Insulin modulates cytokine release and selectin expression in the early phase of allergic airway inflammation in diabetic rats

Abstract

Background: Clinical and experimental data suggest that the inflammatory response is impaired in diabetics and can be modulated by insulin. The present study was undertaken to investigate the role of insulin on the early phase of allergic airway inflammation.

Methods: Diabetic male Wistar rats (alloxan, 42 mg/Kg, i.v., 10 days) and controls were sensitized by s.c. injection of ovalbumin (OA) in aluminium hydroxide 14 days before OA (1 mg/0.4 mL) or saline intratracheal challenge. The following analyses were performed 6 hours thereafter: a) quantification of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and cytokine-induced neutrophil chemoattractant (CINC)-1 in the bronchoalveolar lavage fluid (BALF) by Enzyme-Linked Immunosorbent Assay, b) expression of E- and P- selectins on lung vessels by immunohistochemistry, and c) inflammatory cell infiltration into the airways and lung parenchyma. NPH insulin (4 IU, s.c.) was given i.v. 2 hours before antigen challenge.

Results: Diabetic rats exhibited significant reduction in the BALF concentrations of IL-1beta (30%) and TNF-alpha (45%), and in the lung expression of P-selectin (30%) compared to non-diabetic animals. This was accompanied by reduced number of neutrophils into the airways and around bronchi and blood vessels. There were no differences in the CINC-1 levels in BALF, and E-selectin expression. Treatment of diabetic rats with NPH insulin, 2 hours before antigen challenge, restored the reduced levels of IL-1beta, TNF-alpha and P-selectin, and neutrophil migration.

Conclusion: Data presented suggest that insulin modulates the production/release of TNF-alpha and IL-1beta, the expression of P- and E-selectin, and the associated neutrophil migration into the lungs during the early phase of the allergic inflammatory reaction.

Figure 1

IL-1β, TNF-α and CINC-1 concentrations in the bronchoalveolar lavage fluid of ovalbumin (OA) sensitized non-diabetic and diabetic rats 6 hours after OA (experimental) or saline (control) intratracheal instillation. Insulin (NPH, 4 IU/rat s.c.) was given to diabetic rats 2 hours before OA challenge. Values are means ± SEM for 5 to 7 animals in each group. *P < 0.001 comparing OA challenged with the control in the corresponding group (diabetic or non-diabetic). ^†P < 0.0001 comparing diabetic rats treated vs non-treated with insulin. ^‡P < 0.001 comparing OA challenged with the diabetic or non-diabetic group.

Figure 2

Quantitative evaluation of the immune staining for E- and P- selectins on lung microvessels of ovalbumin (OA) sensitized non-diabetic and diabetic rats 6 hours after OA (experimental) or saline (control) instillation. Insulin (NPH, 4 IU/rat s.c.) was given to diabetic rats 2 hours before OA challenge. Values are means ± SEM for 8 samples/rat, 3 animals/group. Analyses were performed by using the software image-pro Plus, version 4.1, Media Cybernetics. *P < 0.001 comparing OA challenged with the control in the corresponding group (diabetic or non-diabetic). ^†P < 0.0001 comparing diabetic rats treated vs non-treated with insulin. ^‡P < 0.001 comparing OA challenged with the diabetic or non-diabetic group.

Figure 3

E-selectin lung tissue microphotographs. The immune staining for E-selectin on lung microvessels of ovalbumin (OA) sensitized non-diabetic and diabetic rats 6 hours after OA (experimental) or saline (control) instillation. Insulin (NPH, 4 IU/rat s.c.) was given to diabetic rats 2 hours before OA challenge. Values are means ± SEM for 8 samples/rat, 3 animals/group. Analyses were performed by using the software image-pro Plus, version 4.1, Media Cybernetics. The microphotographs of lung tissue were obtained from control non-diabetic rats non-sensitized and instilled with saline (CNSSAL) or sensitized and instilled with saline (CSENSAL) or sensitized and instilled with OA (CSENOA), diabetic rats non-sensitized instilled with saline (DNSSAL) or sensitized and instilled with OA (DSENOA), and insulin treated diabetic rats sensitized instilled with OA (DSENOA+I). *Indicates the vessel lumen. Lung sections (8 μm) were stained for the detection of E-selectin (original magnification × 1500). *P < 0.001 comparing OA challenged with the control in the corresponding group (diabetic or non-diabetic). ^†P < 0.0001 comparing diabetic rats treated vs non-treated with insulin. ^‡P < 0.001 comparing OA challenged with the diabetic or non-diabetic group.

Figure 4

P-selectin lung tissue microphotographs. The immune staining for P-selectin on lung microvessels of ovalbumin (OA) sensitized non-diabetic and diabetic rats 6 hours after OA (experimental) or saline (control) instillation. Insulin (NPH, 4 IU/rat s.c.) was given to diabetic rats 2 hours before OA challenge. Values are means ± SEM for 8 samples/rat, 3 animals/group. Analyses were performed by using the software image-pro Plus, version 4.1, Media Cybernetics. The microphotographs of lung tissue were obtained from control non-diabetic rats non-sensitized and instilled with saline (CNSSAL) or sensitized and instilled with saline (CSENSAL) or sensitized and instilled with OA (CSENOA), diabetic rats non-sensitized instilled with saline (DNSSAL) or sensitized and instilled with OA (DSENOA), and insulin treated diabetic rats sensitized instilled with OA (DSENOA+I). *Indicates the vessel lumen. Lung sections (8 μm) were stained for the detection of P-selectin (original magnification × 1500). *P < 0.001 comparing OA challenged with the control in the corresponding group (diabetic or non-diabetic). ^†P < 0.0001 comparing diabetic rats treated vs non-treated with insulin. ^‡P < 0.001 comparing OA challenged with the diabetic or non-diabetic group.