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. 2006 Dec;169(6):2137-47.
doi: 10.2353/ajpath.2006.060539.

In CD4+ T-cell-induced diabetes, macrophages are the final effector cells that mediate islet beta-cell killing: studies from an acute model

Boris Calderon  1 , Anish Suri, Emil R Unanue
Affiliations

In CD4+ T-cell-induced diabetes, macrophages are the final effector cells that mediate islet beta-cell killing: studies from an acute model

Boris Calderon et al. Am J Pathol. 2006 Dec.

Abstract

To understand better how diabetogenic CD4+ T cells induce islet beta-cell death and cause diabetes, a transfer model of acute diabetes using the diabetogenic CD4+ BDC2.5 T-cell clone was established. Transfer of activated BDC T cells into NOD.scid mice resulted in diabetes within a week, characterized by strong inflammatory reaction. Electron micrographs of pancreas depicted macrophages in close contact with beta cells that exhibited signs of apoptosis. Transfer into irradiated recipients inhibited inflammation and the development of diabetes, demonstrating an obligatory role for leukocytes. Selective depletion of neutrophils or natural killer cells had no effect on diabetes induced by BDC2.5 T cells. In contrast, in vivo depletion of phagocytic cells by injection of liposomes containing clodronate abolished diabetes, although inflammation remained present and was characterized mainly by neutrophil infiltration. Treatment with clodronate-liposomes did not affect the antigen-presenting cells within the pancreas. Last, activated macrophages isolated from infiltrated pancreas exhibited cytolytic activity toward primary islet beta cells. Taken together, these results demonstrate that activated macrophages are the key cells mediating islet beta-cell death induced by activated CD4+ T cells.

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Figures

Figure 1
Figure 1
A: Con A-activated BDC T cells were transferred intravenously into NOD.scid recipients, which were then followed for the onset of diabetes. Depending on the numbers of injected T cells [106 (n = 15), 3 to 4 × 106 (n = 80), 5 to 10 × 106 (n = 40)], diabetes developed between 4 to 10 days. Mice were considered diabetic on two consecutive blood glucose measurements of ≥250 mg/dl. B: H&E staining of pancreas at day 3 after T-cell transfer (4 × 106) into NOD.scid mice. Note the early peri-insulitic lesion composed of neutrophils, macrophages, and lymphocytes. C: H&E staining of pancreas at day 7 after T-cell transfer (4 × 106). Note the extensive infiltration of islets with macrophages, neutrophils, and lymphocytes that disrupt the normal islet architecture. D: Same islet as in C stained for insulin (red). Note the decreased numbers of insulin-positive β cells that correlated with the onset of diabetes. Scale bars = 20 μm.
Figure 2
Figure 2
Electron microscopy analysis of acute diabetic mice at day 4 after BDC T-cell transfer. A: Infiltrated islet including two β cells (black arrows) showing empty vesicles and surrounded by macrophages (white arrows) in close contact with β cells. Small micrograph shows a normal β cell from a normal NOD.scid mouse; note the conserved cytoplasm, mitochondrias, and insulin granules. B: Higher magnification of a β cell in close relationship to a macrophage. C: Higher magnification of a β cell that exhibits mitochondrial swelling (arrow). D: Infiltrated islet showing a β cell undergoing apoptosis (arrow). E: Higher magnification of a macrophage from the previous image showing the close contact with a β cell and also what appears to be an insulin-like granule inside a vacuole (arrow). F: Apoptotic nuclei and insulin-like granule inside a vesicle of a macrophage (arrows).
Figure 3
Figure 3
Distribution of islet-infiltrating leukocytes from NOD.scid mice at day 7 after BDC T-cell transfer (diabetic) and from control NOD.scid mice that did not receive any T cells (normal). Leukocytes were recovered from pancreatic islets of six mice in each group and analyzed by flow cytometry for the various leukocyte markers. The total numbers of islet-infiltrating leukocytes recovered per mouse from diabetic versus normal mice were 5.9 × 105 and 6.3 × 103, respectively. Normal NOD.scid mice contain low numbers of neutrophils, macrophages, and dendritic cells.
Figure 4
Figure 4
A: NOD.Rag1−/− mice irradiated with 650 rads received 107 activated BDC T cells and were followed for diabetes development. Although control unirradiated NOD.Rag−/− mice developed diabetes in 5 days (n = 4), irradiated mice showed a delayed onset of diabetes until day 19 after cell transfer (n = 8). B: H&E staining of an islet from an irradiated mouse at day 5 after cell transfer. Note the lack of insulitis as evidenced by few leukocytes around the islet. C: H&E staining of an islet from an irradiated recipient mouse that became diabetic at day 19 after BDC T-cell transfer. Islet shows severe infiltration with loss of β-cell mass. Scale bar = 20 μm.
Figure 5
Figure 5
Cytotoxicity assay of chromium-labeled YAC-1 target cells in the presence of peritoneal exudate cells recovered at day 4 after BDC T-cell transfer. Experimental mice received either an isotype control antibody or the anti-NK cell-depleting antibody treatment. A: Mice that received anti-asialo GM1 antibody treatment. B: Mice that received anti-NK1.1 antibody treatment. Effector/target ratios of 100:1, 75:1, 50:1, and 25:1 were incubated for 4 hours. Each point represents one experimental mouse.
Figure 6
Figure 6
A: Macrophage depletion in the acute diabetes model by CLOD-LIP treatment. NOD.scid recipients were treated with CLOD-LIP or PBS-LIP on days 1, 2, 3, and 4 after transfer of 107 activated BDC T cells. Although all of the PBS-LIP-treated mice developed diabetes in 6 days (eight of eight), CLOD-LIP-treated mice exhibited a markedly reduced and delayed incidence of diabetes (5 of 15 by day 30). B: Splenocyte analysis by flow cytometry of treated mice at day 6 after cell transfer. CLOD-LIP-treated mice exhibited >99% depletion of phagocytic cells in spleen. C and D: H&E analysis showing an example of an islet with abundant neutrophils in the phagocyte-depleted mice at day 6 after T-cell transfer. E and F: Islet of a phagocyte-depleted mouse free of diabetes at day 6 showing a high number of apoptotic bodies, probably from dead neutrophils. G: Immunofluorescence staining for insulin (red) and nuclear staining (blue) in the pancreatic islets at day 6 after transfer of activated BDC T cells in a PBS-LIP-treated mouse. Note the lack of insulin-positive cells in the presence of leukocytic infiltrate. H: Nondiabetic mouse treated with CLOD-LIP. Note the presence of insulin-positive cells in the presence of leukocyte infiltration. Scale bar = 20 μm.
Figure 7
Figure 7
A: Immunofluorescence staining for F4/80+, CD11b+, and CD11c+ leukocytes (red) in NOD.scid mice after 4 days of liposome treatment (CLOD-LIP or PBS-LIP) and analyzed at day 6. No visual differences in the numbers of APCs were observed between both groups. Nuclear stain is blue. B: Indicated are the data comparing the number of APCs per field between PBS-LIP- or CLOD-LIP-treated mice (n = 4 per group). Each dot represents the APCs in one field per each mouse (number in parentheses). The bar represents the mean counts. C: Indicated are the means of APCs per field from each mouse in each of the two groups, derived from the data of B. No statistical difference was observed between means of PBS-LIP and CLOD-LIP groups (Mann-Whitney U-test) (P = 0.3429). Scale bar = 40 μm.
Figure 8
Figure 8
Cytotoxicity assay of chromium-labeled β cells in the presence of enriched phagocytes from acute diabetic pancreata. CD11b+- and CD11c+-enriched phagocytes were co-cultured with the chromium-labeled β cells at an effector/target ratio of 50:1 (5 × 105:1 × 104). Chromium release was measured at 0, 4, and 12 hours of incubation, and lysis percentage was calculated.
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