Type 1 diabetes predisposes to enhanced gingival leukocyte margination and macromolecule extravasation in vivo

Diabetes and chronic periodontal disease exhibit a bidirectional relationship centered on an enhanced inflammatory response manifested both locally and systemically. The severity and progression of periodontal tissue destruction in such conditions has been attributed to both bacterial and host contributions (1–6). Several studies have examined the impact of chronic hyperglycemia on periodontal structures and have demonstrated an enhanced innate inflammatory response leading to microvascular damage, extracellular matrix degradation and periodontal bone loss (5–9). Even though numerous epidemiological studies have confirmed that diabetes is a significant risk factor for periodontal disease, especially when poorly controlled, the mechanisms that link the two diseases are not fully understood. In vivo examination of leukocyte behavior in postcapillary venules is essential for understanding the patho-biology of conditions associated with abnormal inflammatory responses such as Alzheimer’s disease, inflammatory bowel disease, ischemia–reperfusion injury and diabetic complications, including periodontal disease (10,11). Development of accurate assessment technologies and reproducible methodologies to understand these spatial and temporal relationships in vivo has increased in recent years. In order to characterize intravascular cell–cell interactions, new in vitro preparations, such as three-dimensional collagen gel matrices, monolayers of endothelial cells bathed with flowing solutions to mimic forces experienced by cells in vivo, and clusters of re-aggregated tissue fragments, have been developed. (12). Although they offer many advantages over previous assays, these models still lack intact blood and lymphatic vessels, which makes them unsuitable for investigation of leukocyte trafficking. Optical techniques offer cellular and even subcellular levels of resolution without ex vivo cell manipulation. Real-time imaging offers a powerful diagnostic tool for the evaluation of both endothelial cell and leukocyte functions in living organisms. Fluorescence video-microscopy using leukocyte-labeling dyes has been previously used to evaluate leukocyte behavior in vivo (12–17). Gingival intravital microscopy is a noninvasive alternative that requires no surgical preparation to access the microvasculature, thereby eliminating false positive results due to surgical trauma and exposure of tissues to the outer environment. Furthermore, the preparation time is reduced and this eliminates sensitive steps that can introduce variability and make the technique less reproducible. The gingival marginal tissue in the labial mandibular interincisal area offers the advantage of superficially located postcapillary venules within a thin structure that can be easily observed using classic optical microscopic techniques combined with epifluorescence. Mouse models for human diabetes have been used to understand the complex impact of prediabetic metabolic changes and chronic hyperglycemia on specific tissues in vivo. The Akita mouse is a model of type 1 diabetes with early-onset hyperglycemia. It is a heterozygote carrier of a missense point mutation in the Ins2 gene (C96Y), leading to synthesis of an abnormal pro-insulin peptide that progressively induces proteotoxicity and endoplasmic reticulum (ER) stress in the pancreatic β-cells, eventually leading to their apoptosis and hypoinsulinemia. Akita mice develop hyperglycemia as early as 4 wk of age. Males develop polyuria, polydipsia, polyphagia and neurological deficiencies at 8 wk of age (18,19) and show the first signs of nephropathy and retinopathy at 12 wk. The advantage of the Akita mouse over other models of diabetes is consistent high blood glucose levels without the complicating factors of obesity and autoimmune response against β-cells. Leukocyte pre-activation or priming by diabetes has been demonstrated (20); however, the molecular mechanism by which chronic hyperglycemia leads to leukocyte activation is not known. The first step in leukocyte recruitment at sites of inflammation is mediated by selectins. The leukocyte cell surface ligand P-selectin glycoprotein ligand-1 (PSGL-1 or CD162) binds P-selectin (CD62P) on the surface of endothelial cells, leading to leukocyte rolling along endothelial surfaces. L-Selectin (CD62L) on leukocytes also contributes to neutrophil recruitment once initiated by P-selectin, although this may be primarily mediated by a ligand different from PSGL-1 (21). A recent review summarizes the roles of PSGL-1 in both margination and activation of leukocytes (22). Most importantly, the binding of P-selectin to PSGL-1 is absolutely required for activation of β2-integrins on leukocytes, notably LFA1 (CD11a/CD18), for slow rolling and attachment to endothelial cells (23). The aim of the present study was to evaluate the impact of chronic hyperglycemia on early inflammatory vascular dynamics and leukocyte–endothelial cell interactions in the gingiva and to elucidate diabetes-induced changes in cell-surface molecules responsible for initiating leukocyte– endothelial adhesion. Since microvascular damage is a key step in the onset of diabetic complications, defining leukocyte–endothelial interactions in conditions of chronic hyperglycemia will further our understanding of periodontal inflammation in diabetes.