Combined coinhibitory and costimulatory modulation with anti-BTLA and CTLA4Ig facilitates tolerance in murine islet allografts.

Complex interactions between positive and negative cosignaling receptors ultimately determine the fate of the immune response. The recently identified coinhibitory receptor, B and T lymphocyte attenuator (BTLA), contributes to regulation of autoimmune and potentially alloimmune responses. We investigated the role of BTLA in a fully major histocompatibility complex-mismatched mouse islet transplant model. We report that anti-BTLA mAb (6F7) alone does not accelerate graft rejection. Rather, while CTLA4Ig alone improved allograft survival, the addition of anti-BTLA mAb to CTLA4Ig led to indefinite (>100 days) allograft survival. Immediately after treatment with anti-BTLA mAb and CTLA4Ig, islet allografts showed intact islets and insulin production despite a host cellular response, with local accumulation of Foxp3+ cells. We clearly demonstrate that combined therapy with anti-BTLA mAb and CTLA4Ig mice induced donor-specific tolerance, since mice accepted a second donor-specific islet graft without further treatment and rejected third party grafts. CTLA4Ig and anti-BTLA mAb limited the initial in vivo proliferation of CFSE-labeled allogeneic lymphocytes, and anti-BTLA mAb enhanced the proportion of PD-1 expressing T cells while depleting pathogenic BTLA+ lymphocytes. We conclude that targeting the BTLA pathway in conjunction with CTLA4Ig costimulatory blockade may be a useful strategy for promoting immunological tolerance in murine islet allografts.

A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption.

BACKGROUND: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. AIMS: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. METHODS: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive (59)Fe. RESULTS: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. CONCLUSIONS: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.

Relationship between intestinal iron-transporter expression, hepatic hepcidin levels and the control of iron absorption.

Hepcidin is an anti-microbial peptide predicted to be involved in the regulation of intestinal iron absorption. We have examined the relationship between the expression of hepcidin in the liver and the expression of the iron-transport molecules divalent-metal transporter 1, duodenal cytochrome b, hephaestin and Ireg1 in the duodenum of rats switched from an iron-replete to an iron-deficient diet or treated to induce an acute phase response. In each case, elevated hepcidin expression correlated with reduced iron absorption and depressed levels of iron-transport molecules. These data are consistent with hepcidin playing a role as a negative regulator of intestinal iron absorption.

Anatomy and three-dimensional reconstructions of the brain of a bottlenose dolphin (Tursiops truncatus) from magnetic resonance images.

Cetacean (dolphin, whale, and porpoise) brains are among the least studied mammalian brains because of the formidable challenge of collecting and histologically preparing such relatively rare and large specimens. Magnetic resonance imaging offers a means of observing the internal structure of the brain when traditional histological procedures are not practical. Furthermore, internal structures can be analyzed in their precise anatomic positions, which is difficult to accomplish after the spatial distortions often accompanying histological processing. In this study, images of the brain of an adult bottlenose dolphin, Tursiops truncatus, were scanned in the coronal plane at 148 antero-posterior levels. From these scans a computer-generated three-dimensional model was constructed using the programs VoxelView and VoxelMath (Vital Images, Inc.). This model, wherein details of internal and external morphology are represented in three-dimensional space, was then resectioned in orthogonal planes to produce corresponding series of virtual sections in the horizontal and sagittal planes. Sections in all three planes display the sizes and positions of major neuroanatomical features such as the arrangement of cortical lobes and subcortical structures such as the inferior and superior colliculi, and demonstrate the utility of MRI for neuroanatomical investigations of dolphin brains.

Unexpected characteristics of the IFN-gamma reporters in nontransformed T cells.

Analysis of the IFN-gamma promoter has primarily been conducted by transient expression of reporter constructs in transformed cells. However, the activity of cis elements may differ when expressed transiently compared with their activity within native chromatin. Furthermore, the transcription factors and signaling mechanisms in transformed cells may differ from those in normal T cells. To analyze IFN-gamma promoter regulation in normal T cells, we developed a novel retroviral bottom-strand reporter system to allow the chromatin integration of promoter regions in primary developing T cells. As controls, both the IL-2 and IL-4 promoters were inducible in this system, with the IL-4 reporter having Th2-specific activity. Strikingly, the IFN-gamma promoter exhibited constitutive activity in both Th1 and Th2 subsets, in contrast to the behavior of the endogenous IFN-gamma gene, which is inducible only in Th1 cells. In mapping this activity, we found that the AP-1/GM-CSF site in the distal promoter element is the most critical element for the constitutive activity. Transgenic reporter lines for the IFN-gamma promoter confirmed the constitutive behavior of the isolated IFN-gamma promoter. This constitutive activity was resistant to inhibition by cyclosporin A and was independent of Stat4 and p38 mitogen-activated protein kinase. These results suggest that IFN-gamma promoter regulation may require cis elements residing either downstream or >3.4 kb upstream of the transcriptional start site, involving repression of constitutive activity.

Magnetic resonance imaging and three-dimensional reconstructions of the brain of a fetal common dolphin, Delphinus delphis.

To demonstrate the kinds of data that can be obtained non-destructively and non-invasively from preserved museum specimens using modern imaging technology the head region of a whole body fetal specimen of the common dolphin, Delphinus delphis, aged 8-9 months post-conception, was scanned using Magnetic Resonance Imaging (MRI). Series of scans were obtained in coronal, sagittal and horizontal planes. A digital three-dimensional reconstruction of the whole brain was prepared from the coronal series of scans. Sectional areas and three-dimensional volumes were obtained of the cerebral hemispheres and of the brainstem-plus-cerebellum. Neuroanatomical features identified in the scans include the major sulci of the cerebral hemispheres, well-differentiated regions of gray and white matter, the mesencephalic, pontine, and cervical flexures, the "foreshortened" appearance of the forebrain, and the large auditory inferior colliculi. These findings show that numerous features of the fetal common dolphin brain can be visualized and analyzed from MRI scans.

IL-18-stimulated GADD45 beta required in cytokine-induced, but not TCR-induced, IFN-gamma production.

Interleukin-12 (IL-12) and IL-18 induce synergistic transcription of interferon gamma (IFN-gamma) that is T cell receptor (TCR)-independent, not inhibited by cyclosporin A and requires new protein synthesis. To characterize this pathway, we screened for genes that are induced in IL-12- and IL-18-treated T helper type 1 cells. GADD45 beta, which activates mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase kinase 4 (MEKK4), was induced by IL-18 and augmented by IL-12. GADD45 beta expression in naïve CD4+ T cells activated p38 MAPK and selectively increased cytokine-induced, but not TCR-induced, IFN-gamma production. Kinase-inactive MEKK4 and inhibition of the p38 MAPK pathway both selectively inhibit cytokine-induced, but not TCR-induced, IFN-gamma production. Thus, the synergy between IL-12 and IL-18 may involve GADD45 beta induction, which can maintain the MEKK4 and p38 MAPK activation that is necessary for cytokine-induced, but not TCR-induced, IFN-gamma production.

Anatomy and three-dimensional reconstructions of the brain of the white whale (Delphinapterus leucas) from magnetic resonance images.

Magnetic resonance imaging offers a means of observing the internal structure of the brain where traditional procedures of embedding, sectioning, staining, mounting, and microscopic examination of thousands of sections are not practical. Furthermore, internal structures can be analyzed in their precise quantitative spatial interrelationships, which is difficult to accomplish after the spatial distortions often accompanying histological processing. For these reasons, magnetic resonance imaging makes specimens that were traditionally difficult to analyze, more accessible. In the present study, images of the brain of a white whale (Beluga) Delphinapterus leucas were scanned in the coronal plane at 119 antero-posterior levels. From these scans, a computer-generated three-dimensional model was constructed using the programs VoxelView and VoxelMath (Vital Images, Inc.). This model, wherein details of internal and external morphology are represented in three-dimensional space, was then resectioned in orthogonal planes to produce corresponding series of "virtual" sections in the horizontal and sagittal planes. Sections in all three planes display the sizes and positions of such structures as the corpus callosum, internal capsule, cerebral peduncles, cerebral ventricles, certain thalamic nuclear groups, caudate nucleus, ventral striatum, pontine nuclei, cerebellar cortex and white matter, and all cerebral cortical sulci and gyri.

Role of the Stat4 N domain in receptor proximal tyrosine phosphorylation.

Stat4 is activated by the cytokines interleukin 12 and alpha interferon (IFN-alpha) and plays a significant role in directing development of naïve CD4(+) T cells to the Th1 phenotype. Signal transducers and activators of transcription (STAT) proteins undergo phosphorylation on a conserved tyrosine residue, resulting in homo- and heterodimerization, nuclear translocation, and DNA binding. Stat4 can bind to single IFN-gamma-activated sites (GASs) as a dimer or bind two tandem GASs as a pair of STAT dimers, or tetramer, stabilized through N-terminal domain (N domain) interactions between dimers. We uncovered an unexpected effect of the Stat4 N domain in controlling the proximal activation of Stat4 by tyrosine phosphorylation at activated receptor complexes. Mutation of the N domain at tryptophan residue W37, predicted to interrupt N domain dimer formation, unexpectedly prevented IFN-alpha-induced tyrosine phosphorylation of the Stat4 monomer, blocking dimer formation and nuclear translocation. Furthermore, N domains appear to exert private STAT functions, since interchanging the N domains between Stat1 and Stat4 prevented receptor-mediated tyrosine phosphorylation in one case and interrupted STAT-specific gene activation in another. Finally, replacement of the N domain of Stat1 with that of Stat4 abrogated the normal Stat2 dependence of Stat1 phosphorylation, again suggesting the domains are not equivalent. Thus, in addition to its role in STAT tetramerization, the conserved STAT N domain appears to participate in very proximal steps of receptor-mediated ligand-induced tyrosine phosphorylation.

Signaling and transcription in T helper development.

The recognition of polarized T cell subsets defined by cytokine production was followed by a search to define the factors controlling this phenomenon. Suitable in vitro systems allowed the development of cytokine "recipes" that induced rapid polarization of naïve T cells into Th1 or Th2 populations. The next phase of work over the past several years has begun to define the intracellular processes set into motion during Th1/Th2 development, particularly by the strongly polarizing cytokines IL-12 and IL-4. Although somewhat incomplete, what has emerged is a richly detailed tapestry of signaling and transcription, controlling an important T cell developmental switch. In addition several new mediators of control have emerged, including IL-18, the intriguing Th2-selective T1/ST2 product, and heterogeneity in dendritic cells capable of directing cytokine-independent Th development.

Recruitment of Stat4 to the human interferon-alpha/beta receptor requires activated Stat2.

Stat4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although Stat4 is activated by interleukin (IL)-12 in both human and murine T cells, Stat4 is activated by interferon (IFN)-alpha only in human, but not murine, CD4(+) T cells. This species-specific difference in cytokine activation of Stat4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of Stat4 recruitment and activation by the human IFN-alpha receptor. Analysis of phosphopeptide binding analysis suggests that Stat4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-alpha receptor complex. Expression of murine Stat4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-alpha-induced Stat4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of Stat4 by the IL-12 receptor, Stat4 activation by the human IFN-alpha receptor occurs through indirect recruitment by intermediates involving Stat2.

Selective loss of type I interferon-induced STAT4 activation caused by a minisatellite insertion in mouse Stat2.

The use of murine systems to model pathogen-induced human diseases presumes that general immune mechanisms between these species are conserved. One important immunoregulatory mechanism involves linkage of innate and adaptive immunity to direct the development of T helper subsets, for example toward subset 1 (TH1) development through STAT4 activation. In analyzing type I interferon signaling, we uncovered a difference between murine and human cells which may affect how these two species control linkage between innate and adaptive immunity. We show that in humans, type I interferons induce TH1 development and can activate STAT4 by recruitment to the IFN-alpha receptor complex specifically via the carboxy-terminus of STAT2. However, the mouse Stat2 gene harbors a minisatellite insertion that has altered the carboxy-terminus and selectively disrupted its capacity to activate STAT4, but not other STATs. This defect in murine Stat2 suggests that the signals leading to STAT4 activation and TH1 development in CD4+ T cells are different between mice and humans.

T helper differentiation proceeds through Stat1-dependent, Stat4-dependent and Stat4-independent phases.

Much of our focus in understanding Th1/Th2 development has been on the signals delivered by IL-12 and IL-4 as final determinants of terminal T cell differentiation. Because extinction of IL-12 signaling in early Th2 development could potentially be important in imprinting a more permanent Th2 phenotype on a population of T cells, we have also examined various parameters regulating the IL-12 signaling pathway. Whereas IL-4 appears to repress functional IL-12 signaling through inhibition of IL-12R beta 2 expression, IFN-gamma in the mouse, and IFN-alpha in the human appear to induce IL-12R beta 2 expression and promote IL-12 responsiveness. We propose that Th1 development can be considered in two stages, capacitance and development. Capacitance would simply involve expression of IL-12R beta 1 and beta 2 subunits, regulated by TCR, IL-4 and IFNs. The second stage, development, we propose is the true IL-12 induced developmental stage, involving expression of Stat4 inducible proteins. In the human, this may also occur via IFN-alpha, which is able to activate Stat4. It is perhaps possible that all of Stat4 actions on Th1 development may be exert directly by Stat4 at the IFN-gamma gene, however we suggest that, more likely, Stat4 may act to induce Th1 development through the induction of other non-cytokine genes, whose stable expression maintains the transcriptional state of a Th1 cell.

Induction of interferon-gamma production in Th1 CD4+ T cells: evidence for two distinct pathways for promoter activation.

IFN-gamma produced by CD4+ T helper 1 (Th1) cells promotes protection against intracellular pathogens. Antigen activation of Th1 cells is an important mode of IFN-gamma induction, but here we analyze a second, antigen-nonspecific pathway capable of inducing full IFN-gamma transcription. IL-12 or IL-18 alone do not induce IFN-gamma mRNA, and only modestly augment antigen-induced IFN-gamma mRNA from Th1 cells. However, IL-12 and IL-18 together fully induce IFN-gamma transcription independently of TCR-activated signals, by a mechanism that does not simply involve Stat4 and NF-kappaB activation, but requires additional protein synthesis. Cyclosporin A inhibits TCR-induced IFN-gamma production, but not IL-12/IL-18-induced IFN-gamma production, biochemically discriminating between these pathways. These results suggest that the two pathways induce IFN-gamma production through functionally segregated but spatially overlapping cis-acting elements, similar to other genes under the control of two or more promoters.

The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway.

Interleukin 12 (IL-12)-induced T helper 1 (Th1) development requires Stat4 activation. However, antigen-activated Th1 cells can produce interferon gamma (IFN-gamma) independently of IL-12 and Stat4 activation. Thus, in differentiated Th1 cells, factors regulated by IL-12 and Stat4 may be involved in IFN-gamma production. Using subtractive cloning, we identified ERM, an Ets transcription factor, to be a Th1-specific, IL-12-induced gene. IL-12-induction of ERM occurred in wild-type and Stat1-deficient, but not Stat4-deficient, T cells, suggesting ERM is Stat4-inducible. Retroviral expression of ERM did not restore IFN-gamma production in Stat4-deficient T cells, but augmented IFN-gamma expression in Stat4-heterozygous T cells. Ets factors frequently regulate transcription via cooperative interactions with other transcription factors, and ERM has been reported to cooperate with c-Jun. However, in the absence of other transcription factors, ERM augmented expression of an IFN-gamma reporter by only 2-fold. Thus, determining the requirement for ERM in Th1 development likely will require gene targeting.

Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse.

Iron is essential for many cellular functions; consequently, disturbances of iron homeostasis, leading to either iron deficiency or iron overload, can have significant clinical consequences. Despite the clinical prevalence of these disorders, the mechanism by which dietary iron is absorbed into the body is poorly understood. We have identified a key component in intestinal iron transport by study of the sex-linked anaemia (sla) mouse, which has a block in intestinal iron transport. Mice carrying the sla mutation develop moderate to severe microcytic hypochromic anaemia. Although these mice take up iron from the intestinal lumen into mature epithelial cells normally, the subsequent exit of iron into the circulation is diminished. As a result, iron accumulates in enterocytes and is lost during turnover of the intestinal epithelium. Biochemical studies have failed to identify the underlying difference between sla and normal mice, therefore, we used a genetic approach to identify the gene mutant in sla mice. We describe here a novel gene, Heph, encoding a transmembrane-bound ceruloplasmin homologue that is mutant in the sla mouse and highly expressed in intestine. We suggest that the hephaestin protein is a multicopper ferroxidase necessary for iron egress from intestinal enterocytes into the circulation and that it is an important link between copper and iron metabolism in mammals.

Tpm1, a locus controlling IL-12 responsiveness, acts by a cell-autonomous mechanism.

Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 x BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.

Smoking, obesity, and hypertension alter the dose-response curve and test sensitivity of carbohydrate-deficient transferrin as a marker of alcohol intake.

Serum carbohydrate-deficient transferrin (CDT) is a specific and comparatively sensitive marker of excessive alcohol use; however, reports of its sensitivity vary according to the population or patient groups studied and their average alcohol intake. We have characterized the dose-response curve between alcohol intake and CDT concentrations in a study of 1400 men and women from a community-based twin registry. Our results show that mean CDT increases with increasing reported alcohol consumption even within the range of alcohol use considered to be nonhazardous. We found significant effects of sex, age, smoking, previous alcohol dependence, body mass index, and diastolic hypertension on the alcohol-CDT dose-response curve. These variables either affect test sensitivity or require adjustment of reference intervals. The results also provide insight into the physiological and biochemical factors that affect CDT concentration.

Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism.

Recently, the transcription factor GATA-3 was shown to be selectively expressed in Th2 but not Th1 cells and to augment Th2-specific cytokines. Here, we show that loss of GATA-3 expression by developing Th1 cells requires IL-12 signaling through Stat4 and does not simply result from an absence of IL-4. Moreover, we demonstrate a novel role for GATA-3 in directly repressing Th1 development distinct from its positive actions on Th2-specific cytokines. GATA-3 inhibits Th1 cytokines by a cell-intrinsic mechanism that is not dependent on IL-4 and that may involve repression of IL-12 signaling. Thus, GATA-3 expression and IL-12 signaling are mutually antagonistic, which facilitates rapid dominance of one pathway during early Th development, producing a stable divergence in cytokine profiles.