Islet Dysfunction in a Novel Transgenic Model of T Cell Insulitis.

The newly established CD3FLAG-mIR transgenic mouse model on a C57Bl/6 background has a FLAG tag on the mouse Insulin Receptor (mIR), specifically on T cells, as the FLAG-tagged mIR gene was engineered behind CD3 promoter and enhancer. The IR is a chemotactic molecule for insulin and the Flag-tagged mIR T cells in the BL/6-CD3FLAGmIR transgenic mice can migrate into the pancreas, as shown by immunofluorescent staining. While the transgenic mice do not become diabetic, there are phenotypic and metabolic changes in the islets. The transgenic islets become enlarged and disorganized by 15 weeks and those phenotypes continue out to 35 weeks of age. We examined the islets by RT-PCR for cell markers, ER stress markers, beta cell proliferation markers, and cytokines, as well as measuring serum insulin and insulin content in the pancreas at 15, 25, and 35 weeks of age. In transgenic mice, insulin in serum was increased at 15 weeks of age and glucose intolerance developed by 25 weeks of age. Passage of transgenic spleen cells into C57Bl/6 RAG-/- mice resulted in enlarged and disorganized islets with T infiltration by 4 to 5 weeks post-transfer, replicating the transgenic mouse studies. Therefore, migration of non-antigen-specific T cells into islets has ramifications for islet organization and function.

Insulin receptor based lymphocyte trafficking in the progression of type 1 diabetes.

The insulin receptor (IR) is a transmembrane receptor which recognizes and binds the hormone insulin. We describe two models that were devised to explore the role of IR over-expression on T-lymphocytes and their chemotactic motility in the progression of type 1 diabetes. FVB/NJ-CD3-3×FLAG-mIR/MFM mice were generated to selectively over-express 3×FLAG tagged murine IR in T-lymphocytes via an engineered CD3 enhancer and promoter construct. Insertion of the 3×FLAG-mIR transgene into FVB/NJ mice, a known non-autoimmune prone strain, lead to a minor population of detectable 3×FLAG-mIR tagged T-lymphocytes in peripheral blood and the presence of a few lymphocytes in the pancreas of the Tg+/- compared to age matched Tg-/- control mice. In order to induce stronger murine IR over-expression then what was observed with the CD3 enhancer promoter construct, a second system utilizing the strong CAG viral promoter was generated. This system induces cell specific IR over-expression upon Cre-Lox recombination to afford functional 3×FLAG tagged murine IR with an internal eGFP reporter. The pPNTlox2-3×FLAG-mIR plasmid was constructed and validated in HEK-Cre-RFP cells to ensure selective Cre recombinase based 3×FLAG-mIR expression, receptor ligand affinity towards insulin, and functional initiation of signal transduction upon insulin stimulation.

Tight association between macrophages and adipocytes in obesity: implications for adipocyte preparation.

OBJECTIVE: To determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level. METHODS: Adipose tissue from diet-induced obese mice was digested by collagenase treatment and fractionated by density centrifugation to obtain an adipocyte floating layer and a pellet of stromal vascular cells. The cellular architecture of the adipocyte-macrophage interaction in both intact white adipose tissue (WAT) and the separated density gradient floating layer fraction was analyzed by confocal immunohistochemistry. Cytokine expression was detected by semi-quantitative real time PCR and immunohistochemical analysis. RESULTS: Three dimensional image analysis of WAT and the separated "adipocyte" floating layer revealed lipid-engorged macrophages, macrophages in contact with lipid droplets and sheath-like assemblies of macrophages surrounding adipocytes. The macrophages immunostained for TNFα and to a lesser extent for the immunoregulatory cytokine IL-10. TNFα staining was associated only with macrophages indicating that macrophages and not adipocytes are the source of TNFα expression in the adipocyte floating layer. CONCLUSION: Macrophages form assemblies that tightly adhere to and cover adipocytes and lipid droplets. TNFα found in low density adipocyte preparations is due to contamination with macrophages.