Effect of human intestinal macrophages on immunoglobulin production by human intestinal mononuclear cells isolated from patients with inflammatory bowel disease.

The effect of macrophages on spontaneous immunoglobulin production by isolated human intestinal mononuclear cells (MNC) is unknown. Depletion of macrophages by adherence to fibronectin or by panning with macrophage-specific monoclonal antibody 3C10 lead to a significant reduction in IgA. IgG and IgM production by intestinal MNC from both normal (n = 10) and inflammatory bowel disease (IBD) (n = 13) mucosa. The reduction in immunoglobulin produced by macrophage-depleted intestinal MNC was greater in IBD patients than in normal controls. There was a significant correlation (r = 0.816, P less than 0.001) between the percentage of macrophages depleted by panning with 3C10 and the reduction in IgG produced by macrophage-depleted intestinal MNC. Addition of either fibronectin-adherent cells or the supernatant from these macrophage-enriched cells enhanced immunoglobulin production in a dose-dependent fashion. A greater increase in IgG production by macrophage-depleted cells was seen when cultured with supernatant from inflamed IBD mucosal cells, compared with that from normal mucosal cells. The soluble factor(s) responsible in the supernatant was acid and heat susceptible but was not affected by freezing and thawing. Addition of recombinant human interleukin-1 beta or human interferon-gamma to cell cultures did not influence immunoglobulin production. Thus, human intestinal macrophages enhance spontaneous immunoglobulin production by isolated intestinal MNC by secreting soluble factor(s) which remain to be fully characterized.     

Gene expression in mononuclear cells from patients with inflammatory bowel disease

OBJECTIVES: Discovery of Nod2 as the inflammatory bowel disease 1 (IBD1) susceptibility gene has brought to light the significance of mononuclear cells in inflammatory bowel disease pathogenesis. The purpose of this study was to examine changes in gene expression in peripheral blood mononuclear cells in patients with untreated Crohn's disease (CD) and ulcerative colitis (UC) as compared to patients with other inflammatory gastrointestinal disorders and to healthy controls. METHODS: We used a 2400 gene cDNA glass slide array (MICROMAX) to examine gene expression in peripheral blood mononuclear cells from seven patients with Crohn's disease, five patients with ulcerative colitis, 10 patients with other inflammatory gastrointestinal disorders, and 22 age- and sex-matched controls. Results. Novel categories of genes differentially expressed in Crohn's disease and ulcerative colitis patients included genes regulating hematopoietic cell differentiation and leukemogenesis, lipid raft-associated signaling, the actin cytoskeleton, and vesicular trafficking. Conclusions: Altered gene expression in mononuclear cells may contribute to inflammatory bowel disease pathogenesis.     

Characterization of antigen-presenting activity of intestinal mononuclear cells isolated from normal and inflammatory bowel disease colon and ileum.

Antigen-presenting activity in mononuclear cells, isolated from normal and inflamed human ileum and colon, has been characterized using allogeneic mixed lymphocyte reaction with resting T cells as responders. Greatest proliferation was induced by fibronectin-adherent (macrophage-enriched) cells, and least by fibronectin non-adherent (macrophage-depleted) cells and by mononuclear cells depleted of macrophages by panning with monoclonal antibody 3C10. When intestinal mononuclear cells and allogeneic T cells were incubated in large numbers, clusters were observed. These clusters contained cells with a dendritic morphology that were strongly HLA-D-positive and which also stained with macrophage-specific monoclonal antibodies 3C10, EMB11 and Y1/82A. These cells were closely associated with proliferating T cells. Studies comparing mononuclear cells isolated from normal and inflamed colonic mucosa suggest that the latter may have enhanced antigen-presenting capacity.     

Interleukin-2 stimulation of blood mononuclear cells from patients with inflammatory bowel disease

The functional capacity of biologically active, high-affinity interleukin-2 receptors (IL-2R) was studied by means of interleukin-2 (IL-2) stimulation of blood mononuclear cells (BMC) from 22 patients with inflammatory bowel disease (IBD) and 24 controls. The spontaneous, as well as the IL-2-induced, proliferative responses were significantly decreased in patients with active IBD, whereas the expressions of biologically inactive, low-affinity IL-2R (i.e. TAC antigen or CD25) were significantly increased in the same BMC cultures. In contrast, no significant differences were seen between patients and controls when BMC were stimulated with a nonspecific mitogen (phytohemagglutinin). The results suggest that a downregulation of IL-2 responsiveness may contribute to decreased BMC proliferation in vitro in active IBD.     

In vitro immunoglobulin secretion by normal human gastrointestinal mucosal tissues, and alterations in patients with inflammatory bowel disease.

Intestinal mucosal immunoglobulin secretion in vitro has been studied by culture of endoscopic biopsy tissues obtained from various sites along the gastrointestinal tract. IgA and IgM were the predominant immunoglobulins produced by intestinal tissues distal to the stomach and secretion of both reached a maximum in the small intestine of normal individuals. In patients with inflammatory bowel disease, characteristic alterations in immunoglobulin production were observed in cultures of large intestinal tissue. In ulcerative colitis (UC), significant reductions in IgA secretion (P less than 0.03) occurred in the rectum during remission and IgM in the colon in active disease (P less than 0.01). However, in active Crohn's disease (CD), rectal IgM secretion was enhanced (P less than 0.004) and IgA diminished (P less than 0.01). IgG secretion was increased throughout the colon in UC especially in distal sites, due largely to significant increases in pre-formed immunoglobulin (P less than 0.02). Similar total increases were observed in colonic tissues from patients with CD, although IgG synthesis in biopsies from rectal sites was normal. These findings suggest that specific abnormalities of intestinal mucosal immunoglobulin synthesis occur in patients with both UC and CD.     

Spontaneous secretion of IgG subclasses by intestinal mononuclear cells: differences between ulcerative colitis, Crohn's disease, and controls

Spontaneous IgG and IgG subclass secretion patterns by isolated intestinal mononuclear cells (MNC) from control and inflammatory bowel disease (IBD) specimens were examined. Intestinal MNC from IBD specimens spontaneously secreted more total IgG than did control intestinal MNC. This increased spontaneous IgG secretion by ulcerative colitis intestinal MNC was primarily due to markedly increased production of IgG1. Slightly increased secretion of IgG3, but not IgG2 by ulcerative colitis intestinal MNC was present when compared with control and Crohn's disease intestinal MNC. In contrast, Crohn's disease intestinal MNC exhibited increased spontaneous secretion of all the IgG subclasses examined, with IgG2 being predominant.     

Chronic inflammatory bowel disease--increased plasminogen activator secretion by mononuclear phagocytes.

The secretion of the neutral protease plasminogen activator (PA) by cultured macrophages (M phi) was studied in hospitalized patients suffering from chronic inflammatory bowel disease (IBD). There was markedly enhanced secretion of PA by M phi derived from circulating monocytes of the IBD population (18) compared to an age-matched population (16) which was not afflicted by intestinal disease (P less than 0 . 001). Mean M phi PA activity was greater in the population of 11 Crohn's disease patients (P less than 0 . 01) than in a group of seven ulcerative colitis sufferers (P less than 0 . 05) when compared to the control population. While both the treated and untreated hospitalized IBD populations showed increased M phi PA specific activity, results for the nine untreated patients (5 . 56 +/- 1.14 units/micrograms M phi DNA) were substantially higher than those found in the treated IBD population (2 . 91 +/- 0 . 62 units/micrograms M phi DNA) (P less than 0 . 01). These findings reflect the activity of M phi in IBD and suggest a means by which tissue injury is mediated in these conditions.     

Increased Toll-like receptor 9 expression by B cells from inflammatory bowel disease patients

Background

Toll-like receptors (TLRs) are important mediators of the innate immune response. Our aim was to evaluate TLR9 expression in peripheral B cells, taken from inflammatory bowel disease (IBD) patients before and after anti-inflammatory treatment. Nineteen patients with IBD (12-crohn’s disease, 7-ulcerative colitis) and 18 healthy controls were included in the study. Disease severity was assessed using the Pediatric/Adults crohn’s disease activity index and the ulcerative colitis activity index as needed. Accordingly, patients were classified as mild, moderate or severe disease. Peripheral B cells isolated from IBD patients, before and after anti-inflammatory treatment, and from the control group, were cultured for 24 h with and without CpG oligodeoxynucleotides (ODN-CpG) 0.5 μM. TLR9 expression by memory B cells (CD19+CD27+) was assessed by flow cytometry.

Results

We found that TLR9 expression by peripheral B cells was significantly higher in IBD patients than that in healthy controls (12.42 ± 9.5 MFI vs. 6.0 ± 2.6 MFI p = 0.02). The addition of ODN-CpG to B cells resulted in a significantly increase of TLR9 expression in B cells from healthy controls (6.5 ± 3.2 MFI vs. 8.8 ± 4.2 MFI p = 0.007). On the contrary, B cells from IBD patients only partly respond to the addition of ODN-CpG after anti-inflammatory treatment (6.3 ± 3.8 vs. 7.3 ± 3.7, p = 0.1). TLR9 expression was positively correlated with IBD disease severity (r = 0.681, p < 0.0001).

Conclusions

TLR9 expression in memory B from IBD patients is elevated and associated with disease severity.     

Increased spontaneous secretion of rheumatoid factor by intestinal lamina propria mononuclear cells from Crohn's disease but not ulcerative colitis patients.

Increased levels of rheumatoid factors (RF) have been observed in the serum of Crohn's disease but not ulcerative colitis patients, and have been proposed to relate to an increased state of intestinal lymphocyte activation. We have therefore examined the spontaneous in vitro secretion of RF by intestinal lamina propria mononuclear cells (MNC) isolated from specimens from control and inflammatory bowel disease (Crohn's disease, ulcerative colitis) patients. Normal intestinal lamina propria MNC spontaneously secrete rheumatoid factors of different isotypes during 14 days of in vitro culture (9.7 ng/ml IgA RF, 11.6 ng/ml IgM RF and 64.6 ng/ml IgA anti-Fc (IgG)). In matched studies intestinal MNC isolated from normal large bowel exhibited significantly greater levels of RF synthesis and secretion in vitro than normal small bowel intestinal MNC. A large increase in spontaneous RF secretion was observed from Crohn's disease intestinal MNC (21.4 ng/ml IgA RF, 21.4 ng/ml IgM RF, and 108.15 ng/ml IgA anti-Fc (IgG)), when compared with normal controls. The amount of RF secreted was dependent on the amount of inflammatory activity of the bowel specimens, from which the MNC were isolated (198.3 ng/ml of IgA anti-Fc(IgG) from involved versus 50.0 ng/ml from matched non-involved tissue). Ulcerative colitis MNC released decreased amounts of RF (7.1 ng/ml IgA RF, 6.2 ng/ml IgM RF, and 42.3 ng/ml IgA anti-Fc(IgG)). These observations using isolated intestinal MNC may explain the findings of RF changes in the sera of inflammatory bowel disease patients. Our observations support the hypothesis of a heightened state of activation in normal intestinal lamina propria MNC, which is further increased in active Crohn's disease. The dissimilarities observed between Crohn's disease and ulcerative colitis may indicate fundamental differences in disease pathophysiology and will lead to further studies exploring intestinal immunoregulatory properties of RF.     

Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease

Previous studies on the frequency of intestinal mast cells and eosinophils in patients with inflammatory bowel disease yielded conflicting results. In the present morphometric study, we quantified mast cells and eosinophils in the lamina propria by histological and immunohistochemical methods in 64 patients suffering from Crohn's disease (33 cases) or ulcerative colitis (31 cases), and in 29 controls. Histological data from 206 biopsies were related to the presence of mucosal inflammation and clinical parameters. The number of eosinophils was increased in patients with inflammatory bowel conditions (mean ±  SE : 331 ± 44/mm²) as compared to controls (258 ± 27/mm²), and was dependent on disease activity and drug treatment. Mean mast cell numbers did not differ between patients and controls. However, a reduced mast cell number was found in toluidine blue-stained sections of actively inflamed tissue areas (143 ± 16/mm², versus 206 ± 18/mm² in non-inflamed tissue). Immunohistochemical studies using antibodies against the granule proteins tryptase and chymase suggest that this decrease in mast cell numbers is due to mast cell degranulation. The present data show that the number of intestinal mast cells and eosinophils is altered in patients with inflammatory bowel diseases, suggesting that both cell types are involved in the pathogenesis of chronic intestinal inflammation.     

Immunoglobulin production by isolated intestinal mononuclear cells from patients with ulcerative colitis and Crohn''s disease.

We studied immunoglobulin production by isolated intestinal mononuclear cells from 25 patients with active inflammatory bowel disease (IBD) and 17 controls undergoing surgical resections for intestinal tumour or other disorders. Normal ileal intestinal mononuclear cells spontaneously produced greater amounts of IgA and IgM than did normal colon cells. In cells from patients with IBD there was a significantly reduced IgA production, but production of IgG was enhanced in both colon and ileum. The alteration in IgA and IgG production in IBD was confirmed by comparing the immunoglobulin production by mononuclear cells from inflamed with that from non-inflamed areas of mucosa in six patients with distal ulcerative colitis. The proportion of IgA-containing cells in isolated intestinal mononuclear cells from IBD mucosa was less than normal. However, the proportion of IgG-containing cells from IBD mucosa was not increased in isolated intestinal mononuclear cells although they produced more IgG than normal mucosal cells. Our study showed an altered pattern of immunoglobulin production by intestinal mononuclear cells isolated from patients with inflammatory bowel disease.     

Association between intestinal microbiota and inflammatory bowel disease

Inflammatory bowel disease (IBD), which includes Crohn''s disease (CD) and ulcerative colitis (UC), has emerged as a global disease with high incidence, long duration, devastating clinical symptoms, and low curability (relapsing immune response and barrier function defects). Mounting studies have been performed to investigate its pathogenesis to provide an ever‐expanding arsenal of therapeutic options, while the precise etiology of IBD is not completely understood yet. Recent advances in high‐throughput sequencing methods and animal models have provided new insights into the association between intestinal microbiota and IBD. In general, dysbiosis characterized by an imbalanced microbiota has been widely recognized as a pathology of IBD. However, intestinal microbiota alterations represent the cause or result of IBD process remains unclear. Therefore, more evidences are needed to identify the precise role of intestinal microbiota in the pathogenesis of IBD. Herein, this review aims to outline the current knowledge of commonly used, chemically induced, and infectious mouse models, gut microbiota alteration and how it contributes to IBD, and dysregulated metabolite production links to IBD pathogenesis.     

Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease

Lin Z, Hegarty JP, Cappel JA, Yu W, Chen X, Faber P, Wang Y, Kelly AA, Poritz LS, Peterson BZ, Schreiber S, Fan J‐B, Koltun WA. Identification of disease‐associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease. Overwhelming evidence supports the theory that inflammatory bowel disease (IBD) is caused by a complex interplay between genetic predispositions of multiple genes, combined with an abnormal interaction with environmental factors. It is becoming apparent that epigenetic factors can have a significant contribution in the pathogenesis of disease. Changes in the methylation state of IBD‐associated genes could significantly alter levels of gene expression, potentially contributing to disease onset and progression. We have explored the role of DNA methylation in IBD pathogenesis. DNA methylation profiles (1505 CpG sites of 807 genes) of matched diseased (n = 26) and non‐diseased (n = 26) intestinal tissues from 26 patients with IBD [Crohn's disease (CD) n = 9, ulcerative colitis (UC) n = 17] were profiled using the GoldenGate? methylation assay. After an initial identification of a panel of 50 differentially methylated CpG sites from a training set (14 non‐diseased and 14 diseased tissues) and subsequent validation with a testing set (12 non‐diseased and 12 diseased tissues), we identified seven CpG sites that are differentially methylated in intestinal tissues of IBD patients. We have also identified changes in DNA methylation associated with the two major IBD subtypes, CD and UC. This study reports IBD‐associated changes in DNA methylation in intestinal tissue, which may be disease subtype‐specific.     

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.     

帕金森病人外周血单个核细胞分泌IL-2的实验研究

目的 探讨帕金森病病人中外周血单个核细胞(PBMC)分泌IL-2的能力.方法 采用酶联免疫吸附法(ELISA)分别检测体外培养的PBMC上清液、及PBMC接受刀豆蛋白(ConA)刺激后上清液中IL-2含量.结果 PD Ⅰ期患者中未受刺激的PBMC分泌的IL-2显著高于PDⅡ-Ⅲ期患者(P＜0.05),接受ConA刺激后,以PD Ⅱ-Ⅲ期患者的PBMC生成IL-2最低,显著低于正常对照组(P＜0.05),刺激后同刺激前相比各组IL-2都有升高,但仅健康对照组刺激前后有显著差异(P＜0.05).结论 帕金森病患者的外周血单个核细胞的分泌IL-2能力存在异常.     

Kinetics of cytokine secretion by mononuclear cells of the blood from rheumatoid arthritis patients are different from those of healthy controls.

Mononuclear cells from peripheral blood (PBMC) of rheumatoid arthritis (RA) patients and healthy controls were incubated with alpha-CD3. Cytokine secretion from 2 h to 72 h of incubation was measured by ELISA. There were no significant differences in secretion of T cell derived IL-2 and IL-4 in cultures from RA patients and controls. The macrophage-derived cytokines, IL-1 beta and tumour-necrosis factor-alpha (TNF-alpha) were secreted with a steep increase of concentration during the first 16 h of incubation by PBMC from RA patients. PBMC from healthy controls secreted both cytokines at a constantly rising rate with a maximum for TNF-alpha at 48 h and for IL-1 beta at 72 h. Interferon-gamma (IFN-gamma) is secreted in significantly reduced concentrations by PBMC from untreated RA patients compared with controls. Gold-salt treatment led to a slightly delayed and enhanced secretion of TNF-alpha and IL-1 beta, an enhanced secretion of IL-2 and a restored secretion of IFN-gamma.     

Immunopathology of human inflammatory bowel disease

Conclusions Mucosal pIgA, and thereby secretory immunity, are disfavored in IBD as revealed by decreased J-chain expression and strikingly increased IgG production by the mucosal immunocytes in both UC and CD. Moreover, a significant shift from IgA2 to the less-resistant IgA1 subclass takes place. Preferential overproduction of IgG1 is seen in UC; and apical deposits of this isotype on the surface epithelium, together with activated complement, is suggestive of a cytotoxic attack related to a brushborder antigen. Comparison of identical twins, discordant with regard to clinical expression of UC, further suggests that this IgG1 response is genetically determined. The IBD lesions furthermore contain many recently recruited and activated T cells as well as monocyte-like macrophages (L1⁺CD14⁺) with elevated capacity for production of proinflammatory cytokines (IL-1 and TNF-) and ROM. Together all these changes reflect less restricted extravasation of precursor cells because of altered expression of adhesion molecules on the microvasculature. Isolated and cultured intestinal endothelial cells subjected to proinflammatory cytokines show enhanced ICAM-1 expression as well as induction of functional E-selectin and MHC class II molecules with antigen-presenting properties.The immunopathology of IBD thus involves severely altered mucosal homeostasis, apparently reflecting break of tolerance to the indigenous microbial flora, and in addition it appears to include an autoimmune component in UC. The origin of abrogated immunological tolerance at the mucosal level may be alterations both in leukocyte extravasation and in antigen-presenting mechanisms induced by aberrant expression of endothelial and epithelial MHC class II molecules, as well as changed profiles of co-stimulatory molecules on macrophages. A shift from B7.2 on mucosal resident APC to B7.1 on the recently recruited macrophages, may particularly disfavor Th2 immune responses and their associated down-regulatory cytokines. In conclusion, perturbation of a tightly controlled cytokine network with abnormal crosstalk between several mucosal cell types, is probably the first step of a progressive immunopathological development in IBD, but the initiation of this series of events remains undefined.