Selective resistance of mucosal T-cell activation to immunosuppression in Crohn''s disease

BACKGROUND & AIMS: The inappropriately high state of T-cell activation found in Crohn's disease could be due to failure to respond to inhibitory signals. We tested the hypothesis that Crohn's disease mucosal T-cells are resistant to the immunosuppressive action of interleukin4. PATIENTS: Patients with Crohn's disease, ulcerative colitis, and other malignant and non-malignant conditions undergoing bowel resection. METHODS: The effect of interleukin-4 on lamina propria mononuclear cells from Crohn's disease, ulcerative colitis and control mucosa was assessed on various T-cell functions: interleukin-2-induced cytotoxicity, soluble interleukin-2 receptor and interleukin-2 production, and expression of mRNA for interleukin-2R and interferon-gamma. RESULTS: Cytotoxicity of control and ulcerative colitis cells was markedly decreased by interleukin-4, whereas Crohn's disease cells failed to be inhibited. Addition of interleukin-4 to interleukin-2-stimulated cultures decreased soluble interleukin-2R production significantly less in Crohn's disease and ulcerative colitis than control cells. In the same cultures, residual levels of interleukin-2 were significantly increased in control and ulcerative colitis, but not Crohn's disease cultures. Finally, Crohn's disease cells were significantly more resistant to interleukin-4-mediated inhibition of spontaneous and interleukin-2-induced expression of interleukin-2Ralpha and interferon-gamma mRNA compared to control cells. CONCLUSIONS: The effector function, receptor expression and cytokine production of Crohn's disease mucosal T-cells are resistant to interleukin4-mediated inhibition. Failure to respond to down-regulatory signals may contribute to persistent T-cell activation and chronicity of inflammation in Crohn's disease.     

The importance of peripheral immune cells in inflammatory bowel disease.

Background/aims: Inappropriate down regulation of an activated immune system is considered as the main pathogenetic mechanism in inflammatory bowel disease. Migration of circulating cells to a diseased intestine is considered as an important factor in the pathogenesis of inflammatory bowel disease. We aimed to evaluate some features of circulating immune cells in inflammatory bowel disease. Methods: Twenty-two control, 29 Crohn's disease and 17 ulcerative colitis patients were studied. CD2, CD3, CD4, CD8, CD11b, CD11c, CD25, CD45RA, CD45RO, CD54 and HLA DR on the surface of peripheral blood lymphocytes and CD11b, CD11c, CD45RA and CD45RO on the phagocytes were researched with two-color immunofluorescence flow cytometry. Results: The percentages of CD2+ and CD4+ lymphocytes were found significantly reduced in ulcerative colitis. CD3+ and CD8+ lymphocytes in inflammatory bowel disease were higher than in controls. CD45RA+ lymphocytes were found significantly decreased in ulcerative colitis and active Crohn's disease. CD45RO+ lymphocytes and CD45RO+, CD11b+ and CD11c+ phagocytes were significantly increased in Crohn's disease. Conclusions: We demonstrated that there were significant differences between ulcerative colitis and Crohn's disease in the expression of some important surface markers on the peripheral blood immune cells. It seems that circulating CD11b-CD11c and CD45RA-CD45RO expressing phagocytes are important in inflammatory bowel disease and may be useful in distinguishing Crohn's disease from ulcerative colitis. These findings may give us some clues about the immunopathogenesis of inflammatory bowel disease.     

Flow cytometric analysis of peripheral blood lymphocytes in ulcerative colitis and Crohn''s disease.

Using two colour immunofluorescence with fluorescein isothiocyanate and phycoerythrin labelled monoclonal antibodies, multi-parameter flow cytometry was used to examine the antigenic characteristics of peripheral blood lymphocytes in whole blood of patients with ulcerative colitis and Crohn's disease who were not taking immunosuppressive drugs. The numbers of CD4+ and CD8+ lymphocytes in patients with ulcerative colitis and Crohn's disease remained unchanged so that the CD4/CD8 ratio was the same as that of normal control subjects. In Crohn's disease there were many activated T cells (CD3+, CD25+). Although natural killer cells in active Crohn's disease were lower than in normal control subjects, cytotoxic T lymphocytes, as defined by CD3+, CD16+, did not differ in patients with inflammatory bowel disease compared with normal control subjects. For B cell subsets, there were differences in Leu-1+ B cells, Leu-8+ B cells, Fc epsilon R+B cells (Leu-16+, Leu-20+), and activated B cells (Leu-12+, Leu-21+) between patients with inflammatory bowel disease and normal control subjects. These differences are compatible with local activation of B cells in the inflamed colon.     

Soluble interleukin 2 and CD8 and CD4 receptors in inflammatory bowel disease.

Serum levels of soluble interleukin 2 receptor (sIL-2R) have been proposed as a clinical marker of inflammatory bowel disease. The source of sIL-2R in patients with Crohn's disease and ulcerative colitis is unknown, and other soluble receptors have not been investigated. In the present study, sIL-2R and soluble CD8 and CD4 levels were measured in plasma and culture supernatants of peripheral blood and intestinal mucosal mononuclear cells from patients with inflammatory bowel disease, surgical controls, and healthy subjects. Level of plasma sIL-2R was significantly higher in patients with Crohn's disease and ulcerative colitis than in healthy volunteers. Intestinal cells always produced more sIL-2R than peripheral cells. Spontaneous sIL-2R production by mucosal cells was significantly elevated in Crohn's disease but not in ulcerative colitis supernatants compared with levels of surgical controls. Soluble CD8 and CD4 were poor indicators of systemic or mucosal immunity. A positive correlation was found between plasma sIL-2R and spontaneous production by intestinal cells of patients with Crohn's disease and surgical control patients, whereas ulcerative colitis plasma sIL-2R correlated with spontaneous production by peripheral cells. The association of plasma or spontaneous sIL-2R levels with the degree of intestinal inflammation was weak, and there was a wide overlap with control values. Therefore, caution should be used before considering sIL-2R an accurate marker of inflammatory bowel disease activity.     

Differential expression of CD25 (interleukin-2 receptor) on lamina propria T cells and macrophages in the intestinal lesions in Crohn's disease and ulcerative colitis.

Many interleukin-2 receptor (CD25) bearing cells can be identified by alkaline phosphatase immunohistochemistry in the diseased intestinal lamina propria of children with Crohn's disease or ulcerative colitis, but rarely in normal intestine. In both diseases, the CD25+ cells are present as aggregates in the lamina propria below the epithelium, and constitute a large proportion of the lamina propria mononuclear cells. In Crohn's disease, but not ulcerative colitis, CD25+ cells are abundant in the submucosa. The CD25+ cells in Crohn's disease are 58-88% CD3+, CD4+, CD8-, indicating that they are T cells, whereas in ulcerative colitis the CD25+ cells are greater than 80% CD3-, CD4+, HLA-DR+, indicating that they are macrophages. Thus, differential expression of CD25 on T cells and macrophages serves to distinguish the immunologic lesions in ulcerative colitis and Crohn's disease.     

Coexpression of CD4 and CD8 on peripheral blood T cells and lamina propria T cells in inflammatory bowel disease by two colour immunofluorescence and flow cytometric analysis.

Using two colour immunofluorescence with fluorescein isothiocyanate and phycoerythrin labelled monoclonal antibodies and multiparameter flow cytometry, we investigated the coexpression of CD4 and CD8 antigens on peripheral blood lymphocytes and lamina propria lymphocytes of patients with ulcerative colitis and Crohn's disease and normal control subjects. Both the absolute number and the proportion of peripheral blood CD4+, CD8+ cells in inflammatory bowel disease were small but significantly increased compared with those in normal control subjects. Peripheral blood lymphocytes activated with phytohaemagglutinin showed appreciably increased coexpression of CD4+, CD8+. These CD4, CD8 positive cells were large and granular. Thus the increased number of peripheral blood CD4+, CD8+ cells in inflammatory bowel disease suggests that chronic immune activation occurs not only in the active state of the disease but also in remission. The proportion of CD4+, CD8+ cells in the lamina propria was greater than in peripheral blood in normal subjects, suggesting chronic immune stimulation of the local immune system. This was also seen in patients with Crohn's disease or inactive ulcerative colitis. The proportion of CD4+, CD8+ cells was, however, significantly less in the lamina propria of patients with active ulcerative colitis. Whether this implies a possible defect in mucosal immunoregulation in active ulcerative colitis cannot be determined from these results.     

Cytokine production in patients with inflammatory bowel disease.

The production of cytokines in peripheral blood mononuclear leukocytes of patients with inflammatory bowel disease was investigated. T cell subset analysis and differential white blood cell counts were also performed. Thirty five patients with ulcerative colitis, 14 with Crohn's disease, and 15 age matched healthy volunteers were studied. No differences were observed in T cell subsets and OKT4/OKT8 ratios in patients with ulcerative colitis or Crohn's disease compared with controls. Interleukin 1 beta production was significantly increased in active ulcerative colitis and Crohn's disease, compared with values in controls, but returned to control levels in the inactive stages. In addition, in active ulcerative colitis and Crohn's disease, there were significant correlations between the interleukin 1 beta production and the ulcerative colitis activity index or Crohn's disease activity index. Interleukin 2 production was also significantly increased in the active ulcerative colitis and significantly correlated to the activity index, but there was no change in Crohn's disease patients compared with controls. Gamma interferon production in patients was the same as that in controls. This study suggests that the interleukin 1 beta and 2 values in peripheral mononuclear leukocytes of active untreated inflammatory bowel disease are indicators of the disease states of ulcerative colitis or Crohn's disease, or both.     

Intestinal immune reactivity to interleukin 2 differs among Crohn's disease, ulcerative colitis, and controls

When stimulated by the lymphokine interleukin 2, human intestinal mucosal mononuclear cells mediate lymphokine-activated killer cell activity. When supplied with optimal doses of exogenous interleukin 2, lamina propria mononuclear cells isolated from inflammatory bowel disease and control tissue display comparable levels of cytotoxicity in vitro. However, cultures of Crohn's disease- and ulcerative colitis-derived cells contain significantly decreased interleukin 2 activity, suggesting that in vivo the availability of interleukin 2 may be limited, perhaps resulting in impaired cytotoxic function. To test this hypothesis, lamina propria mononuclear cells from inflammatory bowel disease and control patients were stimulated to produce endogenous interleukin 2, which was then used to induce autologous lymphokine-activated killer cells. When tested against K562 and Daudi target cells, Crohn's disease cells, despite producing only one-third of the amount of interleukin 2 generated by control cells, exhibited comparable levels of cytotoxicity. In contrast, ulcerative colitis cells produced substantially less interleukin 2 and exhibited remarkably low lymphokine-activated killer cell activity. When the same cells were supplied with an amount of human recombinant interleukin 2 equivalent to the average titer found in control cultures, similar results were obtained, and Crohn's disease cells even showed a significantly greater cytolytic activity than controls. These results suggest that the observed differences in lymphokine-activated killer cell activity cannot be attributed to the level of interleukin 2 alone, and that response to this lymphokine is different among Crohn's disease, ulcerative colitis, and control intestine. In Crohn's disease, there is either an increased number of interleukin 2-responsive cells or an exacerbated reactivity to interleukin 2. In ulcerative colitis, a loss of interleukin 2-responsive cells, a hyporesponsiveness to interleukin 2, or both might be present. In conclusion, this study demonstrates that reactivity to interleukin 2 distinguishes inflammatory bowel disease from control intestinal mononuclear cells, and, under appropriate experimental conditions, it can be used to uncover abnormalities of intestinal immunity.     

T lymphocyte subsets in inflammatory bowel disease: peripheral blood

Peripheral blood T lymphocytes and T lymphocyte subsets have been quantified in 28 patients with ulcerative colitis and 26 with Crohn's disease by an indirect immunofluorescence technique using monoclonal antibodies: OKT3, which detects all peripheral blood T lymphocytes; OKT4 (T cells of helper phenotype); and OKT8 (T cells of supressor-cytotoxic phenotype). Eighteen normal subjects and 16 patients with a variety of non-inflammatory gastrointestinal disorders were studied as controls. No significant differences were found between patient and control groups in the proportions of circulating T lymphocytes or their subsets. When compared with normal subjects, absolute numbers of T lymphocytes were reduced in patients with active ulcerative colitis or Crohn's disease (p less than 0.05). OKT4+ T cell numbers were reduced in ulcerative colitis, whether active (p less than 0.02) or inactive (p less than 0.05) and in active Crohn's disease (p less than 0.05) Numbers of OKT8+ T cells were reduced in active Crohn's disease (p less than 0.01). There were no differences in T lymphocyte numbers between the patient groups and the disease control subjects. The OKT4+:OKT8+ ratio in patients with inflammatory bowel disease did not differ from that in controls. No relation was found between any of the parameters studied and disease activity, site, or extent of disease, or treatment with sulphasalazine or corticosteroids. The presence of Ia-like, HLA-DR antigens on T cells was detected using a double marker immunofluorescence technique. In control subjects up to 7% of OKT3+ cells were HLA-DR+. In only three patients was the proportion of HLA-DR+ cells greater than in controls. These results indicate that the pathogenesis of ulcerative colitis or Crohn's disease does not depend upon an alteration in the proportion of circulating T lymphocytes nor upon an imbalance of T lymphocyte subsets as defined by monoclonal antibodies. The reduction in T lymphocyte numbers may result from mucosal infiltration. The findings also suggest that circulating T lymphocytes are not activated.     

Immunohistochemical characterization, distribution, and ultrastructure of lymphocytes bearing T-cell receptor gamma/delta in inflammatory bowel disease

Phenotypic characterization and distribution of gamma/delta T lymphocytes in the intestinal mucosa were investigated in ulcerative colitis and Crohn's disease by immunohistochemistry. The ratio of delta(+) cells to CD3(+) cells in the intraepithelial space of colon was decreased in Crohn's disease (13%) and strikingly decreased in ulcerative colitis (8%) compared with the control (36%). Delta(+) cells in the lamina propria were also decreased, particularly in the distal ileum of Crohn's disease (4%), compared with the control (15%). On the contrary, the cells gathered at the severe inflammatory sites with other inflammatory cells, including beta(+) cells, and were densely distributed in the T-cell zone around lymphoid follicles. Phenotypic characterization showed that delta(+) lamina proprial lymphocytes of colon were mainly CD4(-)CD8(-) in the control (80%) and Crohn's disease (59%). However, in ulcerative colitis, CD4(-)CD8(-) delta(+) lymphocytes were rarely found (3%). This reflects the difference of immunologic background between the two diseases. Immunoelectron microscopically, these cells in inflammatory bowel disease were rich with vesicular structures in cytoplasms, whereas those in the control group contained electron-opaque granules. The decrease and the morphological change may be closely related to the weakness of mucosal defense.     

The sentinel node technique is useful for studies of intestinal immunology in inflammatory bowel disease patients

OBJECTIVE: Mesenteric lymph nodes may play a crucial role in the pathogenesis of human inflammatory bowel disease. We have used the sentinel node technique to analyze mesenteric lymph nodes draining inflammatory lesions sentinel nodes and corresponding site of inflamed bowel in patients with ulcerative colitis or Crohn's disease. METHODS: Thirty-two patients undergoing surgical treatment of inflammatory bowel disease were included. Sentinel nodes were identified intraoperatively. The T cells were harvested from the mesenteric lymph nodes and characterized by flow cytometry. RESULTS: The distribution of CD4CD62L (homing-marked) and CD4CD69 (activated) T cells was studied in mesenteric lymph nodes draining inflammation, nodes draining normal intestine, blood, and mucosa. The turnover of T cells was markedly increased in the lymph nodes connected to inflammatory segments. The immunologic activity of the sentinel lymph nodes correlated with the degree of intestinal inflammation. CONCLUSION: Mesenteric sentinel nodes provide important information about locoregional immunology and pathogenesis in inflammatory bowel disease.     

Immunohistological study of T-lymphocytes (total and helper) and B-lymphocytes of the colonic mucosa in children with Crohn's disease and non-specific ulcerative colitis

For one to understand better the immunological mechanism in the colonic mucosae of inflammatory bowel disease, 19 children with ulcerative colitis and 12 children with Crohn's disease were studied. The inflammatory cells identified by the immunoperoxidase technique and specific markers for total T lymphocytes (CD3), helper T lymphocytes (CD4) and B lymphocytes (CD20) in differents evolutionary periods of the disease were analysed. PI (before treatment), PII (until two years of treatment) and PIII (with more than two years of treatment). In general, all marker studied in this research, presented higher averages if comparing to the control group with different degrees of significance in the different studied periods. The marker CD3, of the total T lymphocyte, increased in the epithelial (superficial and glandular) and in the lamina propria of the involved mucosae in relation to the controlled ones, was diffusely distributed over the mucosae, concentrating itself around the microgranulomas and the nodules lymphoids. In Crohn's disease, PI, its average differed significantly from the controlled ones. Similar behave was seen in the marker CD20 of the lymphocytes in the involved mucosae, whole averages were superior in relation to the controlled ones; thought, only in the PI of the Crohn's disease this difference had been significant. The immunochromatic cells CD3 distributed themselves diffusely over the involved mucosae, concentrating themselves in the interior of the lymphoides nodules and in the injured areas, near the macrophages. The marker CD4, of helper T lymphocyte also presented in the involved mucosae high averages in relation to the controlled mucosae, but without estatistic significance. The CD4 cells distributed themselves diffusely over the whole thickness of the involved mucosae, concentrating themselves around the lymphoids nodules and microgranulomas. These results confirm the participation of these elements in the pathogenesis of inflammatory bowel disease, but did not permit the establishment of any discriminatory criteria between ulcerative colitis and Crohn's disease. The results of positive correlation between the increased of CD4 and CD20 in ulcerative colitis and CD3 and CD20 in Crohn's disease show clearly the immunoregulation action of the CD4+ cells over clonal expansion and the differenciation of the B lymphocytes from plasmocytes and the IgG syntese.     

炎症性肠病患者CD27-CD70共刺激途径的激活研究

目的 探讨CD27-CD70共刺激途径在炎症性肠病患者外周循环和肠黏膜中的表达,比较炎症性肠病患者CD27-CD70表达与正常对照者间的差异.方法 共纳入62例克罗恩病(CD)、64例溃疡性结肠炎(UC)患者和56名正常对照者.应用酶联免疫吸附试验分析炎症性肠病患者和正常对照者血浆中CD27-CD70蛋白的表达;SYBR-green实时PCR法分析炎症性肠病患者和正常对照者外周血单个核细胞中CD27-CD70 mRNA的表达;免疫组化法分析炎症性肠病患者和正常对照者肠黏膜组织CD27-CD70蛋白的表达.结果 CD和UC患者血浆中CD27-CD70的表达均显著高于正常对照者(P值均<0.05).ROC曲线的分析表明,CD27-CD70对区分CD患者和正常对照者以及区分UC患者和正常对照者具有显著的诊断价值(P值均<0.05).CD患者血浆中CD27和CD70水平与内镜下疾病活动性(EDAI)无关(r值分别=0.055和0.024,P值分别为0.673和0.852),且UC患者血浆中CD27和CD70水平与EDAI无关(r值分别=0.077和0.021,P值分别为0.547和0.869).CD患者和UC患者外周血单个核细胞CD27和CD70 mRNA表达均显著高于正常对照者外周血单个核细胞中mRNA的表达(P值均=0.000).免疫组化实验表明CD患者和UC患者肠黏膜组织CD27和CD70的表达均显著高于正常对照者(P值均=0.000).结论 炎症性肠病患者血浆、外周血单个核细胞和肠黏膜中均存在CD27-CD70途径的激活,但血浆中CD27-CD70的水平不能反映内镜下疾病活动程度.选择性干预CD27-CD70共刺激通路可能成为缓解或减轻炎症性肠病进程的有益尝试.     

Increased activation of isolated intestinal lamina propria mononuclear cells in inflammatory bowel disease

Normal human lamina propria lymphocytes are in a heightened state of activation compared with peripheral blood with regard to cell-surface activation antigen expression (transferrin receptor, interleukin-2 receptor, 4F2) and the increased spontaneous secretion of immunoglobulins in vitro. This study evaluates the cell-surface expression of activation-associated antigens in different subpopulations of isolated colonic lamina propria mononuclear cells in inflammatory bowel disease. In pilot studies using three-color flow cytometry, autofluorescence was observed that was emitted by unstained lamina propria mononuclear cells, which interfered with both the sensitivity and the specificity of the analyses. Because a major portion of the intestinal lymphocyte populations of interest were autofluorescent, a method to remove autofluorescence signals was developed by designing a computer program for the subtraction of autofluorescence from the emissions of each individual cell. This technique increases both the sensitivity and specificity of flow-cytometric analyses of intestinal lamina propria mononuclear cells. Using fluorescence-activated cell-sorter analyses with subtraction of autofluorescence on a single-cell basis, increased expression of lymphocyte activation antigens (interleukin-2 receptor, transferrin receptor, 4F2) was found on the cell surface of isolated intestinal B cells, T cells, CD4+ T cells, and CD8+ T cells in both Crohn's disease and ulcerative colitis. Therefore, markedly increased intestinal lymphocyte activation is a major immunological alteration in inflammatory bowel disease and includes all lymphocyte subpopulations investigated in this study. In addition, 5-aminosalicylic acid, which is used for the treatment of intestinal inflammation in inflammatory bowel disease, inhibits the expression of cell-surface activation antigens on mitogen-activated peripheral blood lymphocytes in a dose-dependent manner. These observations suggest that lymphocyte activation may play an important role in underlying immune processes that lead to chronicity and perpetuation of inflammatory bowel disease and may implicate an additional mechanism for the therapeutic action of 5-aminosalicylic acid.     

Lamina propria T cells in Crohn's disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis

BACKGROUND & AIMS: Normal human lamina propria lymphocytes manifest increased unstimulated apoptosis compared with peripheral lymphocytes, which are enhanced after stimulation via the CD2 activation pathway. This activation-induced apoptosis down-regulates cell expansion and cytokine production. In previous studies, it was shown that lamina propria T cells from patients with Crohn's disease and ulcerative colitis manifest abnormal proliferation and cytokine production. It was therefore of interest to determine if such cells also showed abnormal patterns of apoptosis. METHODS: Apoptosis was evaluated by propidium iodide staining of cells followed by flow cytometric analysis. Fas expression and Bcl-2 levels in cells were evaluated by immunofluorescence. RESULTS: Lamina propria lymphocytes from patients with Crohn's disease and ulcerative colitis as well as from 2 patients with diverticulitis showed defective CD2 pathway-induced apoptosis. Studies of the mechanisms of this defect focusing on cells from patients with Crohn's disease showed that Crohn's disease lamina propria lymphocytes from inflamed tissues express the same amount of cell surface Fas but are less sensitive to Fas-mediated apoptosis than control cells. In addition, lamina propria lymphocytes from inflamed Crohn's disease tissues manifest increased expression of Bcl-2 after CD2 pathway stimulation and elevated Bcl-2 levels in cultures of unstimulated T cells. CONCLUSIONS: T cells isolated from areas of inflammation in Crohn's disease, ulcerative colitis, and other inflammatory states manifest decreased CD2 pathway-induced apoptosis. Studies of cells from inflamed Crohn's disease tissue indicate that this defect is accompanied by elevated Bcl-2 levels. These changes are probably caused by the chronic inflammation and may aggravate the underlying disease processes that are present.     

Deficiency of Invariant NK T Cells in Crohn's Disease and Ulcerative Colitis

The aim of this study was to investigate whether immunoregulatory invariant NK T cells are deficient in Crohn's disease or ulcerative colitis. Blood was collected for flow cytometry from 106 Crohn's disease, 91 ulcerative colitis, and 155 control subjects. Invariant NK T cells were assessed by Vα24 and (α-galactosylceramide/CD1d tetramer markers. Intracellular cytokine was measured after in vitro anti-CD3 antibody stimulation. Vα24+ T cells were quantified in ileocolonic biopsies as mRNA by real-time PCR and by immunofluorescence. Circulating invariant NK T cells were 5.3% of the control levels in Crohn's (P < 0.001) and 7.9% of the control levels in ulcerative colitis (P < 0.001). Interleukin-4 production was impaired in Crohn's disease and ulcerative colitis. Intestinal Vα24 mRNA expression was 7% in Crohn's disease (P < 0.05) and 9% in ulcerative colitis (P < 0.05). Intestinal Vα24+ T cells were 23% in Crohn's disease but not reduced in ulcerative colitis. We conclude that invariant NK T cells are deficient in Crohn's disease and in ulcerative colitis.     

The proportion of CD40+ mucosal macrophages is increased in inflammatory bowel disease whereas CD40 ligand (CD154)+ T cells are relatively decreased, suggesting differential modulation of these costimulatory molecules in human gut lamina propria

BACKGROUND: Signal transduction through binding of CD40 on antigen-presenting cells and CD40 ligand (CD154) on T cells appears to be crucial for mutual cellular activation. Antibodies aimed at blocking the CD40-CD154 costimulatory pathway dampen the severity of experimental colitis. To elucidate the microanatomical basis for signaling through this costimulatory pathway in human inflammatory bowel disease, we studied in situ the cellular distribution of these 2 molecules on lamina propria macrophages and T cells, respectively. METHODS: Colonic specimens from 8 patients with ulcerative colitis and 8 with Crohn's disease, 8 small bowel specimens of Crohn's disease, and histologically normal control samples (6 from colon and 6 from small bowel) were included. Multicolor immunofluorescence in situ staining was performed to determine the percentage of subepithelial macrophages expressing CD40 and that of lamina propria T cells expressing CD154 while avoiding cells in lymphoid aggregates. RESULTS: The proportion of subepithelial CD40CD68 macrophages was significantly increased in normal colon compared with normal small bowel and showed further elevation in both colon and small bowel afflicted with inflammatory bowel disease. In addition, on a per-CD68-cell basis, CD40 expression was significantly increased in severely inflamed compared with moderately inflamed colonic specimens. Conversely, the proportion of CD154 T cells was similar in colon and small bowel, and interestingly, it was significantly reduced in colonic inflammatory bowel disease. CONCLUSIONS: Our findings suggested that modulation of CD40 expression by subepithelial macrophages and CD154 by lamina propria T cells is inversely modulated in the human gut.