Flow cytometric measurement of intracellular migration inhibition factor and tumour necrosis factor alpha in the mucosa of patients with coeliac disease

There is increasing evidence that proinflammatory cytokines contribute to many of the small intestinal features in coeliac disease. The aim of the study was to investigate the expression of two proinflammatory cytokines, migration inhibition factor (MIF) and tumour necrosis factor alpha (TNF-alpha) in duodenal biopsy specimens from patients with coeliac disease on a gluten-free diet and normal control subjects. A flow cytometric system was used to analyse intracellular protein levels of MIF and TNF-alpha in freshly isolated cells from duodenal biopsies taken from 12 patients with treated coeliac disease and 10 healthy control subjects. From the biopsy specimens, single cell suspensions of the epithelium and lamina propria were prepared using EDTA/DTT and enzymes. Intracellular cytokine expression was studied in intraepithelial lymphocytes (IELs), lamina propria T cells (LP T) and intestinal epithelial cells using different surface labelling antibodies. MIF protein was constitutively expressed in IELs, LP T cells and epithelial cells from normal intestinal mucosa. In contrast, although TNF-alpha was found in LP T cells, this cytokine was virtually undetectable in either IELs or epithelial cells. In coeliac disease, intracellular levels of MIF were significantly higher in epithelial cells compared with control subjects (P = 0.005). Raised levels of TNF-alpha were found in epithelial cells (P = 0.03) as well as IELs (P = 0.045) from coeliac patients compared with controls. The findings from this study show up-regulated expression of MIF and TNF-alpha in IELs and epithelial cells of histologically normal mucosa in patients with coeliac disease. Increased expression of proinflammatory cytokines in cells occupying the epithelial layer could help explain the rapidity with which the coeliac mucosa may respond to gluten challenge.     

Jejuna of patients with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine.     

The proportion of Th1 cells, which prevail in gut mucosa, is decreased in inflammatory bowel syndrome

T lymphocytes and their cytokines have an important role in the regulation of immune responses in the gut and in the pathogenesis of intestinal inflammation such as in Crohn's disease. The aim of this study was to analyse the Th1/Th2 cytokine profile (IFN-gamma, IL-2, IL-4 and IL-10) in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) in Crohn's disease (CD) and ulcerative colitis (UC) in relation to healthy controls (C). Colonic and ileal biopsy specimens were obtained from controls (n = 13) and patients with CD (n = 32). Colonic biopsies were obtained from patients with UC (n = 11). Intracytoplasmic IFN-gamma, IL-2, IL-4 and IL-10 were determined by flow cytometry after PMA-ionomycin stimulation in IEL and LPL. In colonic LPL, a significant proportional decrease of IFN-gamma and IL-2 producing CD3+ cells was observed in patients with CD and UC compared to controls. In ileal LPL, a similar tendency was found although differences were not significant. In IEL no differences in cytokine profiles could be observed. Flow cytometric analysis of intracytoplasmic cytokines at single cell level showed a proportional decrease of IFN-gamma and IL-2 producing T cells in colonic lamina propria in patients with inflammatory bowel disease.     

In vitro activated CD4+ T cells from interferon-gamma (IFN-gamma)-deficient mice induce intestinal inflammation in immunodeficient hosts.

To investigate the role of IFN-gamma in the immunopathogenesis of inflammatory bowel disease (IBD), severe combined immunodeficient (SCID) mice were transplanted with in vitro activated CD4+ T cells from either wild-type (WT) or IFN-gamma-deficient (IFN-gammaKO) BALB/c mice. In vitro, the two types of T cells displayed comparable proliferation rates and production of tumour necrosis factor-alpha (TNF-alpha), IL-2, IL-4 and IL-10 after concanavalin A (Con A) stimulation. When transplanted into SCID mice, WT CD4+ blasts induced a lethal IBD, whereas IFN-gammaKO blasts induced a less severe intestinal inflammation with moderate weight loss. Intracellular cytokine staining of lamina propria lymphocytes (LPL) revealed comparable fractions of CD4+ T cells positive for TNF-alpha, IL-2 and IL-10 in the two groups of transplanted SCID mice, whereas a two-to-three-fold increase in the fraction of IL-4-positive cells was found in IFN-gammaKO-transplanted SCID mice. Flow cytometric analyses showed strong up-regulation of MHC class II expression of colonic epithelial cells of WT-CD4+ T cell-transplanted compared with IFN-gammaKO-transplanted SCID mice. A significantly higher fraction of CD4+ LPL were found to enter the cell cycle, i.e. to incorporate bromo-deoxy-uridine, and to undergo apoptosis in vivo in WT-transplanted compared with IFN-gammaKO-transplanted SCID mice. These data point towards an important role for IFN-gamma in the development of IBD in SCID mice. The inflammation might be initiated and subsequently enhanced by the ability of IFN-gamma to induce de novo MHC class II expression in the colonic epithelium, a change which could lead to increased antigen processing and production of local proinflammatory cytokines, CD4+ T cell turnover and thereby to exaggeration of disease.     

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.     

Increase in T‐Cell Subsets of Oral Mucosa: a Late Immune Response in Patients with Treated Coeliac Disease?*

BACKGROUND AND AIMS: In coeliac disease, the gut involvement is gluten-dependent. Following the introduction of a gluten-free diet, inflammatory cell infiltration decreases in the small intestinal mucosa. Our hypothesis was that the oral mucosa might mirror the changes found in coeliac disease similarly to the mucosa of the small intestine. Thus, the number of inflammatory cells in the oral mucosa would decrease in patients with coeliac disease on a gluten-free diet. METHODS: The distribution CD45RO+ and CD3(+) T cells, T-cell subpopulations (CD4(+), CD8(+), T-cell receptor (TCR)alpha beta+ and TCR gamma delta+ cells) and HLA DR expression were studied in the buccal mucosa of 15 untreated and 44 gluten-free diet treated coeliac disease patients, and of 19 controls. All 15 patients with untreated coeliac disease were immunglobulin (Ig)A endomysial antibody positive and all 44 patients on gluten-free diet except one were endomysial antibody negative, as were all control subjects. RESULTS: Untreated coeliac disease patients did not differ from controls in the densities of CD45RO+ cells, CD3(+) cells or of T-cell subsets. In contrast, in treated coeliac disease patients, a significant increase in the numbers of mast cells, CD3(+) and CD4(+) lymphocytes was found in the lamina propria of oral mucosa as compared with patients with untreated coeliac disease and controls. The increase in CD3(+) T cells was in part owing to an increase in lymphocytes expressing no TCR. No differences were found in the expression of human leucocyte antigen (HLA) DR in the epithelium or in the lamina propria in the patient groups studied or in the controls. In treated coeliac disease patients only a few TCR gamma delta+ T cells were found intraepithelially and in the lamina propria, but these cells were not detected in the lamina propria of oral mucosa of patients with untreated coeliac disease or in the controls. CONCLUSIONS: The infiltration of T cells into oral mucosa was increased in treated coeliac disease patients in spite of adherence to a gluten-free diet. Because the CD3(+) T cell count was higher than those of the TCR alpha beta+ and TCR gamma delta+ T cells, there must be other cells involved, probably natural killer (NK) cells. The increase in T-cell subsets in the treated coeliac disease patients seems not to result from poor dietary compliance, but might occur as a late immune response in coeliac disease and reflect chronic immunologic stimulation followed by regeneration of memory T cells.     

Detection of endogenous cytokines in sera or in lymph nodes obtained from patients with sarcoidosis.

To investigate the possible role of endogenous cytokines in the immunopathogenesis of sarcoidosis, a study of cytokines in lymph nodes obtained from patients with active pulmonary sarcoidosis was carried out using immunoperoxidase staining and radioimmunoassays (RIA). Cells stained for interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), which appeared to be CD3+ cells and CD14+ cells, respectively, were seen scattered around granulomas. In contrast, cells positive for interleukin-1 beta (IL-1 beta) were located in CD11c+ cells within granulomas. Lymph nodes of patients with sarcoidosis contained significantly higher amounts of IFN-gamma, TNF-alpha and IL-1 beta than control lymph nodes. The levels of IFN-gamma and TNF-alpha in extracts of lymph nodes were correlated with the histological status of the granulomas. IFN-gamma was detected in all stages, while the highest level of TNF-alpha was obtained from lymph nodes containing many active granulomas. The level of serum IFN-gamma was always lower than in lymph node extract and correlated significantly with either serum angiotensin-converting enzyme or serum lysozyme. Patients with positive gallium-67 uptake or bilateral hilar lymphadenopathy had high levels of either serum IFN-gamma or lysozyme. These results suggest that quantitative analyses of IFN-gamma and TNF-alpha in sera and lymph nodes may serve to elucidate the pathophysiology of sarcoidosis.     

Immunohistologic assessment of cytokine production of infiltrating cells in various forms of leprosy.

The aim of this study was to determine cytokines in human leprosy lesions by means of immunohistologic examination. Cryostat sections of skin biopsies from 57 patients with various forms of leprosy were immunostained according to the APAAP method, using monoclonal antibodies against interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and, in addition, against CD 1 antigen. Granulomas in biopsies of untreated patients with tuberculoid leprosy showed large amounts of cells positive for IL-1 beta, TNF-alpha, IFN-gamma, and CD 1, whereas no positive signals could be detected in untreated patients with lepromatous leprosy. However, in those biopsies obtained from lepromatous leprosy patients undergoing chemotherapy, positive staining for cytokines as well as subepidermal Langerhans cells increased to a detectable amount. Remarkably, in tuberculoid leprosy patients, the number of IL-1 beta--positive cells did not vary under therapy, while the number of TNF-alpha and IFN-gamma reactive cells decreased. These results suggest that immunohistologic determination of cytokines in combination with the assessment of subepidermal Langerhans cells in human leprosy lesions may be used as a parameter for the patient's status of cell-mediated immunity under chemotherapeutic treatment.     

Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-1-alpha-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-alpha-producing cells were T lymphocytes (CD3+), while the TNF-alpha-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P<0.001) than that seen in the healthy control animal, while TNF-alpha- and IL-1-alpha-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.     

Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF

BACKGROUND: We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. METHODS: Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RESULTS: RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. CONCLUSIONS: The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.     

Decreased lamina propria effector cell responsiveness to interleukin-10 in ileal Crohn's disease.

We investigated whether a lack of IL-10 production or responsiveness could be involved in Crohn's disease pathogenesis. Lamina propria mononuclear cells, isolated from the ilea of Crohn's disease patients (n = 16) and controls (n = 13), were activated with anti-CD3 mAb in the presence of CD80 transfectants or LPS +/- IFN-gamma. No evidence for deficient IL-10 production by either T cells or macrophages in Crohn's disease was found. However, the efficacy of rhIL-10 to down-regulate IFN-gamma and especially TNF production in cell cultures from the involved tissues of Crohn's disease patients was poor, and the use of an anti-IL-10R mAb even provided evidence for proinflammatory effects of IL-10. This lack of IL-10 effect possibly results from IL-12 activity. We conclude that IL-10 exhibits poor anti- and even potential proinflammatory effects on ileal Crohn's disease lamina propria. These data might explain the lack of therapeutic efficacy when IL-10 is given to Crohn's disease patients.     

Distribution of IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas

In order to examine the immune response at the site of pathology in tuberculosis, we analysed cytokines present in lung granulomas, their associations with each other and with caseous necrosis as well as the phenotype of the cellular infiltrate. Paraffin-embedded tissue from the lungs of seven patients with pulmonary tuberculosis was analysed by immunohistochemistry and in situ hybridization to detect interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) proteins and IL-12p40 mRNA. All seven patients had granulomas staining positive for IFN-gamma, TNF-alpha and IL-12p40, but only four stained positive for IL-4. Cells with the morphology of lymphocytes, macrophages and giant cells expressed TNF-alpha, IFN-gamma and IL-4 protein. Furthermore, CD68-positive myeloid cells expressed IL-12p40 mRNA, as expected, but a subset of CD3-positive lymphocytes also expressed this mRNA. These lymphocytes producing IL-12p40 also stained positive for CD8 but not CD4. A total of 141 granulomas were scored for the presence or absence of cytokine or necrosis and two major associations were identified. The first association was between IFN-gamma and IL-12, with 76% of granulomas staining positive for both cytokines. Unexpectedly, those granulomas positive for IL-4 were always positive for IFN-gamma. The second association was between TNF-alpha and caseous necrosis, where all necrotic granulomas were TNF-alpha positive. This association was modulated by IL-4. Therefore, heterogeneity of cellular infiltrate and cytokine expression is observed between adjacent granulomas in the same patient.     

Transforming growth factor-beta (TGF-beta) and tissue transglutaminase expression in the small intestine in children with coeliac disease

The production of cytokines from T cells and macrophages is of potential importance for the histological changes apparent in coeliac disease (CoD). Small intestinal biopsy specimens from children with CoD and disease control subjects were investigated for their content of cytokines and tissue transglutaminase (tTG). The transforming growth factor-beta1 (TGF-beta1) expression was increased in the lamina propria of children with villous atrophy. In contrast, TGF-beta3 was expressed at a higher level in the epithelium and the lamina propria of the disease control subjects. The tTG expression was increased in the small intestine of CoD patients as compared with that in subjects. Interleukin-4 (IL-4) was detected in the lamina propria of both CoD patients and controls, and some of the investigated biopsy specimens also showed IL-4 expression in the epithelium. We conclude that children with active CoD could have an altered expression of TGF-beta and tTG in the small intestine and that a disturbed regulation of TGF-beta may be of importance in the immune pathogenesis of CoD.     

High TNF-alpha and low IL-2 producing T cells characterize active disease in Takayasu's arteritis

We have investigated intracellular production by T cells and plasma levels of TNF-alpha, IL-2 and IFN-gamma in 12 active and 10 inactive Takayasu's arteritis (TA) patients and 12 healthy controls. The active TA compared to inactive TA and controls had higher TNF-alpha (52.7 +/- 22.3% vs. 32.9 +/- 14.2% and 35.2 +/- 14.5%, respectively; P = 0. 020), lower IL-2 (19.6 +/- 13.2% vs. 36.1 +/- 10.1% and 31.2 +/- 10.3%, respectively; P = 0.010) and comparable IFN-gamma (38.6 +/- 13.9% vs. 34.2 +/- 12.4% and 34.9 +/- 11.1%, respectively; P = 0.581) producing CD3+ T cells. There was no difference in the plasma levels of the cytokines between active TA, inactive TA and controls (TNF-alpha: 79.1 +/- 94.5 vs. 72.9 +/- 120.0 and 9.5 +/- 6.7 pg/ml, P = 0.110; IL-2: 4.3 +/- 4.8 vs. 6.6 +/- 4.7 and 8.6 +/- 4.5 pg/ml, P = 0.094 and IFN-gamma: 10.1 +/- 11.3 vs. 8.8 +/- 8.7 and 8.2 +/- 6.5 pg/ml, P = 0.871, respectively). The data show an important role of these high TNF-alpha and low IL-2 producing T cells in TA.     

Localization of IFN-gamma and IL-6 mRNA in murine intestine by in situ hybridization.

Previous studies have highlighted the importance of CD4+ T cells in regulation of IgA responses and have indicated a functional heterogeneity among these cells between inductive (Peyer's patch) and effector (lamina propria) sites in the intestine. To determine whether these functional differences could be accounted for by differences in cytokine profile of cells in each of these sites, the distribution of mRNA for interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) was investigated by in situ hybridization techniques using 35S-labelled riboprobes. Whereas message for IL-6 is abundant in all regions of the lamina propria from the base of the mucosa to the tips of the villi, very little is expressed in Peyer's patches or in the epithelium. In contrast, message for IFN-gamma is expressed predominantly by cells localized only in the base of the lamina propria and, as with IL-6, very little message was detected in Peyer's patches although occasional strongly positive IFN-gamma cells were observed in the epithelium. These results indicate that, at least in the absence of deliberate intestinal stimulation, functional expression of these cytokines is restricted to effector rather than induction sites in the intestine. This is consistent with our previous observations demonstrating a requirement for T-cell signals in promoting post-extravasation differentiation and proliferation of IgA-committed B cells in vivo and the implications of these findings to the role of the Th1 and Th2 subsets of CD4+ cells in mucosal immune responses is discussed.     

In situ production of gamma interferon, interleukin-4, and tumor necrosis factor alpha mRNA in human lung tuberculous granulomas

Human tuberculous granulomas from five adults undergoing surgery for hemoptysis were analyzed by nonradioactive in situ hybridization for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and interleukin-4 (IL-4) gene expression. All of the patients produced TNF-alpha mRNA. Three patients stained positive for both IFN-gamma and IL-4 mRNA; the other two stained positive for IFN-gamma but not IL-4 mRNA. Heterogeneity between the granulomas was observed in those patients staining positive for both IFN-gamma and IL-4 mRNA; these patients exhibited granulomas having IFN-gamma and not IL-4 mRNA as well as granulomas positive for both cytokine mRNAs. There was no evidence of caseation in these granulomas, and the cytokine patterns may represent events in the evolution of the granuloma. However, in those granulomas exhibiting caseous necrosis, very little IFN-gamma or IL-4 mRNA was observed, implying that progression of the granuloma is accompanied by a down regulation of T-cell responses. TNF-alpha mRNA expression was highest in patients with both IFN-gamma and IL-4 mRNA. Populations of CD68 positive macrophage-like cells within the granulomas produce mRNA for TNF-alpha, IFN-gamma, and IL-4. This implies that macrophages within the tuberculous granuloma may not be dependent on T-cell cytokines for modulation of their function but may be able to regulate their own activation state and that of the surrounding T cells. These findings have implications on the delivery of immunotherapies to patients with tuberculosis.     

Differential upregulation of intercellular adhesion molecule-1 in coeliac disease.

The expression of intercellular adhesion molecule-1 (ICAM-1) was studied on peroral jejunal biopsies from patients with coeliac disease. The biopsies from untreated patients exhibited greater staining of the superficial lamina propria cells compared with treated patients and controls. A gluten challenge in treated patients produced an altered staining pattern within 2 h. The results demonstrate the role of ICAM-1 expression in coeliac disease, providing further evidence for the role of lamina propria cells in the pathogenesis of this condition.