Richard L. Deem

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells.

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cylotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions arc required for killing of human colonic epithelial cells. Cytokine‐containing supernatants were induced by incubating unscparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti‐CD3 and/or K562 target cells for 18h at 37°C. Cytokines were measured by cytotoxicity assays using L929(murine fibroblast) and HT‐29 (human colonic tumour) lines as target cells in combination with blocking anti‐cytokine antibodies. Supernatanis derived from unseparaled, CD4+ (>95% pure) and CD8+ (>90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor‐alpha, respectively). All or nearly all of the eylotoxicity was due to the presence of tumour necrosis factor‐alpha (little or no tumour necrosis factor‐beta was delected). These same supernatanis were cytoloxic (up to 32% lysis at 1/4 dilution) to HT‐29 targets in a 48‐h 111In release assay. Recombinant tumour necrosis factor‐alpha and interferon‐gamma alone produced minimal killing of HT‐29, but together killed the HT‐29 target celts. Anti‐tumour necrosis factor‐alpha or anti‐interferon‐gamma alone blocked killing of HT‐29 target cells by LPL‐derived supernatants, although anti‐tumour necrosis factor‐beta had no effect upon killing of HT‐29. These results demonstrate that human LPL T cells, triggered by addition of anti‐CD3 and target cells, produce tumour necrosis factor‐alpha and interferon‐gamma, both of which are required for optimal killing of HT‐29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.

Human mucosal T-cell cytotoxicity

Non-major histocompatibility complex-restricted cytotoxicity triggered by antibodies to the CD3 component of the human T-cell receptor complex is thought to be an indirect measure of in vivo primed cytotoxic T-cell activity. We have used this technique to examine the lytic activity of freshly isolated T cells from noninflamed human colonic mucosa. Anti-CD3-triggered T-cell (anti-CD3-T) cytotoxicity was found in all mucosal specimens studied. The mucosal anti-CD3-T effectors do not have Fc receptors for immunoglobulin G, and are therefore distinct from T gamma cells, which mediate antibody-dependent cellular cytotoxicity. The surface antigen phenotype of mucosal anti-CD3-Ts is CD2+, CD3+, CD8+, CD4-, CD16-, and Leu7-. In contrast, peripheral blood anti-CD3-T effectors are Leu7+. Although non-major histocompatibility complex-restricted, mucosal anti-CD3-T cytotoxicity has considerable target specificity, which differs from that of natural killer and lymphokine-activated killer cells. The profile of target cell susceptibility and the inhibitory effects of anti-CD45 antibody suggest that the CD45 molecule on the effector cell may be an important determinant of anti-CD3-T sensitivity. As anti-CD3-triggered lysis may be a marker of in vivo primed mucosal T cells of undetermined antigen specificity, this technique might have important implications in inflammatory bowel disease, where the antigen(s) inciting the mucosal immune reactivity is not certain.

Mucosa-Specific Targets for Regulation of IFN-γ Expression: Lamina Propria T Cells Use Different cis-Elements than Peripheral Blood T Cells to Regulate Transactivation of IFN-γ Expression

Activation of lamina propria (LP) T cells via the CD2 pathway enhances IFN-γ (IFN-γ) secretion with further enhancement after CD28 coligation. The molecular mechanisms regulating IFN-γ expression in LP T cells remain unknown. Previous studies in PBL and T cell lines identified cis- and trans-regulatory elements in TCR-mediated expression of IFN-γ. This study examines CD2 and PMA/ionophore-responsive IFN-γ promoter elements. Activation of LPMC via CD2-induced IFN-γ secretion and a parallel up-regulation of mRNA expression. CD28 coligation enhanced mRNA stability without up-regulating transcription as measured by nuclear run-on. Transfection of a −2.7-kb IFN-γ promoter-reporter construct into PBL and LP mononuclear cells (LPMC) revealed significant promoter activity after CD2 activation, with additional transactivation after CD2/CD28 costimulation in PBL, but not in LPMC. Functional analysis using truncated promoter fragments identified distinct cis-regulatory regions selectively transactivating IFN-γ expression in PBL compared with LPMC. In PBL, CD2 activation elements reside within the −108- to +64-bp region. However, in LPMC the upstream region between −204 and −108 bp was essential. Transfection of the proximal and distal AP-1-binding elements, as well as TRE/AP-1 constructs, revealed functional activation of AP-1 subsequent to CD2 signaling, with activation critical in PBL but diminished in LPMC. Electromobility shift analysis using oligonucleotides encompassing the proximal, distal, and BED/AP-1-binding regions failed to demonstrate selective transactivation after CD2 signaling of LPMC. This report provides evidence that activation of LPMC results in transactivation of multiple promoter elements regulating IFN-γ expression distinct from those in PBL.

Distinct IFNG methylation in a subset of ulcerative colitis patients based on reactivity to microbial antigens

Background: High antibody reactivity toward microbial antigens in Crohns disease (CD) patients is predictive of a more aggressive disease course. However, few ulcerative colitis (UC) patients exhibit serologic reactivity toward microbial antigens. Mucosal expression of IFN‐&ggr; plays a pivotal role in inflammatory bowel disease (IBD) pathogenesis. Recent genome‐wide association studies (GWAS) surprisingly link UC, but not CD, risk loci to IFNG. We recently demonstrated that mucosal T cells from IBD patients exhibit distinct patterns of IFNG methylation compared to controls. This study evaluated the relationship between IFNG methylation and serologic and clinical profiles in peripheral T cells from IBD patients. Methods: DNA from peripheral T cells of 163 IBD patients (91 CD and 64 UC) and 42 controls was analyzed for methylation of eight IFNG sites. Serum markers ASCA, OmpC, I2, CBir, and pANCA were measured by enzyme‐linked immunosorbent assay (ELISA). IFN‐&ggr; secretion was measured by ELISA. Results: IBD patients requiring surgery exhibited reduced IFNG methylation compared to nonsurgical patients (P < 0.02). Enhancement of IFN‐&ggr; secretion (P < 0.003), along with high antibody responses toward multiple microbial antigens (P < 0.017) in UC, but not CD, patients was correlated with decreased IFNG methylation. pANCA levels were not correlated with IFNG methylation. Conclusions: Levels of IFNG methylation were correlated with immune response to microbial components and expression of IFN‐&ggr; in UC patients. Serological and epigenetic markers identify a subset of UC patients with an expression profile of a key TH1 pathogenic cytokine. These data may provide a useful tool to classify a more homogeneous subset of UC patients, allowing for improved diagnostics and targeted therapeutics. (Inflamm Bowel Dis 2011;)

Distinct Methylation of IFNG in the Gut

Mucosal expression of proinflammatory cytokines plays a pivotal role in inflammatory bowel disease (IBD) pathogenesis. Epigenetic remodeling of chromatin via DNA methylation regulates gene expression. In this study, IFNG DNA methylation was analyzed within the mucosal compartment in both normal and IBD populations and compared to its peripheral counterparts. Overall IFNG methylation (across eight CpG sites) was significantly lower in lamina propria (LP) T cells compared to peripheral blood (PB) T cells. No methylation differences were detected when comparing PB T derived from normal to IBD patients. However, LP T-cell DNA derived from IBD patients displayed different levels of IFNG methylation of the upstream regulatory regions compared to DNA from normal controls. In fact, IFNG DNA promoter methylation levels functionally correlate with IFNG mRNA expression in unstimulated T cells, using quantitative real-time PCR. A 5% decrease in promoter methylation status is associated with nearly a 3-fold increase in IFNG expression. Likewise, methylation of the single -54 bp IFNG SnaB1 site strongly inhibited IFNG promoter expression. These results suggest that the epigenetic methylation status of IFNG may play a mechanistic role in the modulation of cytokine secretion in the mucosa.

Enhancer Role of STAT5 in CD2 Activation of IFN-γ Gene Expression

IFN-γ is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-γ promoter: 1) an IL-12-mediated STAT4 binding site at −236 bp; 2) a newly identified IL-2-induced STAT5 binding element at −3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized −3.6-kb IFN-γ STAT5 binding region. CD2 signaling of human PBMC results in activation of the −3.6-kb IFN-γ promoter, whereas mutation of the −3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-γ STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-γ mRNA expression, secretion, and nucleoprotein binding to the IFN-γ STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-γ expression.

Enhanced peripheral blood T-cell cytotoxicity in inflammatory bowel disease

Monoclonal antibodies to the CD3 component of the T-cell antigen receptor can trigger antigen-specific cytotoxic T cells to elicit nonantigen-specific cytotoxicity, possibly by mimicking or bypassing the requirement for antigen triggering. We have used this technique to investigate the possible presence ofin vivo primed cytotoxic T cells, of unknown antigen specificity, in peripheral blood of patients with inflammatory bowel disease. Peripheral blood lymphocytes, which were depleted of background natural killer (NK) activity (CD16−), from patients with Crohns disease exhibited significantly enhanced levels of anti-CD3-triggered T-cell cytotoxicity compared with lymphocytes from normal subjects. Enhanced lytic activity was also found in some patients with ulcerative colitis and in patients with ulcerative colitis postcolectomy. These results were not influenced by treatment or disease activity. There was no correlation between the anti-CD3-triggered T lytic activity and the NK activity in normal subjects or in patients with inflammatory bowel disease. The surface antigen phenotype of the anti-CD3-triggered T killer cell was CD3+, CD8+, CD16−, and Leu 7+. The results provide indirect evidence for increased activity of a subpopulation of cytotoxic T cells, of unknown antigen specificity, in inflammatory bowel disease. Increased activity in patients with ulcerative colitis postcolectomy suggests that this might reflect a fundamental immunological disturbance.

CD2 mediates activation of the IFN-γ intronic STAT binding region in mucosal T cells

The pathways leading to activation of mucosal lamina propria (LP) T cells differ from those of peripheral T cells. LP T cells exhibit enhanced IFN‐γ secretion when activated through the CD2 pathway. This study demonstrates CD2 signaling is followed by activation of STAT proteins in both peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC), although, distinct differences exist in regulation of IFN‐γ promoter gene expression. Both PBMC and LPMC exhibit enhanced secretion and transactivation of the –2.7 kb IFN‐γ promoter region following CD2 signaling, but the IFN‐γ STAT‐binding region (within the first intron) serves as an orientation‐independent enhancer of promoter activity only in LPMC. Mutation of the STAT site impairs enhancer activity. In LPMC, but not PBMC, CD2 mediates binding of STAT1 and STAT4 to the IFN‐γ intronic element. Unstimulated LMPC exhibit low levels of phosphotyrosine‐STAT4 and STAT1 and phosphoserine‐STAT1, which increase substantially following CD2 activation. In PBMC, CD2‐mediated phosphorylation is primarily restricted to enhanced levels of phosphotyrosine‐STAT1. Thus, these results indicate that both common as well as unique molecular mechanisms are involved in CD2 signaling and activation of the STAT pathway in LP T cells which are critical for regulation of IFN‐γ expression in the gut.

Role of mucosal T-cell-generated cytokines in epithelial cell injury

While several factors contribute to the etiopathogenesis of inflammatory bowel disease (IBD), the immune system mediates the tissue damage [1-8]. Two mechanisms of immune-mediated tissue injury which are not mutually exclusive appear likely in IBD: (1) direct autoimmune injury due to a breakdown in immunologic tolerance, or (2) nonspecific bystander injury due to a defect in mucosal immune regulation. Evidence reviewed elsewhere [1, 2] suggests the existence of humoral and cellular reactivity against intestinal epithelial cells in patients with IBD, but the significance of autoimmunity as a primary or secondary phenomenon is not clear. More likely, the damage occurs as an indirect innocent bystander. This hypothesis implies the existence of some defect in the regulation of mucosal immunity leading to an uncontrolled response to a variety of exogenous antigens (virus, bacteria, or protein). Such an event would lead to the generation of effector cells within the efferent mucosal compartment or lamina pro-

An IFNG SNP with an estrogen-like response element selectively enhances promoter expression in peripheral but not lamina propria T cells

This study examines mucosa-specific regulatory pathways involved in modulation of interferon-γ (IFN-γ) in lamina propria T cells. Previous studies identified mucosa-specific CD2 cis-elements within the −204 to −108 bp IFNG promoter. Within this region, a single-site nucleotide polymorphism, −179G/T, imparts tumor necrosis factor-α stimulation of IFNG in peripheral blood lymphocytes, and is linked with accelerated AIDS progression. We discovered a putative estrogen response element (ERE) introduced by the −179T, which displays selective activation in peripheral blood mononuclear cells (PBMC) vs lamina propria mononuclear cells (LPMC). Transfection of PBMC with constructs containing the −179G or −179T site revealed CD2-mediated enhancement of the −179T compared to −179G allele, although, in LPMC, a similar level of expression was detected. Electrophoretic mobility shift assay (EMSA) analysis demonstrated CD2-mediated nucleoprotein binding to the −179T but not the −179G in PBMC. In LPMC, binding is constitutive to both −179G and −179T regions. Sequence and EMSA analysis suggests that the −179T allele creates an ERE-like binding site capable of binding recombinant estrogen receptor. Estrogen response element transactivation is enhanced by CD2 signaling, but inhibited by estrogen in PBMC but not in LPMC, although expression of estrogen receptor was similar. This is the first report to describe a potential molecular mechanism responsible for selectively controlling IFN-γ production in LPMC.