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1α,25-Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL-10-secreting CD4+ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3+ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3+ and IL-10+ CD4+T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL-10 at more moderate levels, with little coexpression of these molecules. The Foxp3+ and IL-10+ T-cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL-10. 1α25VitD3 enables the selective expansion of Foxp3+ Treg cells over their Foxp3− T-cell counterparts. Equally, 1α25VitD3 maintains Foxp3+ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4+Foxp3+ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL-10+ and Foxp3+ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.

CD4(+) T cells producing interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) are reported in chronic infections. However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined. We showed that development of IL-10-producing Th1 cells required high T cell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation. Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells. Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 was a common requirement for production of IL-10 by all Th cell subsets. IL-10 expression also correlated with c-maf expression. Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production.

The molecular mechanisms that regulate expression of the immunosuppressive cytokine IL-10 remain poorly understood. In this study, by measuring sensitivity to DNase I digestion, we show that production of IL-10 by primary mouse bone marrow-derived macrophages stimulated through pattern recognition receptors was associated with chromatin remodeling of the IL-10 locus. We also demonstrate that the IL-10 locus is remodeled in primary Th2 cells and IL-10-producing regulatory T cells that have been differentiated in vitro. Strikingly, a novel DNase I-hypersensitive site (HSS-4.5) was identified in stimulated macrophages, but not in T cells. We show that hyperacetylated histones were recruited to this site in stimulated macrophages. Furthermore, HSS-4.5 is highly conserved and contains a putative NF-κB binding site. In support of a function for this site, NF-κB p65/RelA was recruited to HSS-4.5 in vivo and its activation was required for optimal IL-10 gene expression in LPS-stimulated macrophages.

Indicator dilution curves of 51Cr-EDTA and 125I-insulin injected into perfusate entering the rat liver in vivo are used as a basis for developing a mathematical model of insulin distribution and uptake within the organ. EDTA is not taken up by liver cells and therefore serves as a "volume marker" whose dilution curve reflects the characteristics of perfusate flow through the organ and through the cannulae. These two components are modelled separately but the same approach is used in each case, that is, the minimum number of delayed exponential terms sufficient to reproduce the dilution curves is determined and used as the basis for modelling. This allows the cannula, vascular and sinusoidal volumes to be identified and described as corresponding compartmental configurations. The shape of the insulin dilution curve is additionally influenced by binding to, and uptake by, liver cells. Binding of insulin to receptors on hepatocyte plasma membranes and subsequent internalization of the insulin-receptor complex is modelled by the introduction of additional compartments but this is found to be insufficient unless non-specific binding is also taken into account. The accuracy of determination of the rate constants for insulin-receptor dissociation and for endocytosis is improved by a sudden reduction in the pH of the perfusate about 100 sec after injection of the insulin bolus. This releases any residual receptor bound insulin and is modelled by a sudden shift in the insulin-receptor dissociation rate constant. Matching of the complete model to individual pairs of 51Cr-EDTA and 125I-insulin dilution curves allows vascular and sinusoidal volumes to be determined together with the binding and endocytic rate constants. Use of the model to investigate the effect of substances that modify these rate constants is briefly illustrated in the case where the liver is preperfused with 5 mM indomethacin. The model can also be used to simulate the internal distribution and uptake of insulin with any nominated input function and any set of parameters; this is illustrated by comparing the impulse response in the normal case and that in which indomethacin has been preperfused. Although the present study is confined to insulin, the model and methodology that is developed should be applicable to other ligands for which the hepatocyte carries specific receptors.

Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-β, and it is modulated by activation of the aryl hydrocarbon receptor (AhR). In this study, we show that differentiation of Th17 cells, but not Th1 or induced regulatory T (iT reg) cells, is increased by endogenous AhR agonists present in culture medium. Th17 development from wild-type mice is suboptimal in the presence of the AhR antagonist CH-223191, similar to the situation in AhR-deficient mice, which show attenuated IL-17 production and no IL-22 production. The presence of natural AhR agonists in culture medium is also revealed by the induction of CYP1A1, a downstream target of AhR activation. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells. The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation. Our data emphasize that AhR activation plays an essential part in the development of Th17 cells and provide a rational explanation for the poor in vitro polarization of Th17 cells that is reported in the majority of publications for both mouse and human cells.

Summary Influenza A virus causes worldwide epidemics and pandemics and the investigation of memory T helper (Th) cells that help maintain serological memory following infection is important for vaccine design. In this study we investigated CD94 and NKG2 gene expression in memory CD4 T-cell clones established from the spleens of C57BL/10 (H-2b) and BALB/c (H-2d) mice infected with influenza A virus (H3N2). CD94 and NKG2A/C/E proteins form heterodimeric membrane receptors that are involved in virus recognition. CD94 and NKG2 expression have been well characterized in natural killer (NK) and cytotoxic T cells. Despite CD94 being potentially an important marker for Th1 cells involved in virus infection, however, there has been little investigation of its expression or function in the CD4 T-cell lineage and no studies have looked at in-vivo-generated Th cells or memory cells. We show in this study that in-vivo-generated CD4 Th1 cells, but not Th2 cells, exhibited full-length CD94 and NKG2A gene expression following activation with viral peptide. For NKG2A, a novel ‘short’ (possibly redundant) truncated isoform was detectable in a Th2 cell clone. Another member of the NK receptor family, NKG2D, but not NKG2C or E, was also differentially expressed in Th1 cells. We show here that CD94 and NKG2A may exist as multiple isoforms with the potential to distinguish helper T-cell subsets.

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+ CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+ CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL-protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+ CD4+ T cells further up-regulate the IL-2R alpha chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+ CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+ CD4+ T cells is not a passive phenomenon but instead initiates--in conjunction with signals through the TCR--their differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo.

Regulatory T cells (TReg) control immune responses to self and nonself Ags. The relationship between Ag-driven IL-10-secreting TReg (IL-10-TReg) and naturally occurring CD4+CD25+ TReg is as yet unclear. We show that mouse IL-10-TReg obtained using either in vitro or in vivo regimens of antigenic stimulation did not express the CD4+CD25+ TReg-associated transcription factor Foxp3. However, despite the absence of Foxp3 expression, homogeneous populations of IL-10-TReg inhibited the in vitro proliferation of CD4+CD25− T cells with a similar efficiency to that of CD4+CD25+ TReg. This inhibition of T cell proliferation by IL-10-TReg was achieved through an IL-10-independent mechanism as seen for CD4+CD25+ TReg and was overcome by exogenous IL-2. Both IL-10-TReg and CD4+CD25+ TReg were similar in that they produced little to no IL-2. These data show that Foxp3 expression is not a prerequisite for IL-10-TReg activity in vitro or in vivo, and suggest that IL-10-TReg and naturally occurring CD4+CD25+ TReg may have distinct origins.

Indomethacin inhibits autophosphorylation of the insulin receptor. The lack of a consensus as to whether phosphorylation of the insulin receptor is necessary for its endocytosis prompted an investigation into the effects of indomethacin on the uptake and subcellular processing of insulin in the perfused rat liver. Indomethacin did not affect total uptake of insulin by the liver, but there was a concentration dependent inhibition of transfer from the plasma membrane to the endosomes (1mM, 32% inhibition; 5mM, 90% inhibition). Compartmental analysis showed the endocytic rate constant to be inhibited by 82% at 5mM indomethacin (0.0084 to 0.0015 sec-1). The similarity between the level of inhibition of autophosphorylation and the inhibition of endocytosis suggest that phosphorylation of the receptor is necessary for endocytosis. Indomethacin at 5mM completely abolished efflux of insulin degradation products from the perfused liver, suggesting that internalisation is an absolute requirement for degradation.

The binding and uptake of insulin in perfused rat liver has been investigated with specifically labelled 125I-A14-tyrosyl insulin as a tracer and compared with a commercially available iodo-insulin preparation. The commercial preparation did not show saturation uptake kinetics and the clearance from the perfusate remained low and constant throughout a wide concentration range. A14 labelled insulin showed saturation kinetics and high clearance at low carrier concentration, falling rapidly with increasing carrier concentration and reaching a steady state value of 1 ml/min. These results emphasize the importance of using specifically labelled insulin in physiological and biochemical studies of hepatic insulin metabolism. Perfusion with A14 tyrosine-labelled insulin at 4 degrees C showed apparent saturation with binding to the plasma membrane fraction. Perfusion at 37 degrees C also showed apparent saturation with uptake predominantly to the ligandosome fraction. These results implicate the plasma membrane-ligandosome pathway in the hepatic uptake of insulin at both physiological and pharmacological concentrations of the hormone.

The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied. 1 Methylamine, ammonium chloride and dansyl cadaverine but not chloroquine reduce the total amount of insulin endocytosed. 2 Ammonium chloride, dansyl cadaverine and chloroquine but not methylamine inhibit subsequent degradation and/or translocation of degradation products. 3 None of the weak bases changed the species of the degradation products found within the endocytic vesicles. 4 Kinetic analysis of intravesicular degradation indicates that dissociation from the receptor is the ratelimiting process in degradation. 5 Chloroquine and dansyl cadaverine but not methylamine or ammonium chloride showed specific inhibition of insulin degradation in isolated endocytic vesicles. 6 The effect of chloroquine and dansyl cadaverine on the kinetics of degradation suggest that they are acting by switching the receptor into a tight-binding conformation thereby slowing dissociation.

The specific staining observed in acinar cells raises an interesting point. These cells now appear to synthesize both secretory component and proteins such as amylase, both of which eventually appear in saliva. It is thought that the majority of secreted proteins are transported direct from the Golgi complex in secretory granules to the apical surface of the cell, where they are then secreted by exocytosis. However, in order that secretory component can bind IgA to form the secretory IgA complex found in saliva, it must travel to the basolateral membrane of the cell in order to react with the IgA found in the interstitial fluid, before being secreted at the apical surface. Thus it would seem that salivary glands must possess a protein-sorting mechanism not apparent in the previously studied gut or hepatocyte models. As such it is a useful system in which to compare possible mechanisms of IgA transport and also to extend our general understanding of any form of co-ordination that may be operative between the component sites of IgA secretion. C. S. is an M.R.C. research student. Financial support was provided by the Medical Research Council and the Wellcome Trust.