Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.

Natural killer T (NKT) cells are an abundant subset of liver lymphocytes activated by lipid antigens presented on CD1d molecules that are expressed by cholangiocytes. We aimed to determine if bile from patients with chronic liver diseases contains antigenic lipids that can activate NKT cells. Using murine invariant (24.7, 24.8 and DN32.D3) and non-invariant (14S.6, 14S.7 and 14S.10) NKT hybridomas we investigated the presence of lipid antigens in bile collected from the gallbladder of patients undergoing liver transplantation due to end-stage liver disease. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing. We found that the patient bile samples contain antigens that activate both invariant and non-invariant NKT hybridomas (24.7, 24.8, DN32.D3, 14S.6, 14S.7 and 14S.10), as demonstrated by activation of at least one hybridoma by eight of 10 bile samples. Activation at high dilutions suggests that some antigens are highly potent. We used the non-invariant NKT hybridoma 14S.6 to screen 21 additional patient bile samples for NKT-reactivity and demonstrated that 12 of 21 bile samples resulted in activation, three of which gave a strong activation. Four of 12 activating bile samples contained microbial DNA. Our results reveal an immunological pathway that could be of critical importance in biliary immunology.

Evidence that FcRn mediates the transplacental passage of maternal IgE in the form of IgG anti-IgE/IgE immune complexes.

BACKGROUND: The mechanism(s) responsible for acquisition of maternal antibody isotypes other than IgG are not fully understood. This uncertainty is a major reason underlying the continued controversy regarding whether cord blood (CB) IgE originates in the mother or fetus. OBJECTIVE: To investigate the capacity of maternal IgE to be transported across the placenta in the form of IgG anti-IgE/IgE immune complexes (ICs) and to determine the role of the neonatal Fc receptor (FcRn) in mediating this process. METHODS: Maternal and CB serum concentrations of IgE, IgG anti-IgE, and IgG anti-IgE/IgE ICs were determined in a cohort of allergic and non-allergic mother/infant dyads. Madin-Darby canine kidney (MDCK) cells stably transfected with human FcRn were used to study the binding and transcytosis of IgE in the form of IgG anti-IgE/IgE ICs. RESULTS: Maternal and CB serum concentrations of IgG anti-IgE/IgE ICs were highly correlated, regardless of maternal allergic status. IgG anti-IgE/IgE ICs generated in vitro bound strongly to FcRn-expressing MDCK cells and were transcytosed in an FcRn-dependent manner. Conversely, monomeric IgE did not bind to FcRn and was not transcytosed. IgE was detected in solutions of transcytosed IgG anti-IgE/IgE ICs, even though essentially all the IgE remained in complex form. Similarly, the majority of IgE in CB sera was found to be complexed to IgG. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that human FcRn facilitates the transepithelial transport of IgE in the form of IgG anti-IgE/IgE ICs. They also strongly suggest that the majority of IgE in CB sera is the result of FcRn-mediated transcytosis of maternal-derived IgG anti-IgE/IgE ICs. These findings challenge the widespread perception that maternal IgE does not cross the placenta. Measuring maternal or CB levels of IgG anti-IgE/IgE ICs may be a more accurate predictor of allergic risk.

CD1d is involved in T cell-intestinal epithelial cell interactions.

We assessed the role of the nonclassical class I molecule, CD1d, in the interaction between intestinal epithelial cells and T cells. In a mixed lymphocyte reaction (MLR) system where the stimulator cells were irradiated normal intestinal cells, the anti-CD1d monoclonal antibody (mAb) 3C11 inhibited T cell proliferation. In contrast, no inhibition was seen when mAb 3C11 was added to conventional MLR cultures (non T cell stimulators). Furthermore, no inhibition was seen when either airway epithelial cells were used as stimulator cells or lamina propria lymphocytes were used as responder cells. These latter two conditions along with a conventional MLR favor CD4+ T cell proliferation. However, we have previously shown that normal intestinal epithelial cells stimulate CD8+ T cells under similar culture conditions. Thus, CD1d expressed on intestinal epithelial cells may be an important ligand in CD8+ T cell-epithelial cell interactions.

Distribution of dominant T cell receptor beta chains in human intestinal mucosa.

The majority of human intestinal intraepithelial lymphocytes (iIELs) are CD8+ T cells that use the T cell receptor (TCR)-alpha/beta. Previous studies have shown that iIELs isolated from segments of small intestine or colon contain one or several dominant alpha/beta T cell clones. It is not known whether these clones expand only locally in response to a particular antigen or whether they are widely distributed throughout the intestine. To address this question, iIELs were purified from near the proximal and distal margins in a series of intestinal resections for noninflammatory diseases. TCR-beta expression was then assessed by semiquantitative polymerase chain reaction amplification, analysis of N-region length, and DNA sequencing. The previously described oligoclonal expansion of iIELs was confirmed in each sample. Identical dominant clones were identified in the proximal and distal samples from most cases, including samples taken from sites as distant as the transverse and sigmoid colon or rectum. Distinct clones were found in only one case with samples from the terminal ileum and transverse colon. These results demonstrate that a relatively small number of widely dispersed T cell clones comprise the majority of cells in the human intestinal mucosa.

The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease.

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.

Expression of murine CD1 on gastrointestinal epithelium.

Cluster of differentiation 1 (CD1) in humans is a family of major histocompatibility complex (MHC) class I-like molecules expressed on the surface of immature thymocytes, Langerhans cells, and a subpopulation of B cells. The only function identified for human CD1 is as a ligand recognized by a subpopulation of T lymphocytes. In order to study the distribution and function of these molecules in the mouse, a murine CD1 complementary DNA was expressed in mouse fibroblasts and used to produce monoclonal antibodies. These antibodies revealed prominent expression of murine CD1 only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. Low levels of expression were also detected on thymocytes and peripheral lymphocytes. The gastrointestinal distribution of murine CD1 suggests that this molecule may be important in epithelial immunity.

Oligoclonal expansion and CD1 recognition by human intestinal intraepithelial lymphocytes.

A human intestinal intraepithelial lymphocyte (IEL) T cell line was established from jejunum to characterize the structure and function of the alpha beta T cell antigen receptors (TCRs) expressed by this population. Single-sided polymerase chain reaction (PCR) amplification cloning and quantitative PCR amplification of the TCR chains from the cell line and from fresh IELs demonstrated that IELs were oligoclonal. The IEL T cell line exhibited CD1-specific cytotoxicity and a dominant IEL T cell clone was CD1c-specific. Thus, human jejunal intraepithelial lymphocytes are oligoclonal and recognize members of the CD1 gene family.

Beta 2-microglobulin-independent MHC class Ib molecule expressed by human intestinal epithelium.

A major histocompatibility complex class Ib protein, CD1d, is expressed by human intestinal epithelial cells (IECs) and is a ligand for CD8+ T cells. CD1d was found to be expressed on the surface of human IECs as a 37-kilodalton protein that was beta 2-microglobulin (beta 2M) independent with no N-linked carbohydrate. Transfection into a beta 2M- cell line confirmed that CD1d could be expressed at the cell surface in the absence of beta 2M. These data indicate that IECs use a specialized pathway for CD1d synthesis and that a beta 2M-independent class Ib protein may be the normal ligand for some intestinal T cells.

Blocking FcRn in humans reduces circulating IgG levels and inhibits IgG immune complex-mediated immune responses.

The neonatal crystallizable fragment receptor (FcRn) functions as an intracellular protection receptor for immunoglobulin G (IgG). Recently, several clinical studies have reported the lowering of circulating monomeric IgG levels through FcRn blockade for the potential treatment of autoimmune diseases. Many autoimmune diseases, however, are derived from the effects of IgG immune complexes (ICs). We generated, characterized, and assessed the effects of SYNT001, a FcRn-blocking monoclonal antibody, in mice, nonhuman primates (NHPs), and humans. SYNT001 decreased all IgG subtypes and IgG ICs in the circulation of humans, as we show in a first-in-human phase 1, single ascending dose study. In addition, IgG IC induction of inflammatory pathways was dependent on FcRn and inhibited by SYNT001. These studies expand the role of FcRn in humans by showing that it controls not only IgG protection from catabolism but also inflammatory pathways associated with IgG ICs involved in a variety of autoimmune diseases.

CD1 expression on antigen-presenting cells.

CD1 proteins present self and microbial glycolipids to CD 1-restricted T cells, or in the case of CD1d, to NKT cells. The CD1 family in humans consists of group I proteins CDla, CDlb, CDlc, and CDle and the group II protein CDld. Rodents express only CDld, but as CD1d is broadly expressed and traffics to all endosomal compartments, this single CD1 family member is thereby able to acquire antigens in many subcellular compartments. A complete understanding of the CD 1 family requires an appreciation of which cells express CD1 and how CD1 contributes to the unique function of each cell type. While group I CD 1 expression is limited to thymocytes and professional APCs, CD1d has a wider tissue distribution and can be found on many nonhematopoietic cells. The expression and regulation of CD1 are presented here with particular emphasis on the function of CD1 in thymocytes, B cells, monocytes and macrophages, dendritic cells (DCs), and intestinal epithelial cells (IECs). Altered expression of CD 1 in cancer, autoimmunity, and infectious disease is well documented, and the implication of CD 1 expression in these diseases is discussed.

Activation of peroxisome proliferator-activated receptor gamma suppresses mast cell maturation involved in allergic diseases.

BACKGROUND: Mast cells play a central role in allergic and inflammatory diseases. Several reports indicated role of peroxisome proliferator-activated receptor gamma (PPARgamma) on mast cell function. However, there is no report about the role of PPARgamma on differentiation of mast cells from the progenitors. In this study, we investigated the role of PPARgamma in regulating bone marrow-derived mast cell maturation and the therapeutic implications for mast cell-related diseases such as atopic or contact dermatitis. METHODS: We used in vitro cell culture system for mast cell differentiation from bone marrow-progenitors using specific ligands and lentiviral-mediated short hairpin RNA of PPARgamma, and in vivo murine dermatitis models. RESULTS: Activation of PPARgamma inhibited the maturation of bone marrow progenitors into connective tissue-type mast cells (CTMCs) through up-regulation of GATA-4 and GATA-6 resulting in a decrease in expression of histidine decarboxylase and mast cell histamine content. In comparison, the differentiation of bone marrow progenitors into CTMCs was significantly accelerated by the knockdown of PPARgamma expression by lentiviral-mediated short hairpin RNA. Peroxisome proliferator-activated receptor gamma ligand administration to mice inhibited the maturation of mast cells resulting in attenuation of atopic and contact dermatitis via diminishment of the number of mature mast cells. CONCLUSION: Our results indicate that PPARgamma is one of master regulators on mast cell maturation and potentially useful for the therapy in various disorders involving mast cell activation.

Mucosal T-cell immunoregulation varies in early and late inflammatory bowel disease.

BACKGROUND AND AIMS: Crohn's disease is a life-long form of inflammatory bowel disease (IBD) mediated by mucosal immune abnormalities. Understanding of the pathogenesis is limited because it is based on data from adults with chronic Crohn's disease. We investigated mucosal T-cell immunoregulatory events in children with early Crohn's disease. METHODS: Mucosal biopsies and T-cell clones were derived from children experiencing the first attack of Crohn's disease, children with long-standing Crohn's disease, infectious colitis, and children without gut inflammation. RESULTS: As in acute infectious colitis, interleukin (IL) 12 induced T cells from early Crohn's disease to acquire a strongly polarised T helper (Th) type 1 response characterised by high IFN-gamma production and IL12Rbeta2 chain expression. Th1 polarisation was not induced in clones from late Crohn's disease. Mucosal levels of IL12p40 and IL12Rbeta2 messenger RNA were significantly higher in children with early than late Crohn's disease. These results demonstrate that susceptibility to IL12-mediated modulation is strongly dependent on the stage of Crohn's disease. CONCLUSIONS: At the onset of Crohn's disease mucosal T cells appear to mount a typical Th1 response that resembles an acute infectious process, and is lost with progression to late Crohn's disease. This suggests that mucosal T-cell immunoregulation varies with the course of human IBD. Patients with the initial manifestations of IBD may represent an ideal population in which immunomodulation may have optimal therapeutic efficacy.

Natural killer T cells mediate inflammation in the bile ducts.

Cholangiocytes function as antigen-presenting cells with CD1d-dependent activation of natural killer T (NKT) cells in vitro. NKT cells may act both pro- and anti-inflammatory in liver immunopathology. We explored this immune pathway and the antigen-presenting potential of NKT cells in the bile ducts by challenging wild-type and Cd1d-/- mice with intrabiliary injection of the NKT cell activating agent oxazolone. Pharmacological blocking of CD1d-mediated activation was performed with a monoclonal antibody. Intrabiliary oxazolone injection in wild-type mice caused acute cholangitis with significant weight loss, elevated serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin, increased histologic grade of cholangitis and number of T cells, macrophages, neutrophils and myofibroblasts per portal tract after 7 days. NKT cells were activated after intrabiliary injection of oxazolone with upregulation of activation markers. Cd1d-/- and wild-type mice pretreated with antibody blocking of CD1d were protected from disease. These findings implicate that cells in the bile ducts function as antigen-presenting cells in vivo and activate NKT cells in a CD1d-restricted manner. The elucidation of this biliary immune pathway opens up for potentially new therapeutic approaches for cholangiopathies.

A major histocompatibility complex class I-related Fc receptor for IgG on rat hepatocytes.

Intestinal epithelial cells of the neonatal rat and mouse have been shown to express a major histocompatibility complex (MHC) class I-like Fc receptor, or FcRn, which transports IgG in an apical to basolateral direction. Previous studies have suggested the possible expression of this receptor beyond the neonatal period within the liver. Since bile contains high levels of IgG, we sought to determine whether the FcRn was functionally expressed by adult rat hepatocytes. Using primers specific for FcRn, which did not cross hybridize with MHC class I transcripts, FcRn DNA was amplified by reverse transcriptase polymerase chain reaction from RNA of adult rat hepatocytes. This RNA contained functional FcRn transcripts as it encoded a beta 2-microglobulin-associated cell surface protein as determined by immunoprecipitation of biotinylated cell surface proteins with a polyclonal anti-FcRn specific antiserum. Western blotting of hepatocyte canalicular (apical) and sinusoidal (basolateral) plasma membranes with an FcRn-specific monoclonal antibody further confirmed the protein expression and suggested that FcRn was enriched on the canalicular surface membranes. FcRn, on the surface of hepatocytes, was biologically functional as it bound Fc fragments of IgG at pH 6.0 but not 8.0, which is the same pH dependence observed for FcRn in rat neonatal enterocytes. Thus, FcRn is functionally expressed outside of the neonatal period on the canalicular cell surface of adult hepatocytes. This suggests that hepatocyte FcRn may bind luminal IgG, providing a potential functional communication between parenchymal immune cells and bile.

Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line.

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

Animal models of mucosal inflammation and their relation to human inflammatory bowel disease.

Animal models of inflammatory bowel disease (IBD) have been useful in the identification of those immune responses uniquely involved in IBD pathogenesis and in defining the important roles of environmental influences, such as normal luminal bacterial flora and the genetic composition of the host, in modifying IBD-associated inflammation. Recent studies have focused particular attention on CD4+ T cells which produce excessive quantities either of Th1 cytokines (IFN-gamma and TNF) directed by IL-12 or of a Th2 cytokine (IL-4), relative to the production of suppressive cytokines such as IL-10 and transforming growth factor beta. Such insights will be extremely beneficial in the development of novel approaches to the control of IBD-type inflammation, such as the use of anticytokine therapies and gene therapy, and finally, in the identification of the genetic abnormalities and the antigens driving the inflammation that underlies the human disease.

PPARgamma and inflammatory bowel disease: a new therapeutic target for ulcerative colitis and Crohn's disease.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that is known to play a central role in adipocyte differentiation and insulin sensitivity. Through work in several animal models of intestinal inflammation, it is now recognized that PPARgamma also inhibits tissue injury associated with immune activation. These studies point to PPARgamma as a novel anti-inflammatory mediator with broad therapeutic potential.

Human CD1d associates with prolyl-4-hydroxylase during its biosynthesis.

Recent studies have shown that the CD1 family of proteins present various glycolipid antigens to subsets of T cells. CD1d is expressed on human intestinal epithelial cells (IEC) and exists in two biochemical forms: 37-kDa, beta2-microglobulin (beta2m) independent, nonglycosylated, and 47-kDa, beta2m dependent, glycosylated forms. The biosynthetic pathways and the mechanisms of generation of these two biochemically distinct forms of CD1d in human IEC are unknown. Using a human colonic cell line, T84, transfected with CD1d, the biosynthesis of CD1d was investigated. Pulse-chase metabolic labeling studies of T84 transfected with wild type CD1d demonstrated that CD1d was a stable protein over a 4-day chase period. During the first 24 h of the chase, a novel 65-kDa glycoprotein was co-immunoprecipitated with CD1d. Microsequencing of this protein identified the glycoprotein as the alpha and beta subunits of the resident endoplasmic reticulum protein, prolyl-4-hydroxylase (P4H), an enzyme responsible for hydroxyl modification of proline residues. To study if either one or both biochemical forms of CD1d contained hydroxyproline residues, amino acid composition analysis of the 37 and 48 kDa was performed, and demonstrated that only the 37-kDa, but not the 48-kDa form of CD1d, contained hydroxyproline residues. These studies demonstrate that CD1d exhibits a prolonged association with P4H and that the 37-kDa form contains hydroxyproline residues. This suggests that P4H association with CD1d during its biosynthesis results in a novel post-translational modification of CD1d.

Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites.

Antigen-mediated cross-linking of Immunoglobulin E (IgE) bound to mast cells/basophils via FcɛRI, the high affinity IgE Fc-receptor, is a well-known trigger of allergy. In humans, but not mice, dendritic cells (DCs) also express FcɛRI that is constitutively occupied with IgE. In contrast to mast cells/basophils, the consequences of IgE/FcɛRI signals for DC function remain poorly understood. We show that humanized mice that express FcɛRI on DCs carry IgE like non-allergic humans and do not develop spontaneous allergies. Antigen-specific IgE/FcɛRI cross-linking fails to induce maturation or production of inflammatory mediators in human DCs and FcɛRI-humanized DCs. Furthermore, conferring expression of FcɛRI to DCs decreases the severity of food allergy and asthma in disease-relevant models suggesting anti-inflammatory IgE/FcɛRI signals. Consistent with the improved clinical parameters in vivo, antigen-specific IgE/FcɛRI cross-linking on papain or lipopolysaccharide-stimulated DCs inhibits the production of pro-inflammatory cytokines and chemokines. Migration assays confirm that the IgE-dependent decrease in cytokine production results in diminished recruitment of mast cell progenitors; providing a mechanistic explanation for the reduced mast cell-dependent allergic phenotype observed in FcɛRI-humanized mice. Our study demonstrates a novel immune regulatory function of IgE and proposes that DC-intrinsic IgE signals serve as a feedback mechanism to restrain allergic tissue inflammation.

Cloning of the gene encoding the mouse homologue of the human calcium signal-modulating ligand.

A cDNA clone, representing the mouse homologue of the recently described gene encoding the human calcium signal-modulating ligand, was isolated from a mouse thymus library. This clone exhibits extensive conservation of the primary nucleotide and deduced amino-acid sequences that, when considered with a similar secondary protein structure, transcript size and distribution of expression, suggests a similarity in function.