Compartmentalized expression of Th1 and Th17 cytokines in pediatric inflammatory bowel diseases.

BACKGROUND: Interleukin (IL)-23, IL-17A, IL-17F, and interferon-gamma (IFN-γ) are important mediators of inflammatory colitis and are potential therapeutic targets in inflammatory bowel disease (IBD). Their expression profile in the different parts of normal noninflammatory intestine is unclear and their changes during pathology have not yet been addressed in pediatric IBD patients. METHODS: We quantified the transcriptional expression of IL-23, IL-12, IL-17A, IL-17F, IL-6, and IL-10 in healthy, noninflammatory duodenum, ileum, and colon and in inflamed and noninflamed biopsies of pediatric patients with Crohn's disease (CD) and ulcerative colitis (UC). RESULTS: In healthy tissue, expression of IL-17A is highest in the ileum, with IFN-γ expression lowest in the colon. Compared to healthy sections, CD patients displayed increased IL-12p35 and IFN-γ levels in noninflamed ileum and colon, respectively. Modifications of cytokine expression between noninflamed and inflamed tissues was characterized by increased IL-17A in UC colon, IFN-γ in CD colon, and IL-17A, IFN-γ and IL-6 in CD ileum. Elevated IL-17A levels were positively correlated with IFN-γ in both inflammatory CD and UC but IL-17A and IFN-γ were correlated with IL-23p19 in CD ileum only. CONCLUSIONS: The expression of Th1 and Th17 cytokines varies along the intestine, indicating local specific regulation mechanisms. However, the cytokine expression patterns in the same tissue depends on the pathology, with a Th1 or a Th17 profile in the colon of CD and UC patients, respectively, and a Th1/Th17 profile in the ileum of CD patients. This indicates overlapping but distinct immune mechanisms driving intestinal inflammation in these two pathologies.

Microbial induction of CARD15 expression in intestinal epithelial cells via toll-like receptor 5 triggers an antibacterial response loop.

With the discovery of CARD15 as susceptibility gene for Crohn's disease (CD) a first link to a potential defect in the innate immune system was made. In this work we aimed to analyze enterocyte NOD2/CARD15 expression and regulation in response to bacterial motifs and the consequences of the most common CD-specific CARD15 mutation on antibacterial responses of normal intestinal epithelial cells (IEC). Under normal conditions, IEC lines and ileal enterocytes did not express NOD2/CARD15 mRNA or protein, contrary to IEC derived from inflammatory CD sections. In vitro analyses revealed that the simple contact with non-pathogenic commensal E. Coli K12 was sufficient to induced NOD2/CARD15 mRNA and protein in human IEC (HIEC). We identified bacterial flagellin interacting with TLR5 as major motif in this regulation of NOD2/CARD15. E. Coli mutants not expressing flagellin (DeltaFliC) failed to induce CARD15. Similarly, in HIEC transfected with a plasmid encoding dominant negative TLR5, no CARD15 induction was observed after K12 contact. Isolated TLR2 or TLR4 stimulation had no or only a marginal effect on NOD2/CARD15 expression. NOD2/CARD15 negative HIEC were unresponsive to muramyl dipeptide (MDP), but once NOD2/CARD15 was induced, HIEC and Caco2 cells responded to intra or extracellular MDP presentation with the activation of the NFkB pathway. IEC transfected with the Crohn-specific CARD15 mutant (F3020insC, FS) failed to activate NFkB after MDP-challenge, in contrast to CARD15WT IEC. In response to MDP, IEC induced a massive antibacterial peptide (ABP) response, seen in the apical release of CCL20. This was completely abolished in IEC carrying CARD15FS. These data suggest a critical role of NOD2/CARD15 in the bacterial clearance of the intestinal epithelium while CD-specific mutated NOD2/CARD15 causes an impaired epithelial barrier.

Induction of T lymphocyte apoptosis by sulphasalazine in patients with Crohn's disease.

BACKGROUND: Lamina propria T lymphocytes (LPL) of the intestinal mucosa are chronically activated in Crohn's disease (CD). Defective apoptosis of activated LPL was proposed as a key pathogenic mechanism. In fact, increased expression of antiapoptotic molecules was observed in CD LPL. In the present work, we aimed to analyse the effects and underlying molecular mechanisms of 5-amino salicylic acid (5-ASA) and derivatives on apoptosis of LPL and peripheral blood lymphocytes (PBL) in patients with CD compared with ulcerative colitis (UC) and in non-inflammatory controls. METHODS: PBL and LPL were isolated by Ficoll-Hypopaque gradient centrifugation and the EGTA-collagenase method, respectively. PBL/LPL were stimulated with FasL, 5-ASA, sulphapyridine, and sulphasalazine for 24/48 hours and apoptosis was quantified by flow cytometry (annexin V- propidium iodide method) and immunofluorescence. The molecular mechanisms of drug induced apoptosis were analysed in wild-type and FADD-/- Jurkat T cells using western blots and caspase assays. RESULTS: While PBL displayed a normal apoptosis pattern after Fas stimulation in patients with active CD, LPL from inflammatory areas were highly resistant. Comparable resistance to apoptosis was observed in LPL of UC patients. In contrast with 5-ASA, which did not induce apoptosis in lymphocytes, sulphasalazine proved to be a potent proapoptotic agent. Sulphasalazine induced T lymphocyte apoptosis was independent of the Fas pathway but associated with marked downregulation of antiapoptotic bcl-xl and bcl2, activation of the mitochondrial apoptosis signalling pathway, and subsequent activation of caspase-9 and caspase-3. CONCLUSION: The beneficial effect of sulphasalazine in treating inflammatory bowel disease is at least in part attributable to its proapoptotic effects on LPL which allows potent downregulation of lymphocyte activation.

Molecular cloning and characterization of MT-ACT48, a novel mitochondrial acyl-CoA thioesterase.

While characterizing Eps15 partners, we identified a 48-kDa polypeptide (p48) which was precipitated by Eps15-derived glutathione S-transferase fusion proteins. A search in a murine expressed sequence tag data base with N-terminal microsequences of p48 led to the identification of two complete cDNA clones encoding two isoforms of a 439-amino acid protein sharing 95% nucleic and amino acid identity. Northern blot and immunoblotting studies showed that p48 was ubiquitously expressed. A significant homology (19% identity and 40% similarity) between p48 and rat brain cytosolic acyl-CoA thioesterase was observed in an 80-amino acid C-terminal domain, retrieved from proteins from human, nematode, and plants. The thioesterase function of p48 was further demonstrated against long chain acyl-CoAs in a spectrophotometric assay. Furthermore, data obtained from sequence analysis showed that p48 contained a mitochondrial targeting signal, cleaved in mature protein as assessed by microsequencing. The mitochondrial localization of both endogenous and transfected p48 was confirmed by confocal microscopy. These results indicate that p48, called MT-ACT48 (mitochondrial acyl-CoA thioesterase of 48 kDa), defines a novel family of mitochondrial long chain acyl-CoA thioesterases.

AP-2/Eps15 interaction is required for receptor-mediated endocytosis.

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2-binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2-binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2-binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane-coated pits.

The ear of alpha-adaptin interacts with the COOH-terminal domain of the Eps 15 protein.

The role of Eps15 in clathrin-mediated endocytosis is supported by two observations. First, it interacts specifically and constitutively with the plasma membrane adaptor AP-2. Second, its NH2 terminus shows significant homology to the NH2 terminus of yeast End3p, necessary for endocytosis of alpha-factor. To gain further insight into the role of Eps15-AP-2 association, we have now delineated their sites of interactions. AP-2 binds to a domain of 72 amino acids (767-739) present in the COOH terminus of Eps15. This domain contains 4 of the 15 DPF repeats characteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Epsl5r protein. Precipitation of proteolytic fragments of AP-2 with Eps15-derived fusion proteins containing the binding site for AP-2 showed that Eps15 binds specifically to a 40-kDa fragment corresponding to the ear of alpha-adaptin, a result confirmed by precipitation of Eps15 by alpha-adaptin-derived fusion proteins. Our data indicate that this specific part of AP-2 binds to a cellular component and provide the tools for investigating the functions of the association between AP-2 and Eps15.

The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2.

The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-alpha- and anti-beta-adaptin antibodies but not with anti-gamma-adaptin antibody. In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti-alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein. The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.

Homotypic aggregation of CD103 (alpha E beta 7)+ lymphocytes by an anti-CD103 antibody, HML-4.

One monoclonal antibody, HML-4, directed against the alpha E beta 7 integrin (CD103), an integrin preferentially expressed on human intestinal intraepithelial lymphocytes (IEL), induced the homotypic aggregation of IEL and of a CD103+ MOLT16 cell line. Aggregation was an active adhesion event dependent on an intact cytoskeleton, on tyrosine phosphorylation but not on activation of protein kinase C. It was blocked by four other anti-CD103 antibodies but by none of the antibodies blocking known adhesion lymphocyte pathways. It was associated with a redistribution of the CD103 integrin in the areas of cell-cell contacts. These results indicated that HML-4-induced homotypic adhesion was mediated via CD103 and resulted from the binding of the integrin to an as yet undefined ligand expressed by CD103+ cells. This ligand was distinct from the epithelial ligand of CD103: in contrast with homotypic adhesion, heterotypic adhesion of CD103+ MOLT16 cells on two epithelial intestinal cell lines (DLD1 and HT29) was dependent on the presence of divalent cations, was not enhanced by HML-4, was inhibited by HML-1 but not by the three other antibodies with an inhibitory effect on homotypic adhesion. Finally, the study of conjugates between CD103+ and CD103- sublines derived from the MOLT16 cell line suggested that HML-4-induced homotypic aggregation resulted from homophilic CD103-CD103 interactions.

Enhancement of CD3-induced activation of human intestinal intraepithelial lymphocytes by stimulation of the beta 7-containing integrin defined by HML-1 monoclonal antibody.

Intraepithelial lymphocytes (IEL) form a large population of T cells in close contact with the intestinal lumen and differ from lymphocytes in other lymphoid compartments by their predominant CD8+ phenotype and the strong expression of the recently characterized beta 7-containing integrin defined by the monoclonal antibody (mAb) HML-1. The aim of the present in vitro study was to investigate the possible role of the integrin defined by HML-1 in the activation of human IEL via the CD3-T cell receptor (TcR) pathway. The proliferative response of IEL to optimal concentrations of immobilized OKT3 was found to be similar to that of peripheral blood lymphocytes enriched in CD8+ cells. When co-immobilized with suboptimal concentrations of OKT3, antibodies directed against CD11a, CD29 and the beta 7-containing integrin defined by HML-1 exerted a strong synergistic effect on the proliferative response and on the expression of CD25 and CD71 antigens by human IEL. These data indicate that the CD3-TcR pathway is functional in human IEL and contrast with previous observations suggesting that the CD3-TcR pathway was difficult to elicit in human IEL. Furthermore, the present data show that the immune response of human IEL can be modulated via interactions between integrins expressed by IEL and their respective ligands in the mucosa and suggest that IEL's activation may depend on the level of expression of integrin ligands in the epithelium, particularly of the expression of the as yet unknown ligand for the IEL-specific integrin defined by HML-1.

The human intraepithelial lymphocyte marker HML-1 is an integrin consisting of a beta 7 subunit associated with a distinctive alpha chain.

The membrane antigen defined by the monoclonal antibody (mAb) HML-1 is abundantly expressed on, and largely restricted to, the T cells which populate the intestinal epithelium. We show that the mature form of the antigen is a heterodimer comprising a 150-kDa alpha chain and a 120-kDa beta chain. Direct sequencing of tryptic peptides cleaved from the purified beta chain identified this polypeptide with the integrin beta 7 isotype. cDNA clones coding for the beta 7 chain have recently been isolated from T cell cDNA libraries, but the beta 7 chain had not been identified at the protein level. No information is available concerning the primary structure of the HML-1 alpha chain. We show that this subunit is synthesized as a precursor form that undergoes, like several other integrin alpha subunits, a post-translational cleavage of a peptide bond. Among the 11 human integrin alpha chains previously identified, 10 have biochemical features and/or a distribution different from those of HML-1 alpha. One, VLA alpha 4 (CD49d), has a molecular mass of 150 kDa and is expressed on HML-1+ cells but is not recognized by HML-1 mAb. We conclude that HML-1 is a novel member of the integrin family made of the beta 7 chain and of an as-yet-undescribed human alpha chain characterized by the post-translational cleavage of a 10-kDa peptide. HML-1 is, thus, probably the human counterpart of the mouse antigen M290.

Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H-2-restricted anti-HLA T killer lymphocytes.

The lysis by human and murine anti-HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK- and P815-HTR-TK-. Weak but significant HLA-A11-specific lysis was found occasionally with human CTL on the HLA-A11+ L cells. On the contrary, P815-A11 or P815-A2 cells were lysed strongly and specifically by HLA-A11 or HLA-A2-specific human CTL. The T8+T4- phenotype of the effector cells was confirmed and the reaction was inhibited by anti-HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815-HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human beta 2 microglobulin was irrelevant. Anti-human LFA-1 antibodies abrogated the lysis of P815-A11+ cells showing that the LFA-1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti-HLA CTL have been prepared by immunizing mice against syngeneic HLA-A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA-A11 target cells. This barrier was apparently due to the H-2 restriction of these H-2k anti-HLA murine CTL, as shown by their inability to lyse allogeneic H-2d cells expressing HLA-A11, and by the blocking of their activity by anti H-2k antibodies. By contrast, xenogeneic anti-HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA-1 receptor on these cells; a class I molecule of human origin can be seen as an H-2-restricted minor histocompatibility antigen in another species.

Murine cells expressing an HLA molecule are specifically lysed by HLA-restricted antiviral human T cells.

Class I HLA (histocompatibility locus antigen) molecules are the targets of allospecific cytolytic T lymphocytes (CTL) in graft rejection, and constitute the restricting elements necessary for the interaction between antiviral CTL and virus-infected cells. Cells expressing only one HLA in the absence of other human molecules would provide a remarkable model for studying the function of these molecules. However, HLA+ murine cells transfected with human genes are generally not lysed by allospecific human CTL, and this is ascribed to insufficient HLA expression, lack of human beta 2-microglobulin, alteration of HLA molecules or absence of receptors for human T8 or LFA1 molecules in murine cells. Here we report, for the first time, the specific lysis of virus-infected HLA+ murine cells by HLA-restricted antiviral human CTL. Therefore, these murine cells constitute an excellent model for studying the role of HLA molecules.

Natural suppressor cell inhibiting T killer responses against retroviruses: a model for self tolerance.

We previously reported the characterization of a spontaneous suppressor T cell population (NSC) present in naive mice and able to suppress the cytotoxic response (CTL) against tumor cells induced only by endogenous Gross virus (GLV). In this study we demonstrate the existence of such NSC inhibiting the CTL activity against tumor cells induced by the normally exogenous Moloney virus (M-MLV) in mice of the Mov-13 (V+) strain in which the M-MLV has been artificially endogenized and which express the virus during the embryonal life. These NSC are not found in other Mov strains in which the endogenized M-MLV is not expressed during fetal life. The implication of these data in the mechanism of self tolerance is discussed.

T cells from naive mice suppress the in vitro cytotoxic response against endogenous Gross virus-induced tumor cells.

Syngeneic normal lymphoid cells added in co-culture of immune lymphocytes and tumor cells reveal a suppressive activity inhibiting the generation of cytolytic T lymphocytes. The suppression was specific for the response directed against endogenous virus-induced or x-ray-induced tumor cells expressing endogenous C type virus antigens. Thymocytes, spleen cells, or lymph node cells from naive mice were able to express this suppressive activity. The same cells displayed no suppressive activity on killer cells directed against exogenous C type virus-induced tumor cells. The suppressor cells were Thy-1+, Lyt-1- 2+. Our results strongly suggested that the spontaneous suppressor cells exert their activity by interacting with an early step on the CTL response, probably at the level of the helper T cell function. The suppressive activity was mediated by soluble factor(s) that were antigen specific and possibly H-2 restricted. The possible implications of these spontaneous suppressor T lymphocytes in the development of endogenous virus-induced tumors and their possible implications in tolerance to self antigens are discussed.