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.

Prevalence of an ulcerative colitis-associated CD8+ T cell receptor beta-chain CDR3-region motif and its association with disease activity.

The normal human intestinal mucosa contains clonal T cell expansions. Clonal populations of T cells can be determined through evaluation of the idiotypic, hypervariable region of their T cell receptor (TCR). We have previously reported that there exists a highly conserved TCR pattern among intestinal CD8+ T cells in the majority of ulcerative colitis (UC) patients undergoing colectomy that was not present in normal control individuals. This TCR pattern, or motif, was characterized by particular beta-chain usage (TCRBV3 and TCRBJ1S6) and a defined length in the hypervariable third complementarity determining region (CDR3). The aim of this study was to assess the motif's relationship to disease activity. Subjects were 66 with UC, 19 with Crohn's disease, 14 inflammatory controls, and 6 normal controls. cDNA and gDNA were prepared from colonic biopsies and paraffin blocks, respectively, obtained from study subjects and used to assess TCRBV CDR3 region length and usage, as well as for cloning and sequencing of TCRs. The TCRBV CDR3 region was present in 25 of a series of 48 UC subjects but only 3 of 19 Crohn's disease patients and 3 of 14 inflammatory controls. The motif was more common in UC than either Crohn' s disease or inflammatory controls (chi2 = 7.5, P = 0.006, and chi2 = 4.1, P = 0.04, respectively). The motifs presence was not dependent upon histologic disease activity (either active or inactive UC). Clinical UC disease activity was also not significantly associated with an increased presence of the motif in 14 paired biopsies, which were taken during times of clinical activity or inactivity. There was a trend toward persistence of the motif, as it was present in 6 of 14 subjects over a 3- to 6-month time period. The previously described UC-associated TCRBV CDR3 region motif located in the intestinal CD8+ T-cell subset is found in a significant proportion of UC subjects. The TCR motif does not significantly discriminate active from inactive disease states. The persistent and diffuse nature of this TCR-associated motif in UC suggests that an ongoing T-cell response to a particular antigen(s) is occuring in this disorder.

Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemia-reperfusion injury.

BACKGROUND & AIMS: Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor whose activation has been linked to several physiologic pathways including those related to the regulation of intestinal inflammation. We sought to determine whether PPAR gamma could function as an endogenous anti-inflammatory pathway in a murine model of intestinal ischemia-reperfusion (I/R) injury. METHODS: PPAR gamma-deficient and wild-type mice were examined for their response to I/R procedure. Treatment with a PPAR gamma-specific ligand was also performed. RESULTS: In a murine model of intestinal I/R injury, we observed more severe injury in PPAR gamma-deficient mice and protection against local and remote tissue injury in mice treated with a PPAR gamma-activating ligand, BRL-49653. Activation of PPAR gamma resulted in down-regulation of intercellular adhesion molecule 1 expression by intestinal endothelium and tissue tumor necrosis factor alpha messenger RNA levels most likely by inhibition of the NF-kappa B pathway. CONCLUSIONS: These data strongly suggest that an endogenous PPAR gamma pathway exists in tissues that may be amenable to therapeutic manipulation in I/R-related injuries.

Limited CD4 T-cell diversity associated with colitis in T-cell receptor alpha mutant mice requires a T helper 2 environment.

BACKGROUND & AIMS: T-cell receptor alpha mutant (TCRalpha(-/-)) mice spontaneously develop chronic colitis mediated by CD4(+) TCRalpha(-)beta(+) T cells. The aim of this study was to analyze the mechanisms of expansion of these cells by characterization of the TCRbeta repertoire. METHODS: TCRbeta repertoire was analyzed by reverse-transcription polymerase chain reaction/Southern blot and DNA sequencing. Clonality of T cells was examined in the lymphoid tissues and colons of TCRalpha(-/-) mice and interleukin 4-deficient TCRalpha(-/-) mice. In addition, an in vitro culture system using syngeneic colonic epithelial cells as antigens was used. RESULTS: The clonal expansion of a restricted subset of Vbeta8.2(+) T cells was characterized by conservation of a single negatively charged amino acid residue in the second position of the complementarity-determining region 3 (CDR3). These T cells were observed in the diseased colon and appendix (cecal patch) of TCRalpha(-/-) mice, but not germfree TCRalpha(-/-) mice. Culture of polyclonal T cells from young TCRalpha(-/-) mice with colonic epithelial cells under T helper 2 conditions resulted in the survival of Vbeta8.2(+) T cells characterized by the same CDR3 pattern. In addition, the transfer of the cultivated T cells induced mild colitis in recombination-activating gene 1 mutant mice. CONCLUSIONS: In the TCRalpha(-/-) mice, the development of colitis is associated with the presence of a restricted diversity of Vbeta8. 2(+) T-cell subsets characterized by a specific TCR motif. The limited diversity of lamina propria T cells that are derived from naive T cells expanded by reacting with luminal bacterial antigens is likely caused by the survival of these T cells after stimulation with self-antigens in the presence of a T helper 2 environment.

Activation of natural killer T cells by alpha-galactosylceramide in the presence of CD1d provides protection against colitis in mice.

BACKGROUND & AIMS: CD1d is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of natural killer (NK)1.1(+) T cells. These NK T cells exhibit important immunoregulatory functions in several autoimmune disease models. METHODS: To investigate whether CD1d and NK T cells have a similar role in intestinal inflammation, the effects of the glycolipid, alpha-galactosylceramide (alpha-GalCer), on dextran sodium sulfate (DSS)-induced colitis were examined. Wild-type (WT), CD1d(-/-), and RAG(-/-) mice were examined for their response to either alpha-GalCer or the control analogue, alpha-mannosylceramide (alpha-ManCer). RESULTS: WT mice, but not CD1d(-/-) and RAG(-/-) mice, receiving alpha-GalCer had a significant improvement in DSS-induced colitis based on body weight, bleeding, diarrhea, and survival when compared with those receiving alpha-ManCer. Elimination of NK T cells through antibody-mediated depletion resulted in a reduction of the effect of alpha-GalCer. Furthermore, adoptive transfer of NK T cells preactivated by alpha-GalCer, but not alpha-ManCer, resulted in diminished colitis. Using a fluorescent-labeled analogue of alpha-GalCer, confocal microscopy localized alpha-GalCer to the colonic surface epithelium of WT but not CD1d(-/-) mice, indicating alpha-GalCer binds CD1d in the intestinal epithelium and may be functionally active at this site. CONCLUSIONS: These results show an important functional role for NK T cells, activated by alpha-GalCer in a CD1d-restricted manner, in regulating intestinal inflammation.

Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa.

Fractalkine is a unique chemokine that combines properties of both chemoattractants and adhesion molecules. Fractalkine mRNA expression has been observed in the intestine. However, the role of fractalkine in the healthy intestine and during inflammatory mucosal responses is not known. Studies were undertaken to determine the expression and function of fractalkine and the fractalkine receptor CX3CR1 in the human small intestinal mucosa. We identified intestinal epithelial cells as a novel source of fractalkine. The basal expression of fractalkine mRNA and protein in the intestinal epithelial cell line T-84 was under the control of the inflammatory mediator IL-1beta. Fractalkine was shed from intestinal epithelial cell surface upon stimulation with IL-1beta. Fractalkine localized with caveolin-1 in detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells. Cellular distribution of fractalkine was regulated during polarization of T-84 cells. A subpopulation of isolated human intestinal intraepithelial lymphocytes expressed the fractalkine receptor CX3CR1 and migrated specifically along fractalkine gradients after activation with IL-2. Immunohistochemistry demonstrated fractalkine expression in intestinal epithelial cells and endothelial cells in normal small intestine and in active Crohn's disease mucosa. Furthermore, fractalkine mRNA expression was significantly up-regulated in the intestine during active Crohn's disease. This study demonstrates that fractalkine-CX3CR1-mediated mechanism may direct lymphocyte chemoattraction and adhesion within the healthy and diseased human small intestinal mucosa.

Evidence of T cell receptor beta-chain patterns in inflammatory and noninflammatory bowel disease states.

T cell activation, as defined by expression of relevant cell surface molecules, such as the interleukin-2 receptor (CD25), is increased in many chronic relapsing diseases, including inflammatory bowel disease (IBD). These T cells are generally activated through contact of their clonotypic T cell receptor (TCR) with a peptide antigen presented by a major histocompatibility complex molecule. One of the putative antigenic contact sites for the TCR is the third complementarity determining region (CDR3) of the TCR beta-chain variable region (TCRBV). Therefore, analysis of the TCRBV CDR3 provides insight into the diversity of antigens encountered by a given T cell population. This study evaluated the TCRBV CDR3 usage of the activated intestinal lymphocytes from human subjects with IBD, diverticulitis (inflammatory control), and a normal tissue control. Public patterns, as demonstrated by shared TCRBV CDR3 amino acid sequences of activated intestinal T cell subpopulations, were observed. In particular, a public pattern of TCRBV22, a conserved valine in the fifth position, and use of TCRBJ2S1 or TCRBJ2S5 was present in three of four Crohn's disease subjects while not present in the ulcerative colitis subjects. However, the private patterns of TCRBV CDR3 region amino acid sequences were far more striking and easily demonstrated in all individuals studied, including a normal noninflammatory control. Thus we conclude that selective antigenic pressures are prevalent among an individual's activated intestinal lymphocytes.

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.

Analysis of human common bile duct-associated T cells: evidence for oligoclonality, T cell clonal persistence, and epithelial cell recognition.

The phenotype of T cells associated with the common bile duct (CBD) is unknown. We investigated the hypothesis that they behave like other intraepithelial lymphocytes (IEL). Thus, we sought to determine the phenotype, TCR repertoire, and epithelial recognition of T cells obtained during endoscopic retrograde cholangiopancreatography. Three subjects were studied: two with primary sclerosing cholangitis and one normal control. After establishing a short-term T cell line, cells were 1) stained with mAbs for flow cytometric analysis, 2) analyzed for TCRB chain transcript expression, and 3) used as effector cells for cytotoxicity and proliferation. Flow cytometry revealed that for all the subjects 98% of the T cells were TCR-alpha beta-positive. Immunohistology of the CBD showed that the epithelium and lamina propria contained significant numbers of CD3+ CD43+ CD45RO+ lymphocytes. Complementarity-determining region 3 length displays suggested that the CBD-derived lines were oligoclonal. This was confirmed by cloning and random sequencing of PCR amplification products using TCRBV region family-specific primers; TCRB chain sequences were reiterated in all transcripts analyzed. In one case, two expanded TCRB clones could be identified that were persistent in the bile duct over a 1-yr period. The CBD-derived lines were cytolytic in a redirected lysis assay and caused cytolysis of an intestinal epithelial cell line (Caco-2). This recognition was likely preferential for intestinal epithelial cells, since a CBD-derived line exhibited proliferation to two intestinal epithelial cell lines (HT-29 and Caco-2) but not three other lines (HepG2, human foreskin fibroblast, and KD). We conclude that the CBD contains IELs that share several characteristics with intestinal IELs.

Persistent clonal expansions of peripheral blood CD4+ lymphocytes in chronic inflammatory bowel disease.

It is increasingly recognized that chronic Ag exposure may lead to clonal expansions of T cells, including those within the peripheral blood. Inflammatory bowel disease is a chronic, multisystemic disease of unknown origin that predominantly affects the intestine. We sought to determine whether clonal expansions of T cells are present in the peripheral blood of patients with inflammatory bowel disease by an examination of TCR usage. Positively selected CD4+ and CD8+ peripheral blood T cells were isolated from subjects with active ulcerative colitis, Crohn's disease, and diverticulitis and from normal controls. Analysis of complementarity determining region 3 lengths of 24 TCR-beta chain V region families from CD4+ and CD8+ peripheral blood T cells showed a skewed distribution in the three inflammatory groups, consistent with expansion of T cell clones, in comparison to the normally distributed pattern observed among the control donors. Random sequencing of the PCR amplification products of CD4+ peripheral blood T cells from the subjects with ulcerative colitis, Crohn's disease, and diverticulitis revealed reiterative TCR-beta chain sequences that were not found in the normal donors. In subjects with Crohn's disease, the reiterative TCR-beta chain sequences from the CD4+ peripheral blood T cells were persistent over at least a 1-yr period. The persistently expanded TCR-beta chain sequences of CD4+ peripheral blood T cells were identifiable in genomic DNA isolated from archival tissue of intestine from subjects with Crohn's disease and ulcerative colitis by Southern blotting and direct DNA sequencing. An identical twin pair, concordant for Crohn's disease, shared the same reiterative TCR-beta chain sequences in their CD4+ peripheral blood T cells. These studies show that chronic intestinal inflammation is associated with expansions of CD4+ peripheral blood T cells. Furthermore, in inflammatory bowel disease these T cell clonal expansions are persistent and shared among HLA-identical individuals, implicating a response to specific, persistent, and stimulating Ags in these diseases.