α1-Acid glycoprotein decreases neutrophil migration and increases susceptibility to sepsis in diabetic mice

Abstract

The mechanisms underlying immune deficiency in diabetes are largely unknown. In the present study, we demonstrate that diabetic mice are highly susceptible to polymicrobial sepsis due to reduction in rolling, adhesion, and migration of leukocytes to the focus of infection. In addition, after sepsis induction, CXCR2 was strongly downregulated in neutrophils from diabetic mice compared with nondiabetic mice. Furthermore, CXCR2 downregulation was associated with increased G-protein-coupled receptor kinase 2 (GRK2) expression in these cells. Different from nondiabetic mice, diabetic animals submitted to mild sepsis displayed a significant augment in α1-acid glycoprotein (AGP) hepatic mRNA expression and serum protein levels. Administration of AGP in nondiabetic mice subjected to mild sepsis inhibited the neutrophil migration to the focus of infection, as well as induced l-selectin shedding and rise in CD11b of blood neutrophils. Insulin treatment of diabetic mice reduced mortality rate, prevented the failure of neutrophil migration, impaired GRK2-mediated CXCR2 downregulation, and decreased the generation of AGP. Finally, administration of AGP abolished the effect of insulin treatment in diabetic mice. Together, these data suggest that AGP may be involved in reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice.

FIG. 1.

Mice subjected to alloxan-induced insulin-dependent diabetes died after MS induction. A: Blood glucose levels were measured 10 days after administration of alloxan (diabetic group), alloxan plus insulin treatment (diabetic + INS group), or saline (nondiabetic group); *P < 0.01 compared with nondiabetic group; **P < 0.01 compared with diabetic group. Ten days after alloxan or saline administration, nondiabetic or diabetic mice were subjected to SH, MS, or SS, and survival rate (B), bacterial load in peritoneal cavity lavage (PCL) (C), or blood (D) or neutrophil migration to peritoneal cavity (E) were evaluated 6 h after surgery. The survival rate was determined daily up to 7 days after surgery, and other parameters were determined 6 h after surgery. *P < 0.01 compared with SH nondiabetic group; **P < 0.01 compared with MS nondiabetic group; ∞P < 0.05 compared with MS diabetic group. Diabetic mice were treated with insulin for 5 days (diabetic + INS group). (See research design and methods.) Values are means ± SEM (n = 5) and are representative of three independent experiments.

FIG. 2.

Systemic inflammation in diabetic mice during MS was prevented by insulin treatment. Ten days after alloxan or saline administration, nondiabetic or diabetic mice were subjected to SH, MS, or SS, and 6 h after surgery, seric levels of MIP-2 (A), IL-6 (B), and KC (C) or leukocyte infiltration (D) in lung was determined. *P < 0.01 compared with SH nondiabetic group; **P < 0.05 compared with MS nondiabetic group; ∞P < 0.05 compared with MS diabetic group. Diabetic mice were treated with insulin (INS) for 5 days. (See research design and methods.) Values are means ± SEM (n = 5) and are representative of three independent experiments.

FIG. 3.

Diabetic mice exhibit decreased leukocyte rolling/adherence to mesenteric microcirculation and decreased neutrophil chemotaxis response. Ten days after alloxan or saline administration, nondiabetic or diabetic mice were subjected to SH, MS, or SS, and 3 h after surgery the leukocyte rolling (A) and adhesion (B) were determined in the microcirculation of mesenteric tissue. C: Six hours after surgery, blood neutrophils were isolated and subjected to chemotaxis response to MIP-2 (30 ng/mL) in a Boyden chamber assay. *P < 0.01 compared with SH nondiabetic group; **P < 0.05 compared with MS nondiabetic group; ∞P < 0.05 compared with MS diabetic group. Diabetic mice were treated with insulin (INS) for 5 days. (See research design and methods.) Values are means ± SEM (n = 5) and are representative of three independent experiments.

FIG. 4.

High serum levels of AGP are correlated with inhibition of neutrophil migration in diabetic mice after sepsis. Ten days after alloxan or saline administration, nondiabetic or diabetic mice were subjected to SH, MS, or SS, and 6 h after surgery seric levels of AGP (A) and 4 h after surgery hepatic rate of mRNA AGP/β-actin (B) were evaluated. C: Correlation between number of neutrophils in the peritoneal cavity (y-axis) and seric levels of AGP (x-axis) 6 h after surgery. Thirty minutes before surgery, saline or AGP (50 µg/mice) was administered intravenously, and neutrophil migration to peritoneal cavity (D) or expression of CD62L (F) or CD11b (G) in blood cells with high expression of GR-1 (neutrophils) was determined 6 h after surgery. E: Representative histograms of fluorescence intensity (means of fluorescence intensity [MFI]) of CD62L and CD11b in blood cell with high expression of GR-1 measured by FACSort flow cytometry. Values are means ± SEM (n = 5) and are representative of three independent experiments. *P < 0.01 compared with SH nondiabetic group; **P < 0.05 compared with MS nondiabetic group; ∞P < 0.05 compared with MS diabetic group; •P < 0.05 compared with MS diabetic plus insulin (INS) group.

FIG. 5.

Diabetic mice display reduced CXCR2 expression and GRK2 upregulation after MS. Ten days after alloxan or saline administration, nondiabetic or diabetic mice were treated with saline or AGP (50 μg/mice) intravenously and mice were subjected to SH, MS, or SS. Six hours after surgery, CXCR2 expression in blood cells with high expression of GR-1 (neutrophils) was evaluated by flow cytometry. A: Representative histograms of fluorescence intensity (means of fluorescence intensity [MFI]) of CXCR2 and quantification of expression (means ± SEM). *P < 0.01 compared with SH nondiabetic group; **P < 0.05 compared with MS nondiabetic group; ∞P < 0.05 compared with MS diabetic group; •P < 0.05 compared with MS diabetic plus insulin (INS) group. B: Overlay of nuclei and GRK2 expression in blood neutrophils from different experimental groups. Blue staining by DAPI represents neutrophil nuclei, and red staining represents GRK2 expression in neutrophils (×400). C: Semiquantitative analyses of GRK2 expression showed the means of fluorescence intensity. The number of animals per group was five. Three slices per animal were used, and the means of fluorescence intensity of at least 200 neutrophils per slice was determined. The experiment was repeated three times. Values are means ± SEM. (A high-quality digital representation of this figure is available in the online issue.)