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Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c+ CD8α- DCs (strong CD4+ T cell proliferation inducers) and the CD8α+ CD103+ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c+ CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c+ CD8α- DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c+ CD8α- DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c+ CD8α- DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c+ CD8α- DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.

In non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, plasmacytoid dendritic cells (pDCs) have a diabetes-promoting role through IFN-α production on one hand, while a diabetes-inhibiting role through indoleamine 2,3-dioxygenase (IDO) production on the other. Little is known about the kinetics and phenotype of pDCs in the NOD pancreas during the development of autoimmune diabetes. While para/peri-insular accumulation of conventional dendritic cells (cDCs) could be observed from 4 weeks of age onwards in NOD mice, pDCs only started to accumulate around the islets of Langerhans from 10 weeks onwards, which is concomitant with the influx of lymphocytes. NOD pancreatic pDCs showed a tolerogenic phenotype as assessed by their high expression of IDO and non-detectable levels of IFN-α and MxA. Furthermore, expression of the pDC-attracting chemokines CXCL10 and CXCL12 was significantly increased in the NOD pancreas at 10 weeks and the circulating pDC numbers were increased at 4 and 10 weeks. Our data suggest that a simultaneous accumulation of IDO(+) pDCs and lymphocytes in the pancreas in 10 weeks old NOD mice, which may reflect both an immunogenic influx of T cells as well as a tolerogenic attempt to control these immunogenic T cells.

The NOD mouse is a widely used animal model of autoimmune diabetes. Prior to the onset of lymphocytic insulitis, DCs accumulate at the islet edges. Our recent work indicated that these DCs may derive from aberrantly proliferating local precursor cells. As CD8α(+) DCs play a role in tolerance induction in steady-state conditions, we hypothesized that the autoimmune phenotype might associate with deficiencies in CD8α(+) DCs in the prediabetic NOD mouse pancreas. We studied CD8α(+) DCs in the pancreas and pLNs of NOD and control mice, focusing on molecules associated with tolerance induction (CD103, Langerin, CLEC9A, CCR5). mRNA expression levels of tolerance-modulating cytokines were studied in pancreatic CD8α(+) DCs of NOD and control mice. In the NOD pancreas, the frequency of CD8α(+)CD103(+)Langerin(+) cells was reduced significantly compared with control mice. NOD pancreatic CD8α(+)CD103(+)Langerin(+) DCs expressed reduced levels of CCR5, CLEC9A, and IL-10 as compared with control DCs. These alterations in the CD8α(+)CD103(+)Langerin(+) DC population were not present in pLNs. We demonstrate local abnormalities in the CD8α(+) DC population in the prediabetic NOD pancreas. These data suggest that abnormal differentiation of pancreatic DCs contributes to loss of tolerance, hallmarking the development of autoimmune diabetes.

The non-obese diabetic (NOD) mouse is a widely used animal model for the study of human diabetes. Before the start of lymphocytic insulitis, DC accumulation around islets of Langerhans is a hallmark for autoimmune diabetes development in this model. Previous experiments indicated that an inflammatory influx of these DCs in the pancreas is less plausible. Here, we investigated whether the pancreas contains DC precursors and whether these precursors contribute to DC accumulation in the NOD pancreas. Fetal pancreases of NOD and control mice were isolated followed by FACS using ER-MP58, Ly6G, CD11b and Ly6C. Sorted fetal pancreatic ER-MP58(+) cells were cultured with GM-CSF and tested for DC markers and antigen processing. CFSE labeling and Ki-67 staining were used to determine cell proliferation in cultures and tissues. Ly6C(hi) and Ly6C(low) precursors were present in fetal pancreases of NOD and control mice. These precursors developed into CD11c(+) MHCII(+) CD86(+) DCs capable of processing DQ-OVA. ER-MP58(+) cells in the embryonic and pre-diabetic NOD pancreas had a higher proliferation capacity. Our observations support a novel concept that pre-diabetic DC accumulation in the NOD pancreas is due to aberrant enhanced proliferation of local precursors, rather than to aberrant "inflammatory infiltration" from the circulation., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human chorionic gonadotrophin (hCG) is a hormone produced during pregnancy and present at the implantation site and in the maternal blood. Pregnancy has been proposed to represent a controlled state of inflammation at an early stage at the implantation site and later, systemically extended to the maternal circulation. Earlier, we reported that hCG can inhibit the development of diabetes in NOD mice and LPS-induced septic shock in a murine model. We hypothesize that hCG can contribute to the reduction of inflammation by modifying Mphi function. Here, the TG-induced peritonitis model for inflammation was used to investigate the effect of hCG on cytokine production and cell recruitment in vivo. hCG pretreatment in TG-induced peritonitis increased the number of peritoneal cells, especially PMN and monocytes, compared with mice injected with TG only. This increased cell number was partially explained by increased cell survival induced by hCG. Despite the cellular infiltrate, hCG pretreatment decreased i.p. TNF-alpha, IL-6, PTX3, CCL3, and CCL5 levels. By depleting peritoneal resident Mphi using clodronate liposomes prior to the application of hCG and the TG trigger, we established that Mphi are the main responsive cells to hCG, as the suppressed TNF-alpha and IL-6 production and increased PMN influx are abolished in their absence. Together, these data suggest that hCG contributes to the controlled inflammatory state of pregnancy by regulating Mphi proinflammatory function.

Objectives: In the salivary glands of patients with primary Sjögren Syndrome (pSjS) an accumulation of dendritic cells (DCs) is seen, which is thought to play a role in stimulating local inflammation. Aberrancies in subsets of monocytes, generally considered the blood precursors for DCs, may play a role in this accumulation of DCs. This study is aimed at determining the level of mature CD14lowCD16+ monocytes in pSjS and their contribution to the accumulation of DCs in pSjS., Methods: Levels of mature and immature monocytes in patients with pSjS (n = 19) and controls (n = 15) were analysed by flow cytometry. The reverse transmigration system was used for generation of DCs generated from monocyte subsets. The phenotype of DCs in pSjS salivary glands was analysed using immunohistochemistry. In vivo tracking of monocyte subsets was performed in a mouse model., Results: Increased levels of mature CD14lowCD16+ monocytes were found in patients with pSjS (mean (SD) 14.5 (5.5)% vs 11.4 (3.4)%). These cells showed normal expression of chemokine receptor and adhesion molecules. Mature monocytes partly developed into DC-lysosome-associated membrane glycoprotein (LAMP)+ (19.6 (7.5)%) and CD83+ (16 (9)%) DCs, markers also expressed by DCs in pSjS salivary glands. Monocyte tracking in the non-obese diabetic (NOD) mouse showed that the homologue population of mature mouse monocytes migrated to the salivary glands, and preferentially developed into CD11c+ DCs in vivo., Conclusions: Mature monocytes are increased in pSjS and patient and mouse data support a model where this mature monocyte subset migrates to the salivary glands and develops into DCs.

Type 1 diabetes (T1D) is an autoimmune disease in which a T-cell-mediated attack destroys the insulin-producing cells of the pancreatic islets. Despite insulin supplementation severe complications ask for novel treatments that aim at cure or delay of the onset of the disease. In spontaneous animal models for diabetes like the nonobese diabetic (NOD) mouse, distinct steps in the pathogenesis of the disease can be distinguished. In the past 10 years it became evident that DC and macrophages play an important role in all three phases of the pathogenesis of T1D. In phase 1, dendritic cells (DC) and macrophages accumulate at the islet edges. In phase 2, DC and macrophages are involved in the activation of autoreactive T cells that accumulate in the pancreas. In the third phase the islets are invaded by macrophages, DC and NK cells followed by the destruction of the beta-cells. Recent data suggest a role for a new member of the DC family: the plasmacytoid DC (pDC). pDC have been found to induce tolerance in experimental models of asthma. Several studies in humans and the NOD mouse support a similar role for pDC in diabetes. Mechanisms found to be involved in tolerance induction by pDC are inhibition of effector T cells, induction of regulatory T cells, production of cytokines and indoleamine 2,3-dioxygenase (IDO). The exact mechanism of tolerance induction by pDC in diabetes remains to be established but the intrinsic tolerogenic properties of pDC provide a promising, yet underestimated target for therapeutic intervention.

In the early development of type 1 diabetes macrophages and dendritic cells accumulate around the islets of Langerhans at sites of fibronectin expression. It is thought that these macrophages and dendritic cells are derived from blood monocytes. Previously, we showed an increased serum level of MRP8/14 in type 1 diabetes patients that induced healthy monocytes to adhere more strongly to fibronectin (FN). Here we show that MRP8/14 is expressed and produced at a higher level by type 1 diabetes monocytes, particularly after adhesion to FN, creating a positive feedback mechanism for a high fibronectin-adhesive capacity. Also adhesion to endothelial cells was increased in type 1 diabetes monocytes. Despite this increased adhesion the transendothelial migration of monocytes of type 1 diabetes patients was decreased towards the proinflammatory chemokines CCL2 and CCL3. Because non-obese diabetic (NOD) mouse monocytes show a similar defective proinflammatory migration, we argue that an impaired monocyte migration towards proinflammatory chemokines might be a hallmark of autoimmune diabetes. This hampered monocyte response to proinflammatory chemokines questions whether the early macrophage and dendritic cell accumulation in the diabetic pancreas originates from an inflammatory-driven influx of monocytes. We also show that the migration of type 1 diabetes monocytes towards the lymphoid tissue-related CCL19 was increased and correlated with an increased CCR7 surface expression on the monocytes. Because NOD mice show a high expression of these lymphoid tissue-related chemokines in the early pancreas it is more likely that the early macrophage and dendritic cell accumulation in the diabetic pancreas is related to an aberrant high expression of lymphoid tissue-related chemokines in the pancreas.

The non-obese diabetic (NOD) mouse is a widely used animal model for the study of human diabetes. The lymphocytic (peri-)insulitis is preceded by an early accumulation of dendritic cells (DC) around the islets of Langerhans. This DC accumulation is thought to derive from an influx of monocytes attracted by pro-inflammatory chemokines. Besides chemokines, extracellular matrix (ECM) proteins play an important role in the accumulation of leukocytes in tissues. We studied the expression of the chemokines CCL2, CCL5, CXCL10, CCL19 and CCL21 over time in pancreases of NOD and control mice by ELISA on pancreas lysates as well as by immunohistochemistry. In addition, we studied the adhesive capacity of bone marrow-derived DC (BMDC) to ECM components. DC in the NOD pancreas accumulated at sites with an intense expression of fibronectin. In vitro, NOD BMDC showed increased fibronectin adhesion and increased VLA-5 expression. At the time of early DC accumulation (<10 wk), the lymphoid tissue-related chemokines CCL19 and CCL21 were increased. Our findings support the view that the early accumulation of DC around the NOD islets is not the consequence of an enhanced attraction of precursors and immature DC by pro-inflammatory chemokines. It rather might be the consequence of an aberrantly enhanced adhesion and retention of NOD DC.

The accumulation of macrophages (M Phi) and dendritic cells (DC) in the pancreas plays a crucial role in the pathogenesis of autoimmune diabetes. We studied the recruitment of monocytes, M Phi and DC to sites of inflammation, i.e. the peritoneal cavity and a subcutaneously elicited air pouch in the NOD mouse model of autoimmune diabetes. The leukocyte recruitment was studied from 1 to 7 days after injection of thioglycollate (peritoneum), C5a (peritoneum, air pouch), CCL2 and CCL3 (air pouch). C57BL/6 and BALB/c mice served as controls. Morphological and flow cytometric analysis of the recruited cells was performed, IL-1 beta, TNF-alpha, IL-6, IL-12 and IL-10 in exudates measured, and in vitro CCL2-chemotaxis of exudate M Phi (Boyden chamber) determined. NOD mice were strongly impaired in the recruitment of M Phi, DC, monocytes, and granulocytes. Chemokine-injected air pouches of NOD mice showed an increased IL-10 and a decreased IL-1 beta level, while the other cytokines were normally or very lowly expressed. In addition, NOD exudate M Phi displayed an impaired in vitro CCL2-induced migration. Our data show that NOD mice have an impaired ability to recruit leukocytes into sites of inflammation elicited in the peritoneum and the air pouch. A raised IL-10/IL-1 beta ratio at these sites and a deficient migratory capacity of NOD monocytes are important determinants in this impairment.