SUMMARYReceptors for 1,25(OH) 2 vitaminD 3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1-a -hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 m g/ml), interferon-gamma (IFN-g; 500 U/ml) or a combination of both. By quantitative reverse transcriptase-polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1-a -hydroxylase mRNA were detected in freshly isolated cells (18^7 Â 10 26 copies/b -actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three-fold with LPS (NS), but a six-fold increase was seen after IFN-g stimulation (P , 0´05). Combining LPS and IFN-g did not result in a major additional increase, but addition of cyclosporin A further increased levels 2´5-fold both in IFN-g-and combination-stimulated cells (P , 0´05). Time course analysis revealed that upregulation of 1-a -hydroxylase was a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and tumour necrosis factor-alpha. Finally, a defect in 1-ahydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-a -hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH) 2 D 3 , might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.

1,25 dihydroxyvitamin D3, the active form of vitamin D, has immunomodulatory properties in vitro and in vivo. We report that treatment with 1,25 dihydroxyvitamin D3 (5 micrograms/kg on alternate days) prevents the development of clinical diabetes in NOD mice, an animal model of human autoimmune diabetes. Diabetes incidence in female NOD mice at the age of 200 days was reduced to 8% in the 1,25 dihydroxyvitamin D treated group vs 56% in the control group (p < 0.0001). In parallel, treatment with 1,25 dihydroxy-vitamin D3 resulted in a complete normalisation of the capacity to induce suppressor mechanisms in an autologous MLR, which is severely depressed in control NOD mice. The existence of such suppressor cells was confirmed in transfer experiments, whereby cotransfer of splenocytes from 1,25 dihydroxyvitamin D3 treated NOD mice prevented diabetes transfer by splenocytes from diabetic NOD mice into irradiated, 6-8-week-old male NOD mice. Other known immune defects of the NOD mice, such as defective natural killer cell killing of YAC-1 targets and defective thymocyte activation by anti-CD3 were not corrected. The pharmacological doses of 1,25 dihydroxyvitamin D3 were universally well tolerated as reflected by a normal weight gain of the mice. Serum calcium was increased (2.5 +/- 0.2 vs 2.2 +/- 0.2 mmol/l in the control group, p < 0.005), whereas osteocalcin levels nearly doubled and bone calcium content was halved. These findings show that 1,25 dihydroxyvitamin D3 can prevent diabetes in NOD mice, probably through the correction of their defective suppressor function.

Prevention of type 1 diabetes in NOD mice by 1,25-dihydroxyvitamin D 3 [1␣,25(OH) 2 D 3 ] is accompanied by a T-helper (Th) 1/Th2 cytokine shift in the pancreas. The aim of this study was to investigate whether this immune shift also occurs outside of the pancreas and whether it is limited to autoantigen-specific immune responses. NOD mice treated with 1␣,25(OH) 2 D 3 (5 µg/kg every 2 days) or control vehicle were immunized with GAD65 (p524-543) or ovalbumin (OVA) in the rear footpads. First, we examined T-cell proliferation and cytokine production (via enzyme-linked immunosorbent assay) of draining lymph node cells in vitro with or without peptide rechallenge. Although no differences in proliferation were measured between control and 1␣,25(OH) 2 D 3 -treated mice after in vitro GAD65 rechallenge, a marked shift in cytokine secretion profile was seen in 1␣,25(OH) 2 D 3 -treated mice: interleukin-4 was increased (37 ± 5 vs. 21 ± 12 pg/ml in controls, P < 0.005), whereas ␥-interferon levels were decreased (6 ± 3 vs. 9 ± 3 ng/ml in controls, P < 0.05). This shift was absent in OVA-primed mice. Second, we measured cytokine profiles by reverse transcriptase-polymerase chain reaction in popliteal lymph nodes at different time points after priming with GAD65 or OVA in vivo. A marked Th1/Th2 shift occurred in 1␣,25(OH) 2 D 3 -treated mice after in vivo priming with GAD65. Again, this shift was absent after OVA immunization. Finally, we measured cytokine profiles after rechallenge with a panel of autoantigens (GAD65, heat shock protein 65, insulin Bchain) and control antigens (OVA, keyhole limpet hemocyanine, myelin proteolipid protein, tetanus toxin) and confirmed the Th1/ Th2 shift in autoantigen-injected mice but not in control antigen-injected mice. In conclusion, the immune deviation induced by 1␣,25(OH) 2 D 3 in NOD mice can also be induced in the peripheral immune system but is limited to pancreatic autoantigens.

No abstract

Objective. Systemic juvenile idiopathic arthritis (JIA) is an immunoinflammatory disease characterized by arthritis and systemic manifestations. The role of natural killer (NK) cells in the pathogenesis of systemic JIA remains unclear. The purpose of this study was to perform a comprehensive analysis of NK cell phenotype and functionality in patients with systemic JIA.Methods. Transcriptional alterations specific to NK cells were investigated by RNA sequencing of highly purified NK cells from 6 patients with active systemic JIA and 6 age-matched healthy controls. Cytokines (NK cellstimulating and others) were quantified in plasma samples (n 5 18). NK cell phenotype and cytotoxic activity against tumor cells were determined (n 5 10), together with their interferon-g (IFNg)-producing function (n 5 8).Results. NK cells from the systemic JIA patients showed an altered gene expression profile compared to cells from the healthy controls, with enrichment of immunoinflammatory pathways, increased expression of innate genes including TLR4 and S100A9, and decreased expression of immune-regulating genes such as IL10RA and GZMK. In the patients' plasma, interleukin-18 (IL-18) levels were increased, and a decreased ratio of IFNg to IL-18 was observed. NK cells from the patients exhibited specific alterations in the balance of inhibitory and activating receptors, with decreased killer cell lectin-like receptor G1 and increased NKp44 expression. Although NK cells from the patients showed increased granzyme B expression, consistent with intact cytotoxicity and degranulation against a tumor cell line, decreased granzyme K expression in CD56 bright NK cells and defective IL-18-induced IFNg production and signaling were demonstrated.Conclusion. NK cells are active players in the inflammatory environment typical of systemic JIA. Although their cytotoxic function is globally intact, subtle defects in NK-related pathways, such as granzyme K expression and IL-18-driven IFNg production, may contribute to the immunoinflammatory dysregulation in this disease.Systemic juvenile idiopathic arthritis (JIA) is a chronic immunoinflammatory childhood disorder of unknown etiology that is characterized by arthritis and systemic features such as quotidian fever, rash, lymphadenopathy, and serositis (1,2). An interplay of environmental factors and genetic predisposition is considered to underlie the pathogenesis (1). About 10% of systemic JIA patients develop

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the β-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8+ T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV–infected IFN-γ–deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ–deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.

A murine model of minor histocompatibility antigen-mismatched bone marrow transplantation (BMT) was used to study the role of timing of donor lymphocyte infusion (DLI) in eliciting graft-versushost (GVH) and graft-versus-leukemia (GVL) reactivity. We gave DLI at weeks 3 and 12 after BMT and related its ability to induce a GVL effect with (1) evolution of T cell chimeric status and (2) the extent to which DLI could elicit lymphohematopoietic GVH (LHGVH) reactivity. All mice remained free of GVH disease, but only week 3 DLI chimeras exhibited a significant GVL response when challenged with host-type leukemia cells. In these week 3 DLI chimeras, host-reactive T cells were found to proliferate in vivo (5-[and-6]-carboxyfluorescein diacetate, succinimidyl esther [CFSE]-labeled DLI inocula, TCR-V␤6 ؉ T-cell frequency) and T-cell chimerism rapidly converted from mixed into complete donor type, indicating the occurrence of LHGVH reactivity. In week 12 chimeras, DLI elicited none of the activities noted at week 3. Yet, in both instances, splenocytes, recovered following DLI, generated an equally strong antihost proliferative response in a mixed lymphocyte reaction, thereby arguing against a decisive role of regulatory cells. The lack of in vivo LHGVH reactivity after week 12 DLI was associated with a substantially increased level of pre-existing host-type T-cell chimerism. We conclude that elicitation of a GVL effect may require LHGVH reactivity and that the reason why timing of DLI was critical for obtaining LHGVH reactivity and the desired GVL effect may lie in the evolution of chimeric status. A possible direct involvement of residual host-type antigen-presenting cells in eliciting LHGVH reactivity after DLI should be studied using models that allow chimerism analysis in non-T-cell

Summary 1,25 dihydroxyvitamin D3, the active form of vitamin D, has immunomodulatory properties in vitro and in vivo. We report that treatment with 1,25 dihydroxyvitamin 03 (5 gg/kg on alternate days) prevents the development of clinical diabetes in NOD mice, an animal model of human autoimmune diabetes. Diabetes incidence in female NOD mice at the age of 200 days was reduced to 8 % in the 1,25 dihydroxyvitamin D treated group vs 56% in the control group (p < 0.0001). In parallel, treatment with 1,25 dihydroxyvitamin D 3 resulted in a complete normalisation of the capacity to induce suppressor mechanisms in an autologous MLR, which is severely depressed in control NOD mice. The existence of such suppressor ceils was confirmed in transfer experiments, whereby cotransfer of splenocytes from 1,25 dihydroxyvitamin D3 treated NOD mice prevented diabetes transfer by splenocytes from diabetic NOD mice into irradiated, 6-8-week-old male NOD mice. Other known immune defects of the NOD mice, such as defective natural killer cell killing of YAC-1 targets and defective thymocyte activation by anti-CD3 were not corrected. The pharmacological doses of 1,25 dihydroxyvitamin D3 were universally well tolerated as reflected by a normal weight gain of the mice. Serum calcium was increased (2.5+0.2 vs 2.2 + 0.2 mmol/1 in the control group,p < 0.005), whereas osteocalcin levels nearly doubled and bone calcium content was halved. These findings show that 1,25 dihydroxyvitamin D3 can prevent diabetes in NOD mice, probably through the correction of their defective suppressor function. [Diabetologia (1994) 37: 552-558]