratio (c) were determined. Data shown are the mean ± standard error of the mean of five independent experiments performed in duplicate. Statistical significance was determined with two-way analysis of variance with a 2 × 2 design followed by simple effects analysis for TAC and GSH : GSSG ratio and followed by Student–Newman–Keuls (SNK) post-test for total GSH. *P < 0·05 with respect to the control values; ##P < 0·01 with respect to BALB/c mice.