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-1α, 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.

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MUC7 is a low molecular weight monomeric mucin secreted by submandibular, sublingual and minor salivary glands. This mucin has been implicated in the non-immune host defense system in the oral cavity since it binds and agglutinates a variety of oral microbes. To investigate interactions between this mucin and other secretory salivary proteins, a submandibular gland prey library was screened with baits encoding the N- and C-terminal regions of MUC7 in the yeast two-hybrid system. The N-terminal region interacted with several secretory salivary proteins, whereas the C-terminal region did not. Interacting proteins included amylase, acidic proline-rich protein 2, basic proline-rich protein 3, lacrimal proline-rich protein 4, statherin and histatin 1. Formation of complexes between these proteins and the N-terminal region of MUC7 was confirmed in Far Western blotting experiments. Interactions between mucin and non-mucin proteins in saliva could protect complex partners from proteolysis, modulate the biological activity of complexed proteins or serve as a delivery system for distribution of secretory salivary proteins throughout the oral cavity.

Protein-protein interactions are necessary for homeostasis to be maintained and for biological systems to be integrated. Heterotypic complexes occur in saliva, and a complex between MG2 and SIgA has been suggested to promote microbial clearance from the oral cavity. In this study, we used a peptide display library to investigate previously unrecognized heterotypic complexes involving MG2 and other proteins. The library was panned with MG2 12 times, and analyses of clones identified the sequence Ala-Leu-Leu-Cys-, which occurs in salivary lactoferrin. Blotting experiments confirmed that MG2 and lactoferrin form a heterotypic complex in vitro and in vivo. Periodate treatment of MG2 did not affect the interaction. A synthetic lactoferrin peptide containing the motif Ala-Leu-Leu-Cys-blocked the interaction between MG2 and lactoferrin, confirming the specificity of the interaction identified by panning. This complex may enhance the properties of these salivary components in the oral environment.

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