Distribution of T cell subsets and DR antigen positive T cells in peripheral blood and in intestinal mucosa of inflammatory bowel disease

The absolute number of T cell subsets and the rate of T cell DR antigen expression in the peripheral blood and the intestinal mucosa in IBD patients were compared with those in healthy normal controls by two color analysis. The methods used here were flow cytometry for the peripheral blood and immunohistological fluorescent staining for the intestinal mucosa. In peripheral blood lymphocyte (PBL), helper (CD4+ Leu8-) T cells increased in ulcerative colitis (UC), whereas suppressor (CD8+ CD11+) T cells decreased in Crohn's disease (CRD). In lamina propria lymphocyte (LPL) of intestinal mucosa, there were no changes in the proportion of T cell subsets in UC, whereas suppressor (CD8+ CD11+) T cells increased in inflamed mucosa of CRD. DR antigen positive T cells did not increase in PBL, but they increased in LPL of both UC and CRD. In conclusion, there are some differences in distribution of T cell subsets and DR antigen positive T cells between the peripheral blood and the intestinal mucosa in IBD patients. Moreover, the heterogeneity of the distribution of T cell subsets is observed between UC and CRD.     

Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn's disease and control patients

To better define the nature of intestinal T cells, the phenotypes of isolated lamina propria lymphocytes (LPL) were determined in both Crohn's disease patients and control patients using combinations of monoclonal antibodies that have been found to correlate with particular immunoregulatory functions. Isolated LPL and autologous peripheral blood lymphocytes (PBL) were stained with multiple combinations of monoclonal antibodies and studied by dual immunofluorescence flow cytometry. In LPL, compared with PBL, there was a significant increase in the proportion of T cells having the Leu-3+, Leu-8- and Leu-3+, 2H4- phenotypes (associated with helper-inducer function) and a corresponding decrease in the proportion of T cells having the Leu-3+, Leu-8+ and Leu-3+, 2H4+ phenotypes (associated with suppressor-inducer function). It was also found that in LPL, compared with PBL, the percentage of cells with the Leu-2+, Leu-15+ phenotype (associated with suppressor-effector function) was significantly lower. However, the percentage of T cells with the Leu-2+, 9.3+ phenotype (associated with cytolytic function) was similar in PBL and LPL in control patients. There were no major differences comparing Crohn's disease patients with control patients, except that the proportion of Leu-2+, 9.3+ lymphocytes was higher in PBL in Crohn's disease patients. These results show that the lymphocyte subpopulations in the lamina propria differ from those in peripheral blood in having predominantly the phenotypes of helper-inducer and cytolytic T cells, whereas the phenotypes of suppressor-inducer cells and activated suppressor cells are less frequently observed.     

Hyperexpression of inducible costimulator and its contribution on lamina propria T cells in inflammatory bowel disease

BACKGROUND & AIMS: To investigate the role of inducible costimulator (ICOS), a new member of the CD28 family involved in regulation of T-cell activation and chronic intestinal inflammation, we assessed its expression and functional role in patients with inflammatory bowel disease (IBD). METHODS: Expression of ICOS, CD28, and cytotoxic T-lymphocyte antigen (CTLA) 4 on intestinal lamina propria mononuclear cells (LPMC) from patients with ulcerative colitis (UC), Crohn's disease (CD), and normal controls was determined using flow cytometry and immunohistochemistry. Expressions of the ICOS ligand, B7h, on lamina propria B cells, macrophages, and epithelial cells (EC) in the intestinal mucosa were also determined using flow cytometry. The functional costimulatory effect of ICOS on LPMC was assessed by the proliferative response and cytokine production. RESULTS: CD4(+) LPMC expressing ICOS was significantly increased in the inflamed mucosa of IBD patients but not in inflammatory or normal controls. B7h was also significantly up-regulated on B cells, macrophages, and EC in inflamed mucosa of IBD patients. Proliferative responses of anti-CD3/ICOS costimulation were significantly higher compared with those of anti-CD3 monoclonal antibody (mAb) alone. Anti-CD3/ICOS-stimulated-LPMC from UC secreted significantly increased amounts of interleukin (IL)-5 among the 3 groups. In contrast, anti-CD3/ICOS-stimulated-LPMC from CD secreted significantly increased amounts of interferon (IFN)-gamma in the presence of IL-12. CONCLUSIONS: Highly expressed ICOS in activated CD4(+) LPMC of IBD patients contributes to the dysregulated immune responses in IBD. Because ICOS hyperexpression was limited to inflammatory sites in IBD patients, ICOS would be a feasible therapeutic target for the treatment of IBD.     

白细胞介素-23在炎症性肠病的表达升高并诱导促炎细胞因子分泌

目的 通过分析白细胞介素(IL)-23p19及其受体(IL-23R)在炎症性肠病(IBD)患者中的表达,研究IL-23对IBD患者外周血T细胞激活和效应应答的影响,探讨其在疾病发生过程中的免疫病理作用.方法 收集12例克罗恩病(CD)患者、25例溃疡性结肠炎(UC)患者和20名健康者外周血和肠黏膜组织活检标本,使用免疫组化染色和逆转录(RT)-PCR分析IL-23p19表达,分离外周血单个核细胞(PBMC)和肠黏膜固有层内单个核细胞(LPMC),利用流式细胞仪检测IL-23R在CD4+、CD8+T细胞和自然杀伤(NK)细胞表面表达.体外培养PBMC,使用IL-23和抗CD3单抗体外刺激,使用酶联免疫吸附试验检测肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ和白细胞介素(IL)-2分泌.结果 IBD患者炎症肠黏膜组织内IL-23p19蛋白和mRNA表达水平比健康对照者显著升高(P<0.05).IL-23R在IBD患者外周血和肠黏膜固有层组织内CD4+、CD8+T细胞和NK细胞表达水平也比健康者显著升高(P<0.05).体外培养PBMC,使用IL-23刺激,发现IL-23可显著诱导IBD患者,尤其是CD患者PBMC激活,分泌高水平TNF-α、IFN-γ和IL-2(P<0.05).结论 IL-23及其受体在IBD患者炎症肠黏膜组织中表达升高,IL-23可诱导IBD患者淋巴细胞分泌高水平的炎性介质,提示IL-23参与了肠黏膜炎症损伤,阻断IL-23生物学效应可能治疗IBD.     

Duodenal Intraepithelial and Lamina Propria T Lymphocytes in Human Immunodeficiency Virus-Infected Patients with and without Diarrhoea

Background: Diarrhoea is an important problem in human immunodeficiency virus (HIV)-infected patients. Intestinal pathologic conditions may arise from changes in local immunocyte populations. The aims of our study were to establish the histologic features of the duodenal mucosa of HIV-infected patients and to determine a) the phenotype of small-intestinal—intraepithelial (IELs) and lamina propria (LPLs)—lymphocytes; b) their degree of activation and differentiation within the lamina propria; and c) their relation to the presence of diarrhoea. Methods: Distal duodenal biopsy specimens were obtained prospectively from 29 HIV-infected patients—11 patients with diarrhoea (group 1) and 18 patients without diarrhoea (group 2)—and from 42 patients who had neither any risk factor for HIV nor diarrhoea (group 3). Histopathologic and immunohistochemical studies were combined with flow cytometric analysis, after separation of the mucosal intraepithelial compartment from the lamina propria. Results: The median number of IELs and the percentage of γ$dL IELs were both unchanged in HIV-infected patients as compared with controls. In HIV-infected patients LP CD4 cells were decreased, and LP CD8 cells increased. No significant difference was found in the expression of CD25 or CD27 within the LP CD8 populations of HIV-infected patients in groups 1 and 2. Conclusions: These findings suggest that the occurrence of diarrhoea in HIV-infected patients is unrelated to IEL and LPL phenotype.     

A comparison of the expression of lymphocyte activation markers in blood, bronchial biopsies and bronchoalveolar lavage: evidence for an enrichment of activated T lymphocytes in the bronchoalveolar space.

In this study healthy never-smoking subjects (n = 18) were recruited from a population study. Bronchoalveolar lavage (BAL), blood lymphocytes and bronchial biopsies, analysed both in the epithelium and lamina propria, were stained for T and B lymphocytes, natural killer (NK) cells and different subpopulations of T lymphocytes. In BAL, significantly higher proportions of T lymphocytes (CD3), T lymphocyte activation markers; HLA-DR, CD26+, CD49a+, CD54+ and CD69+, helper T (CD3+4+) and memory helper T lymphocytes (CD4+45RO+29+) and memory T lymphocytes (CD3+45RO+) were found, compared to blood. However, the proportion of IL-2 receptor-positive T lymphocytes (CD25+) was lower in BAL than in blood. A previously described higher ratio of CD3+4+/CD3+8+ in BAL than in blood (3.4 vs 1.7; P = 0.001) was confirmed. In bronchial biopsies, we found significantly higher numbers of CD8+ cell profiles per mm2 in the epithelial compared to the lamina propria compartment. We conclude that healthy never-smoking men have higher levels of activated memory T lymphocytes in BAL than in blood, and that the T-cell subpopulations differ in the epithelial compared to the lamina propria compartment in the bronchial mucosa and these compartments should be analysed separately. It is reasonable to think that there is a gradient from blood to the airway lumen where T cells are recruited from blood to take part in the defense towards damaging agents.     

Apoptosis in the intestinal mucosa of patients with inflammatory bowel disease: evidence of altered expression of FasL and perforin cytotoxic pathways

Background and aims Abnormal apoptosis may result in the persistence of activated intestinal T-cells in inflammatory bowel disease (IBD). We investigated apoptosis in distinct mucosal compartments, and the expression of Fas/Fas ligand and perforin in the inflamed and non-inflamed intestinal mucosa of patients with IBD.Methods Colon specimens from 15 patients with ulcerative colitis (UC) and inflamed and non-inflamed mucosa from 15 patients with Crohns disease (CD) were analysed for densities and distribution of apoptotic cells determined by the terminal deoxynucleotidyltransferase-mediated dUDP-biotin nick-end labelling (TUNEL) method. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68, by double immunofluorescence with confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser.Results Colonic lamina propria (LP) and epithelium from patients with UC showed higher rates of apoptosis than controls, but no difference was shown regarding patients with CD. In LP, co-expression of Fas was reduced with T-cells in inflamed CD mucosa, and with macrophages in all patients with IBD. No difference was found in the expression of Fas on B-cells. Rates of FasL-expressing cells in LP were higher in IBD than in controls, with no correlation with the rates of apoptosis. Rates of perforin-expressing cells in LP were greater in UC than in controls, and correlated to the rates of apoptosis. No difference was shown regarding the inflamed and non-inflamed CD mucosa. Rates of FasL and perforin-expressing intra-epithelial lymphocytes showed no difference among groups.Conclusions Increased expression of FasL in IBD colonic LP not parallelled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells. Expression of perforin is correlated to the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in UC.Heitor S.P. Souza and Claudio J.A. Tortori contributed equally to this paper     

Local challenge on oral mucosa with an alpha-gliadin related synthetic peptide in patients with celiac disease

OBJECTIVE: Gluten-derived peptides (e.g., amino-acids 31-49 of alpha-gliadin) have been shown to cause changes typical of celiac disease in the gut. Gluten-derived peptides have mostly been used in in vitro studies. The easiest access to the gastrointestinal system may be the mouth. In the present study we were interested to see whether a synthetic peptide corresponding to amino-acids 31-49 of alpha-gliadin could induce inflammatory changes in the oral mucosa after a local challenge in celiac disease patients. METHODS: The challenge was made by injecting the peptide solution at a concentration of 10 microg/ml submucosally into the oral mucosa of 10 celiac disease patients after a gluten-free diet (GFD) and 12 healthy control subjects. B and CD45RO+ T cells, mast cells, CD3+, CD4+, CD8+ lymphocytes, and alphabeta and gammadelta T-cell receptor-bearing (TcR alphabeta, TcR gammadelta) lymphocytes were counted and HLA DR expression was determined. The expression of CD25 and Ki-67 antigen was also examined. RESULTS: The peptide significantly increased the total number of T cells in the lamina propria of the celiac disease patients. The expression of T-cell activation marker CD25 (IL-2 receptor), but not that of cell proliferation marker Ki-67, was also significantly increased in the lamina propria after peptide challenge. Such a reaction was not observed in the controls. The numbers of CD3+ and CD4+ T cells in the lamina propria were also increased in celiac disease patients after the challenge. The count of TcR gammadelta+ cells was very small in the oral mucosa in celiac disease and showed no increase when the oral mucosa was challenged with the peptide. The expression of HLA DR staining was enhanced after the submucosal peptide challenge in celiac disease; however, the difference was not statistically significant. CONCLUSIONS: The results show that in the celiac disease patients after the peptide challenge the oral mucosal lamina propria responds with a nonproliferative increase of lymphocytes. Thus, submucosal challenge with the peptide 31-49 can be used as an aid in the diagnosis of celiac disease. However, further studies with optimized methodology, including various concentrations of the peptide, adjuvants, other peptides, etc., are warranted, especially because the oral mucosa provides the easiest access to an in vivo peptide challenge in celiac disease.     

Activation and signaling status of human lamina propria T lymphocytes

In this study, proliferative responses of human lamina propria T lymphocytes were examined in vitro. The response of lamina propria T lymphocytes to Sepharose-bound anti-CD3 antibody plus interleukin 2 was significantly lower than the response of autologous peripheral blood T lymphocytes, whereas the responses of lamina propria T lymphocytes to anti-T11(2/3) antibodies plus sheep erythrocytes or anti-CD28 antibody plus interleukin 2 were largely preserved. After coculture with mucosa supernatant, peripheral blood T lymphocytes showed a similar pattern of reactivity as lamina propria T lymphocytes. This reduced reactivity to T-cell antigen receptor stimulation appears to exist at the level of signal transduction, because triggering of CD3 induces low amounts of intracellular inositol 1,4,5-triphosphate and no free calcium increase in lamina propria T lymphocytes when compared with peripheral blood T lymphocytes. This study indicates that the antigen receptor-dependent activation pathway of lamina propria T lymphocytes for proliferation is down-regulated by intestinal mucosa derived factor(s) and that the alternative pathways mediated by CD2 or CD28 are largely preserved. Based on previous data that lamina propria T lymphocytes can provide help to B cells, it is possible that these alternative activation pathways play an important role in T-B cell interaction in the gut.     

白细胞介素-25在炎症性肠病患者中的表达及临床意义

目的 通过检测白细胞介素(IL)-25在炎症性肠病(IBD)患者肠黏膜及血清中的表达水平,探讨其在IBD发病过程中的作用及意义.方法 收集12例溃疡性结肠炎(UC)患者、16例克罗恩病(CD)患者及13例对照者的内镜肠黏膜活检标本,采用荧光定量PCR技术检测肠黏膜内IL-25 mRNA的表达情况,免疫组化技术分析IL-25在肠黏膜中的原位表达;同期收集20例UC、24例CD患者及20名健康对照者血清标本,采用酶联免疫吸附测定(ELISA)检测血清中IL-25水平.结果 与健康对照组相比,UC及CD患者肠黏膜组织内IL-25 mRNA表达显著降低(P＜0.05),UC及CD组间的表达量差异无统计学意义(P＞0.05).免疫组化分析显示IL-25阳性细胞在正常肠黏膜固有层内有较多表达,同时黏膜内的肠上皮细胞也存在IL-25低表达,UC及CD患者肠黏膜IL-25蛋白表达量显著降低(P＜0.05),UC及CD组间的表达量差异无统计学意义(P＞0.05).ELISA显示UC及CD患者血清中IL-25表达量显著低于健康对照组(P＜0.05).结论 IL-25在IBD患者肠黏膜及血清中表达显著降低,提示IL-25表达缺陷与IBD的发生发展密切相关,IL-25有可能成为IBD治疗的新靶点.     

Immunohistochemical analysis of colonic mucosa with ulcerative colitis--activation of lymphocytes and expression of ICAM-1 by lamina propria dendritic cells and macrophages]

The colonic mucosa of 30 patients with ulcerative colitis was analyzed by an immunohistochemical technique. A quantitative evaluation for lymphocyte subsets show significantly increased numbers of CD3+, CD4+, CD8+, and CD28+ cells in ulcerative colitis cases of histological grades 3, 4 and 5 by Matts' classification comparing to normal control cases. CD4/CD8 ratio in each histological grade of ulcerative colitis was not significantly different from those in normal controls and disease controls (infectious colitis cases). However, CD28/CD3 ratio was increased significantly in ulcerative colitis cases of histological grades 3, 4 and 5 comparing to control cases. Most of the lymphocytes were positive for lymphocyte function-associated antigen-1 alpha (LFA-1 alpha). There were increased numbers of S100-beta + dendritic cells and CD68+ macrophages in the luminal area of the lamina propria. Moreover double stainings revealed that most of the S100-beta + dendritic cells and CD68+ macrophages were intercellular adhesion molecule-1 (ICAM-1, a ligand for LFA-1) positive. These findings suggested that the expression of ICAM1 on S100-beta + dendritic cells and CD68+ macrophages is important by the interaction with T cells and T cell antigen recognition.     

Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease

BACKGROUND & AIMS: T-helper (Th)1 cells play a central role in the pathogenesis of tissue damage in Crohn's disease (CD). Interleukin (IL)-12/STAT4 signaling promotes Th1 cell commitment in CD, but other cytokines are needed to maintain activated Th1 cells in the mucosa. In this study, we examined the expression and role of IL-21, a T-cell-derived cytokine of the IL-2 family; in tissues and cells isolated from patients with inflammatory bowel disease. METHODS: IL-21 was examined by Western blotting in whole mucosa and lamina propria mononuclear cells (LPMCs) from patients with CD, ulcerative colitis (UC), and controls. We also examined the effects of exogenous IL-12 on IL-21 production, as well as the effects of blocking IL-21 with an IL-21-receptor Ig fusion protein. Interferon (IFN)-gamma was measured in the culture supernatants by enzyme-linked immunosorbent assay, and phosphorylated STAT4 and T-bet were examined by Western blotting. RESULTS: IL-21 was detected in all samples, but its expression was higher at the site of disease in CD in comparison with UC and controls. Enhanced IL-21 was seen in both ileal and colonic CD and in fibrostenosing and nonfibrostenosing disease. IL-12 enhanced IL-21 in normal lamina propria lymphocytes through an IFN-gamma-independent mechanism, and blocking IL-12 in CD LPMCs decreased anti-CD3-stimulated IL-21 expression. Neutralization of IL-21 in CD LPMC cultures decreased phosphorylated STAT4 and T-bet expression, thereby inhibiting IFN-gamma production. CONCLUSIONS: Our data suggest that IL-21 contributes to the ongoing Th1 mucosal response in CD.     

Exclusive increase of CX3CR1+CD28-CD4+ T cells in inflammatory bowel disease and their recruitment as intraepithelial lymphocytes

BACKGROUND: CX3CL1/Fractalkine (FKN) has been reported to play important roles in various inflammatory diseases. We examined the role of FKN and its receptor CX3CR1 in T-cell migration in the inflammatory bowel diseases (IBDs), ulcerative colitis (UC) and Crohn's disease (CD). METHODS: CX3CR1 expression on peripheral CD4(+) cells from normal controls (NL n = 24) and IBD patients (UC n = 28, CD n = 26) was examined using flow cytometry. CX3CR1(+)CD4(+) T cells were further characterized for surface antigens, cytokine production, and cytotoxic granule release by flow cytometry and ELISA. FKN expression in 53 colonic biopsy specimens (UC n = 20, CD n = 23, NL n = 10) was analyzed by quantitative PCR and immunohistochemistry. Isolated lamina propria and intraepithelial lymphocytes were also analyzed by flow cytometry (UC n = 10, CD n = 10, NL n = 6). RESULTS: CX3CR1(+)CD4(+) cells were increased in IBD while they were virtually absent in controls. Upregulation of CX3CR1 on CD4(+) T cells was positively correlated with disease activity. These unique T cells expressed markers for both effector memory and cytotoxic cells. Interestingly, CX3CR1 was expressed on CD4(+) T cells lacking CD28. CX3CR1(+)CD28(-)CD4(+) cells produced more IFN-gamma and TNF-alpha than CX3CR1(-) counterparts and released cytotoxic granules. FKN mRNA was upregulated in inflamed colonic tissues and robust expression of FKN was immunohistochemically observed on epithelial cells. Although CX3CR1(+) CD4(+) cells could not be detected in the gut, CD28(-)CD4(+) cells were found in IBD mainly as intraepithelial lymphocytes. CONCLUSIONS: FKN/CX3CR1 may contribute to the pathogenesis of IBD through the emergence of unique CX3CR1(+)CD28(-)CD4(+) T cells that can act both as proinflammatory and cytotoxic cells.     

Altered expression of alpha 4 beta 7, a gut homing integrin, by circulating and mucosal T cells in colonic mucosal inflammation.

BACKGROUND AND AIMS: Expression of alpha 4 beta 7 on memory T lymphocytes identifies a cell population that preferentially migrates to the gut. Detection of alpha 4 beta 7 on circulating lymphocytes may permit the identification of specific subsets trafficking between the circulation and the gut in inflammatory bowel diseases. PATIENTS: Samples and clinical details were taken from patients with Crohn's disease (CD), ulcerative colitis (UC), diverticulitis/ infectious colitis, and healthy controls. METHODS: Peripheral blood and lamina propria mononuclear cells were isolated. Cells were labelled with CD3, CD4, CD25, CD45RO or alpha 4 beta 7. RESULTS: Median levels of circulating total memory T cells (CD4+CD45RO+) were increased in CD (p < 0.01) and UC (p < 0.05). However, the proportion of systemic gut homing T cells (CD4+CD45RO+ alpha 4 beta 7+) was decreased in CD (p < 0.05), UC (p < 0.002), and inflammatory controls (p < 0.05). Levels of activated gut homing T cells (CD4+CD25+ alpha 4 beta 7+) were increased in CD (p < 0.01) and UC (p < 0.05). For both CD4+CD45RO+ and CD4+CD25+ cells, the proportion of lymphocytes coexpressing alpha 4 beta 7 was decreased compared with controls. In small and large intestine lamina propria, expression of alpha 4 beta 7+ on CD3+ cells was extensive, although it was decreased in CD (p < 0.03), UC (p < 0.05), and inflammatory controls (p < 0.05). CONCLUSIONS: Circulating and mucosal gut homing lymphocyte populations are changed in patients with colonic inflammation. This may arise due to a dilution effect from recruited naive T cells, or from integrin down regulation. Changes in general CD4+ lymphocyte populations mask more subtle variations in those cells with gut homing potential.     

A functional role of flip in conferring resistance of Crohn's disease lamina propria lymphocytes to FAS-mediated apoptosis

BACKGROUND & AIMS: There is evidence that, in Crohn's disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. METHODS: Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. RESULTS: After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. CONCLUSIONS: Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis.     

Anti-CD2 and anti-CD3 induced T cell cytotoxicity of human intraepithelial and lamina propria lymphocytes.

The effector function of immunocompetent cells in the gut mucosa has not yet been defined. The cytotoxic function of these cells might be important in the normal immune response and could be relevant to the mucosal damage seen in inflammatory conditions. The cytotoxic function of isolated intraepithelial and lamina propria mononuclear cells in six and 18 hour assays after the addition of various stimuli that interact with the human leukocyte antigens CD2 and CD3 on the mucosal effector cells was investigated. T cell phenotypes were determined using CD4, CD8, and HML1 to characterise cells of the appropriate compartments. Anti-CD3 and phytohaemagglutinin can induce toxic activity of lamina propria lymphocytes in most individuals after six hours and in all individuals after 18 hours. Anti-CD2, anti-CD3, and phytohaemagglutinin are similarly effective at triggering lamina propria lymphocytes. Intraepithelial lymphocytes contain predominantly CD8 and HML1 positive T cells, differentiating phenotypically intraepithelial lymphocytes from lamina propria lymphocytes. Intraepithelial lymphocytes are not cytotoxic at six hours, but have a toxic function comparable with lamina propria lymphocytes after 18 hours with all three triggers. Intraepithelial lymphocytes from inflamed mucosa (Crohn's disease and diverticulitis) mediate significantly reduced cytotoxicity in vitro compared with normal mucosa, whereas lamina propria lymphocyte toxicity is not different. Reduced numbers of cytotoxic cells and reduced reactivity to the trigger substances used after in vivo activation or cold target inhibition could explain the observed differences between intraepithelial lymphocytes from inflamed and uninflamed mucosa. Changes in cell mediated cytotoxicity of intraepithelial lymphocytes and lamina propria lymphocytes may be involved in the mucosal damage in these inflammatory conditions.     

Mucosal Expression of Interleukin-6 and Interleukin-8 Messenger RNA in Ulcerative Colitis and in Crohn's Disease

To elucidate the possible role ofproinflammatory cytokines in inflammatory bowel disease,the expression and localization of interleukin (IL)-6and IL-8 mRNAs were examined in colonic biopsy specimens obtained from 10 patients with activeulcerative colitis (UC), 5 with inactive UC, 6 withCrohn's disease (CD), and 5 normal controls. In situhybridization with digoxigenin-labeled probes andimmunohistochemistry for both cytokines were performed. The IL-6mRNA expression was enhanced in the inflamed mucosa in4 of 6 CD patients, while that of UC patients stayed atbaseline. In contrast, IL-8 mRNA expression was apparently augmented (P = 0.044) in 7 of 10active UC and 3 of 6 CD patients (NS). The cell countpositive for IL-8 mRNA per unit area was definitelyincreased in moderate/severe UC when compared to mild UC (53.1 ± 14.4/mm² vs 9.0± 5.1/mm², P = 0.028) according to thedegree of inflammation. IL-6 mRNA positive cells in CDwere preferentially located in deeper lamina propriathan IL-8 mRNA positive cells in UC. Interestingly, IL-8 mRNA wasexpressed in the mucosal epithelial cells in one UCpatient. The patients treated by corticosteroids tendedto show suppressed expression of each mRNA, except one patient with intractable UC. Our data suggestenhanced expression of mucosal IL-6 mRNA in CD and ofIL-8 mRNA in UC by infiltrating mononuclear cells,indicating the distinct participation of each cytokine in the pathogenesis of UC and CD. Moreover,intestinal epithelial cells in UC occasionally exhibitIL-8 mRNA.