The phagocyte NAPDH-oxidase complex consists of several phagocyte oxidase (phox) proteins, generating reactive oxygen species (ROS) upon activation. ROS are involved in the defense against microorganisms and also in immune regulation. Defective ROS formation leads to chronic granulomatous disease (CGD) with increased incidence of autoimmunity and disturbed resolution of inflammation. Because regulatory T cells (Tregs) suppress autoimmune T-cell responses and are crucial in down-regulating immune responses, we hypothesized that ROS deficiency may lead to decreased Treg induction. Previously, we showed that in p47 phox -mutated mice, reconstitution of macrophages (Mph) with ROSproducing capacity was sufficient to protect the mice from arthritis. Now, we present evidence that Mph-derived ROS induce Tregs. In vitro, we showed that Mph ROS-dependently induce Treg, using an NADPH-oxidase inhibitor. This finding was confirmed genetically: rat or human CGD Mph with mutated p47 phox or gp91 phox displayed hampered Treg induction and T-cell suppression. However, basal Treg numbers in these subjects were comparable to those in controls, indicating a role for ROS in induction of peripheral Tregs. Induction of allogeneic delayed-type hypersensitivity with p47 phox -mutated Mph confirmed the importance of Mph-derived ROS in Treg induction in vivo. We conclude that NAPDH oxidase activity in Mph is important for the induction of Tregs to regulate T cell-mediated inflammation.chronic granulomatous disease | NADPH oxidase | neutrophil cytosolic oxidase 1 | redox

Dendritic cells (DCs) play critical roles in immune responses and can be distinguished in two major subsets, myeloid and plasmacytoid DCs. Although the presence of DC in all peripheral organs, including the kidney, has been well documented, no accurate estimates of DC subsets in human kidneys have been reported. This study shows a detailed analysis of DC subsets in cryosections of human renal tissue. The cortex of normal kidneys contains at least two different HLA-DR(+) myeloid DC subtypes characterized by BDCA-1(+)DC-SIGN(+) and BDCA-1(+)DC-SIGN(-). The staining for DC-SIGN completely overlapped with CD68 in the renal interstitium. Unexpectedly, BDCA-2(+)DC-SIGN(-) plasmacytoid DCs are also abundantly present. Both subsets are located in the tubulo-interstitium often with a high frequency around, but rarely observed within glomeruli. Quantification of BDCA-1(+), DC-SIGN(+), and BDCA-2(+) cells in normal human renal tissue (pretransplant biopsy living donors; n=21) revealed that BDCA-1 is about four times as frequently present as BDCA-2. A preliminary cross-sectional analysis of DC in diseased kidneys, including rejection and immunoglobulin A nephropathy, revealed that the number of DC as well as their anatomical distribution might change under pathophysiological conditions. In conclusion, we show that human kidneys contain a dense network of myeloid and plasmacytoid DCs and provide the tools for phenotyping and enumeration of these cells to better understand interindividual differences in immune responses.

Rapamycin (Rapa), a recently introduced immunosuppressive drug, seems to be effective in preventing acute allograft rejection. Although its antiproliferative effect on T lymphocytes has been investigated extensively, its effect on the initiators of the immune response, the dendritic cells (DCs), is not known. Therefore, the effect of Rapa on monocyte-(mo-DCs) and CD34 ؉ -derived DCs in vitro but also on other myeloid cell types, including monocytes and macrophages, was examined. The present study shows that Rapa does not affect phenotypic differentiation and CD40L-induced maturation of mo-DCs. However, Rapa dramatically reduced cell recovery (40%-50%). Relatively low concentrations of Rapa (10 ؊9 M) induced apoptosis in both moDCs and CD34 ؉ -derived DCs, as visualized by phosphatidylserine exposure, nuclear condensation and fragmentation, and DNA degradation. In contrast, Rapa did not affect freshly isolated monocytes, macrophages, or myeloid cell lines. The sensitivity to Rapa-induced apoptosis was acquired from day 2 onward of mo-DC differentiation. Rapa exerts its apoptotic effect via a reversible binding to the cytosolic receptor protein FKBP-12, as demonstrated in competition experiments with FK506, which is structurally related to Rapa. Partial inhibition of Rapa-induced apoptosis was obtained by addition of ZVAD-fmk, which implies caspase-dependent and caspase-independent processes. The fact that Rapa exerts a specific effect on DCs but not on monocytes and macrophages might contribute to the unique actions of IntroductionDendritic cells (DCs) are professional antigen-presenting cells that are specialized in the uptake of antigens and their transport from peripheral tissues to the lymphoid organs. Because of their capacity to stimulate naive T cells, DCs have a central role in the initiation of primary immune responses. 1 Human DCs can be grown in vitro from CD34 ϩ progenitors in medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-␣ 2,3 or from monocytes in the presence of GM-CSF and interleukin (IL)-4 or IL-13. 4 It has been demonstrated also that in vivo monocytes can differentiate into DCs upon transmigration through endothelial layers. 5,6 DCs are thought to play a critical role in diverse facets of immune regulation, ranging from tolerance induction and the prevention of autoimmunity to the induction of antitumor immunity and the protection against infectious agents. 1,7-9 A similar dichotomy of DC functions has been proposed to play a role in allograft transplantation. Various experimental models have demonstrated that DCs are critical for the induction of transplant tolerance. [10][11][12][13] As a consequence DCs have been genetically modified to promote their tolerogenic potential. 14 On the other hand, it is clear that the immunostimulatory potential of DCs may also contribute to disease pathogenesis of allograft rejection. 15 Donor DCs transplanted en bloc with the allograft migrate to the recipient spleen, 16 where they can activate na...

The longevity of dendritic cells (DCs) is a critical regulatory factor influencing the outcome of immune responses. Recently, we demonstrated that the immunosuppressive drug rapamycin (Rapa) specifically induces apoptosis in DCs but not in other myeloid cell types. The present study unraveled the mechanism used by Rapa to induce apoptosis in human monocyte-derived DCs. Our data demonstrate that granulocyte-macrophage colonystimulating factor (GM-CSF) preserves DC survival specifically via the phosphatidylinositol-3 lipid kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway, which is abrogated by Rapa at the level of mTOR. Disruption of this GM-CSF signaling pathway induced loss of mitochondrial membrane potential, phosphatidyl-serine exposure, and nuclear changes. Apoptosis of these nonproliferating DCs was preceded by an up-regulation of the cell cycle inhibitor p27 KIP1 . Overexpression of p27 KIP1 in DCs using adenoviral gene transduction revealed that apoptosis is directly regulated by p27 KIP1 . Furthermore, both overexpression of p27 KIP1 and disruption of the GM-CSF/PI3K/mTOR signaling pathway decreased the expression of the antiapoptotic protein mcl-1. This mTOR/p27 KIP1 /mcl-1 survival seems unique for DCs and may provide novel opportunities to influence immune responses by specific interference with the life span of these cells. IntroductionApoptosis, or programmed cell death, is a physiologic process that is required for the normal development and maintenance of tissue homeostasis. 1 It is an active process that is regulated by gene products, which either block or accelerate programmed cell death. In most cells, the apoptotic program is always ready to be executed unless continuously inhibited by extracellular survival factors. 2 Apoptosis regulates many aspects of immunologic homeostasis, including initiation, magnitude, and termination of immune responses. Dendritic cells (DCs) play a critical role in the diverse facets of immune regulation, ranging from tolerance induction and the prevention of autoimmunity to the induction of antitumor immunity and the protection against infectious agents. 3,4 DCs are the most potent antigen-presenting cells. They play a major role in the uptake, transport, and presentation of antigens and have the unique capacity to stimulate naive T lymphocytes. 5 In addition to their polarizing capacity on contact with naive T cells, 4 they can interact with B cells 6 and natural killer (NK) cells 7 and thus direct the character of the immune response. Although of possible biologic importance to down-regulate immune responses, apoptosis in DCs has been scarcely investigated. Several different death receptors have been identified on DCs, including Fas (CD95), tumor necrosis factor (TNF) receptor, and TNF receptorrelated apoptosis-inducing ligand receptor (TRAIL-R), suggesting a role for death ligand-induced DC apoptosis. 8-10 DC apoptosis can also be triggered by UVB radiation, 11 glucocorticoids, reactive haptens, infectious pathogens, tumor cells, and NK cells...