Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice

Abstract

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1alpha, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.

FIGURE 1

Leukocyte dysfunction in hyperglycemia. A, PMN migration across a cellulose-nitrate membrane in a Boyden chamber in response to fMLP. Zymosan-elicited PMN from WT and Akita mice were incubated with 10 nM fMLP or PBS for 2 h. fMLP-induced migration is expressed as the difference between fMLP-stimulated and unstimulated cell migration in PBS (p = 0.011, n = 6 mice each group). B, The effect of the synthetic chemotactic peptide WKYMVm (1 nM) on zymosan-elicited PMN in a Boyden chamber (p = 0.008, n = 4 mice each group). C, Superoxide release from WT and Akita PMN upon fMLP stimulation. Zymosan-elicited peritoneal PMN were incubated with fMLP (1 μM) or PBS for 5 min. Superoxide release was measured as the superoxide dismutase-inhibitable reduction of cytochrome c and expressed as the difference between fMLP-elicited and unstimulated superoxide release in PBS (p = 0.018, n = 6 mice each group). D, Premature translocation of p47^phox to the cell membrane compartment in unstimulated Akita PMN. Cytoplasmic and membrane-associated proteins were extracted from unstimulated BM PMN and were size-fractionated on SDS-PAGE (80 μg protein/lane). Western blot shows p47^phox immunoreactivity exclusively in the cytoplasm in WT PMN, whereas in Akita PMN a portion of p47^phox is translocated to the cell membrane fraction. gp91^phox is found exclusively in the membrane fraction for both groups. The Western blot is representative of four separate experiments. A, Akita; Memb, membrane-associated protein extract; Cyt, cytoplasmic protein extract.

FIGURE 2

Alveolar bone loss following molar ligation in WT and Akita mice. Periodontal disease was induced by tying a silk ligature around the second maxillary molar (middle tooth) in WT (top left) and Akita (bottom left) mice for 3 wk. Alveolar bone loss was determined by measuring the distance between the CEJ-ABC (white bars). White, Enamel of the tooth crown; light blue, cementum of the exposed root surface; dark blue, alveolar bone of the maxilla. Right panel, Mean ± SD of CEJ-ABC measurements. On nonligated molars, there is no difference in the CEJ-ABC between WT and Akita mice. Ligation induces significantly more alveolar bone loss in Akita compared with WT mice (p = 0.018, n = 7 mice each group).

FIGURE 3

Intravital microscopy revealed elevated numbers of rolling and attached cells in gingival vessels of Akita mice. Left panel, Microscopic still images of rhodamine 6G-labeled leukocytes in the gingival microcirculation in unstimulated WT and Akita mice. Leukocytes rolling on or attached to the endothelium are visible in red; the microvasculature is labeled green using FITC-dextran. Original magnification, ×400. Right panel, The number of rolling and attached leukocytes in gingival venules (20–40 μm in diameter) during a 30-s observation period (rolling cells, p = 0.016 WT vs Akita; attached cells, p = 0.032 WT vs Akita, n = 5 mice each group).