Epithelial expression of interleukin‐37b in inflammatory bowel disease

Interleukin (IL)‐37 is a member of the IL‐1 cytokine family. We investigated IL‐37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL‐37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL‐37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL‐37b mRNA and protein expression were determined by real time‐polymerase chain reaction (PCR) and Western blotting, respectively. IL‐37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL‐37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL‐37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL‐37b mRNA and protein was enhanced by tumour necrosis factor (TNF)‐α. This IL‐37b induction by TNF‐α was mediated by nuclear factor (NF)‐κB and activator protein (AP)‐1 activation. Furthermore, IL‐37b inhibited TNF‐α‐induced interferon‐γ‐inducible protein (IP)‐10 expression significantly in human colonic SEMFs. Epithelial IL‐37b expression was increased in IBD patients, especially UC patients. IL‐37b may be involved in the pathophysiology of IBD as an anti‐inflammatory cytokine and an inhibitor of both innate and acquired immune responses.     

Expression of Toll‐like receptor‐3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2

Anti‐microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti‐microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in‐situ hybridization. Moreover, we examined the regulation of LCN2 in HT‐29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll‐like receptor (TLR)‐3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up‐regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de‐novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT‐29 was enhanced by both interleukin (IL)‐1β and the TLR‐3 ligand poly(I:C), and TLR‐3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.     

HLA-D region antigens on isolated human colonic epithelial cells: enhanced expression in inflammatory bowel disease and in vitro induction by different stimuli.

Colonic epithelial cells (CEC) were isolated from actively inflamed mucosa of IBD patients and checked for HLA-DR, HLA-DP, and HLA-DQ. Half of the freshly isolated CEC from IBD tissue expressed DR, and one third were positive for DP and DQ. Normal human CEC were then cultured for 24 h and their expression of these markers in response to different types of in vitro stimulation was investigated. A significant increase in the expression of DR, DP and DQ was observed in response to the nonspecific mitogen phytohaemagglutinin (PHA), the lymphokine gamma-interferon (gamma-IFN) and the epidermal growth factor (EGF). The enhancement of DR expression was more marked than that of DP and DQ. The effect of gamma-IFN was more rapid and significantly more marked than that of either PHA and EGF for all three antigens. EGF appeared to be more potent than PHA in enhancing the expression of DP and DQ. The data from this study indicate that HLA-D region antigens can be induced on human CEC by different types of stimuli and provide further evidence that the expression of these markers in the colonic epithelium is a normal event the magnitude of which can increase under various circumstances. The data also suggest that the increased expression of HLA-D region antigens by IBD CEC occurs as a result of different mechanisms, and that this expression is an indicator of the active participation of the colonic epithelium to the mucosal inflammatory response.     

Influence of LPS, TNF, TGF-ß1 and IL-4 on the expression of MMPs, TIMPs and selected cytokines in rat synovial membranes incubated in vitro

Synovial membranes are formed by four main types of cells, i.e. fibroblasts, macrophages, epitheliocytes and adipocytes. To study the combined effect of various factors on these cell populations, synovial membranes dissected from rat knee joints were incubated in control medium or medium with lipopolysaccharide (LPS), TNF, TGF-?1 or IL-4 for 12 h. LPS stimulated TNF secretion and both agents stimulated secretion of IL-6. TGF-?1 slightly increased IL-6 secretion. LPS increased the mRNA levels of IL-6, IL-1?, TGF-?1, MMP1a, MMP1b, MMP3, MMP9, MMP13, MMP14, TIMP1 and TIMP3 while the mRNA levels of MMP2, TIMP2 and TIMP4 were significantly decreased. Expression of IL-1?, MMP1a, MMP1b, MMP3, MMP9, MMP13 and TIMP1 increased after TNF treatment, while mRNA levels of MMP2, MMP14, TIMP2, TIMP3 and TIMP4 were decreased. TGF-?1 decreased the mRNA levels of IL-1?, all MMPs, TIMP1, TIMP2, TIMP4 and increased mRNA levels of itself and TIMP3. IL-4 decreased mRNA levels of IL-1?, TGF-?1, MMP2, MMP9, MMP13 and all TIMPs. Only LPS decreased the amount and activity of MMP2. The effect of LPS and cytokines on most of the MMPs and TIMPs produced by whole synovial membrane was in good agreement with previous studies on their action on similar types of cells as those present in synovial membranes, but originating from other tissues. All tested agents decreased MMP2 mRNA expression levels and in the case of LPS also the protein level and its activity determined by zymography, contrary to previous observations on isolated cell populations. This indicates that the response of the organized tissue is an interplay of all components and cannot be deduced from the individual reactions.     

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)‐α, a known agonist of the mitogen‐activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho‐p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex‐ and age‐matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF‐α, interleukin (IL)‐1β and IL‐6 were measured in the organ and cell culture supernatants by enzyme‐linked immunosorbent assay. We found higher levels of phospho‐p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF‐α, IL‐1β and IL‐6 from IBD LPMCs and biopsies. Activated p38α MAPK is up‐regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down‐regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.     

5氟尿嘧啶上调干细胞标记物CD133在结肠癌细胞中的表达

目的: 研究通过5氟尿嘧啶(5-FU)对结肠癌干细胞标记物CD133表达的影响,探讨5-FU对结肠癌干细胞的影响。方法: 用流式细胞仪检测CD133在结肠癌细胞株表面的表达, 磁珠细胞分离的方法分离结肠癌细胞株DLD1中CD133阳性和阴性的细胞群,细胞克隆形成实验检测2群细胞的自我更新能力,新型四唑氮盐方法(MTS)检测2群细胞对5-FU敏感性的差异,qPCR方法检测5-FU处理结肠癌细胞后CD133mRNA水平的变化。结果: 结肠癌细胞株DLD1、HT29、SW480、HCT116、Lovo、RKO细胞表面CD133的表达率分别为30.20%、82.00%、0.34%、91.80%、85.30%、0.28%。DLD1细胞中以CD133为标记有2群明显的细胞,MACS方法分离后阳性细胞群中CD133为87.21%±5.33%, 而阴性细胞群中阴性细胞的比例为84.30%±4.65%;CD133阳性的细胞与未分离及CD133阴性细胞相比,克隆形成能力强(46.33%±4.44% vs 31.00%±2.00%,P<0.05),对5-FU的敏感性下降20%,P<0.01。在DLD1和HT29细胞中,5-FU 1 mg/L上调CD133mRNA水平的表达,从1升为1.684±0.012(P<0.01)、HT29细胞从30.702±0.284升为49.379±0.460(P<0.01)。结论: 与CD133阴性细胞相比CD133阳性细胞克隆形成能力强,对5-FU的敏感性下降;5-FU上调干细胞标记物CD133mRNA水平的表达,CD133阳性的结肠癌干细胞在5-FU的治疗过程中被富集。     

E-cadherin expression in intestinal epithelium.

AIMS--To investigate E-cadherin expression in normal and inflamed intestine, in the colonic adenocarcinoma cell line HT29, in normal fetal intestine, and in a fetal gut organ culture model where a T cell mediated enteropathy can be generated; to determine whether expression of E-cadherin changes in intestinal inflammation. METHODS--Immunohistochemistry was used to determine E-cadherin expression in following tissues: frozen and paraffin wax sections of normal and inflamed intestine; HT29 colonic adenocarcinoma cell line cultured on coverslips in the presence or absence of cytokines; frozen sections of fetal small intestinal tissue (gestational age 11-22 weeks); and frozen sections of cultured fetal gut in which a T cell mediated enteropathy had been induced. RESULTS--E-cadherin was strongly and evenly expressed by the epithelium in all specimens of intestine studied. Although there was no change in inflammation generally, in some cases of Crohn's disease groups of glands with the characteristic morphology of "ulcer associated cell lineage" showed lower expression of E-cadherin. In fetal gut organ cultures epithelial expression of E-cadherin was lower when local T cells were activated with mitogens, compared with control explants. By contrast, the HT29 cell line showed low levels of expression which increased after treatment with conditioned medium from activated tonsil cells. CONCLUSIONS--E-cadherin is strongly and evenly expressed by epithelium in normal and inflamed intestine, although an increase in E-cadherin expression in cytokine treated HT29 cells was observed. E-cadherin expression is reduced in the epithelium adjacent to ulcers (ulcer associated cell lineage), possibly to assist regeneration.     

Hypoxia inducible factor‐1α‐induced interleukin‐33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor‐1α (HIF‐1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC‐derived HIF‐1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF‐1α and IL‐33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)?33 were correlated positively with that of HIF‐1α. A HIF‐1α‐interacting element was identified in the promoter region of IL‐33, indicating that HIF‐1α activity regulates IL‐33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF‐1α‐dependent IL‐33 expression in IEC. Our data thus demonstrate that HIF‐1α‐dependent IL‐33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis.     

Increased Expression of CCL20 in Human Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) constituting Crohn's disease (CD) and ulcerative colitis (UC) is related to a dysregulated T cell response. CCL20 attracts memory T lymphocytes and dendritic cells. We asked whether CCL20 expression is altered in IBD. Colonic biopsies were obtained from 114 subjects with IBD, non-IBD colitis, irritable bowel syndrome, and healthy controls. CCL20 and CCR6 mRNA expression was measured by Taqman-PCR, and protein secretion from colonic explant cultures (CEC) and its regulation by TNF-alpha by ELISA. CCL20, CCR6, and Langerin were identified by immunohistochemistry and immunofluorescence. CCL20 mRNA and protein were severalfold increased in involved CD and UC but not in non-IBD colitis. TNF-alpha increased and anti-TNF-alpha decreased CCL20 release in healthy control CEC but not in involved IBD colonic specimens. CCL20 localized to follicle-associated epithelium, and CCR6 to the adjacent mantle zone of lymphoid follicles. Furthermore, abundant numbers of Langerin(+) immature dendritic cells were identified in the subepithelial space of IBD specimens. CCL20 might regulate the attraction of T lymphocytes and dendritic cells in IBD.     

E1AF expression is associated with extra‐prostatic growth and matrix metalloproteinase‐7 expression in prostate cancer

E1AF is associated with malignant aggressiveness via regulation of matrix metalloproteinases (MMPs), which play pivotal roles in invasion through the degradation of extracellular matrix of tissues surrounding tumors. However, the clinical significance of E1AF and MMPs in patients with prostate cancer is not fully understood. We reviewed 50 tissue samples from patients with T2‐3N0M0 prostate cancer who had undergone radical operation. Expression levels of E1AF, MMP‐1, ‐3, ‐7, ‐9 and ‐14 were determined semiquantitatively by immunohistochemistry. The mean ± SD percentage of E1AF‐stained cancer cells was 8.56 ± 5.22, and it was significantly higher (p < 0.001) than the E1AF‐immunostaining index of normal cells (1.17 ± 0.61). E1AF immunostaining index in pT3 (12.74 ± 4.80) was significantly higher (p < 0.001) than that in pT2 (5.78 ± 3.31). Although E1AF expression correlated with that of MMP‐7 and MMP‐9 (r = 0.47, p < 0.001 and r = 0.41, p = 0.004, respectively), multivariate analysis showed that E1AF correlated with only MMP‐7 expression (OR = 5.81, 95% CI = 1.27–26.59, p = 0.023). Our results demonstrated that increased expression of E1AF is involved in tumor aggression of prostate cancer. This finding may be influenced by regulation of MMP‐7. We speculate that E1AF is a possible target in treatment and prevention of tumor growth in prostate cancer.     

Study of matrix metalloproteinases and their tissue inhibitors in ductal in situ carcinomas of the breast

Aims: To analyse the expression of metalloproteinases (MMPs) and their inhibitors (TIMPs) in ductal carcinoma in situ of the breast (DCIS). Methods and results: An immunohistochemical study was performed in 56 patients with pure DCIS, in 39 with DCIS adjacent to invasive carcinoma (IDC) and 63 patients with T1 IDC, using tissue microarrays and specific antibodies against MMPs and TIMPs. Immunohistochemical results were categorized using a specific software program. The data were analysed by unsupervised hierarchical cluster analysis by each cellular type. IDC showed a higher expression rate of MMP‐7 and TIMP‐1 than pure DCIS, as well as a higher expression rate of MMP‐9 and TIMP‐3 than the DCIS component of mixed cases, whereas pure DCIS showed a higher rate of expression of MMP‐9 and ‐11 and TIMP‐3 than in the DCIS component of mixed cases. Pure DCIS with a periductal inflammatory infiltrate showed significantly higher MMP‐2, ‐14 and TIMP‐1. Dendograms identified two cluster groups with distinct MMP/TIMP expression profiles in neoplastic cells and fibroblastic or mononuclear inflammatory cells surrounding the neoplastic ducts of pure DCIS. Conclusions: The results indicate the distinct variability in MMP/TIMP expression by DCIS, which may be of potential biological and clinical interest in breast cancer.     

The effect of gender and genetic polymorphisms on matrix metalloprotease (MMP) and tissue inhibitor (TIMP) plasma levels in different infectious and non‐infectious conditions

Matrix metalloproteases (MMPs) are increased in different infections due to their role in controlling immune responses and are regulated by tissue inhibitors (TIMPs). Different MMP promoter single nucleotide polymorphisms (SNPs) induce changes in MMP genes, mRNA and protein expression. Gender might also modify MMP plasma levels. In order to determine the weight of these variables on MMP secretion we studied MMP‐1, ‐2, ‐3, ‐8, ‐9, ‐10, ‐13 and TIMP‐1, ‐2, ‐4 plasma levels in 90 patients with severe bacterial sepsis, 102 with anti‐retroviral (ARV)‐treated HIV monoinfection, 111 with ARV‐treated HIV–hepatitis C virus (HCV) co‐infection and 86 non‐infected controls (45 stroke and 41 trauma patients). MMP‐1(‐1607 1G/2G), MMP‐3(‐1612 5A/6A), MMP‐8(‐799C/T), MMP‐9(‐1562 C/T) and MMP‐13(‐77A/G) SNPs were genotyped. MMP‐3 plasma levels were significantly higher in men than in women in each diagnostic group, and MMP‐3 SNP allele 6A carriers also had higher levels than allele 5A carriers, an effect that was magnified by sepsis. Independent predictors of higher MMP‐3 levels were male gender (P = 0·0001), MMP‐3(‐1612 5A/6A) SNP (P = 0·001), higher levels of TIMP‐4 (P = 0·004) and MMP‐8 (P = 0·006) and lower levels of MMP‐1 (P = 0·03) by multivariate analysis. No strong associations with gender or SNPs were observed for other MMPs or TIMPs. In conclusion, male gender and MMP‐3(‐1612 5A/6A) 6A allele carriage increased MMP‐3 plasma levels significantly, especially in patients with severe bacterial sepsis. This confounding gender effect needs to be addressed when evaluating MMP‐3 plasma levels in any infectious or non‐infectious condition.     

Comparative analysis and clinical value of the expression of metalloproteases and their inhibitors by intratumour stromal mononuclear inflammatory cells and those at the invasive front of breast carcinomas

González L O, González‐Reyes S, Marín L, González L, González J M, Lamelas M L, Merino A M, Rodríguez E, Pidal I, del Casar J M, Andicoechea A & Vizoso F
(2010) Histopathology 57, 862–876 Comparative analysis and clinical value of the expression of metalloproteases and their inhibitors by intratumour stromal mononuclear inflammatory cells and those at the invasive front of breast carcinomas Aims: Matrix metalloproteases (MMPs) and their inhibitors (TIMPs) play an essential role in the degradation of stromal connective tissue and basement membrane components. The aim of this study was to determine whether the dynamic analysis of these components can help to predict tumour aggressiveness. Methods and results: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs ‐1, ‐2, ‐7, ‐9, ‐11, ‐13 and ‐14 and TIMPs ‐1, ‐2 and ‐3. More than 5000 determinations on cancer specimens from 124 patients with invasive breast cancer were performed on the tumour centre core as well as on the invasive front. Immunostaining for MMPs/TIMPs on mononuclear inflammatory cells (MICs) was evaluated. To identify specific groups of tumours with distinct expression profiles, data obtained from both MICs populations were analysed by unsupervised hierarchical cluster analysis. When compared with MICs at the invasive front, intratumour MICs more frequently showed expression of MMP‐7 and ‐1 and TIMP‐3, but less frequently expression of MMP‐9 and ‐11 and TIMP‐2. Conclusions: Our data led us to consider the need of further studies in order to identify subsets of MICs and other protein elements of the microenvironment as attractive targets for new therapeutic strategies against cancer.     

两类金属蛋白酶在炎性细胞模型中的变化及中药RDQ的天然抑制效应

目的探讨金属蛋白酶(MPs)家族的MMPs和ADAMs两亚类成分在前体TNF-α(mTNF-α)向成熟型TNF-α(sTNF-α)转化过程所起的作用,MMPs在炎症过程中活性的变化和中药RDQ抗炎保护功效的分子机制,为人工干预炎症过程提供依据和手段。方法以LPS刺激HL-60细胞为天然模型,用细胞学和分子生物学技术在蛋白质和mRNA水平上研究MMPs、ADAM17、TNF-α在炎症过程中的表达和活性变化。结果①LPS刺激后,HL60细胞上清中MMPs的活性随刺激时间延长逐渐减弱,胞浆裂解物中的MMPs则随刺激时间延长活性增加但LPS刺激对MMPs mRNA的表达影响不大;②LPS刺激能使HL-60细胞中ADAM17mRNA和TNF-αmRNA表达在6h同时达高峰;③RDQ能明显降低LPS诱导的ADAM17 mR- NA的增加,并能抑制sTNF-α的分泌。结论①ADAM17对sTNF-α的产生起主导作用;②纯中药注射液热毒清(RDQ)能调节TACE的表达,最终抑制TNF-α的分泌。     

Matrix metalloproteinases,TIMPs and growth factors regulating ameloblastoma behaviour

Siqueira A S, Carvalho M R D, Monteiro A C D, Freitas V M, Jaeger R G & Pinheiro J J V.
(2010) Histopathology 57 , 128–137
Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour Aims: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness ¹ . The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. Methods and results: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non‐invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP‐9 and TIMP‐2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)‐α and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP‐1, ‐2 and EGFR. We found a positive correlation between EGF and TIMP‐1, and between TGF‐α and TIMP‐2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. Conclusions: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.     

Matrix metalloproteinases contribute to the regulation of chemokine expression and pulmonary inflammation in Cryptococcus infection

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that play roles in regulating the immune response in inflammatory processes. Previous studies indicated that different MMPs were involved in the host defence and tissue damage in response to different pathogens. However, the contributions of MMPs during Cryptococcus infection have not been addressed clearly. Here, we examined the expression and activity of MMPs during Cryptococcus infection. Among MMP family members, we found significant increases of MMP‐3 and MMP‐12 mRNA levels and MMP12 zymographic activities in response to C. neoformans but not C. gattii infection. The expression of MMP12 was induced in RAW cells after C. neoformans treatment and in alveolar macrophages purified from C. neoformans‐infected mice. Interestingly, administration of MMP inhibitor GM6001 into C. neoformans‐infected mice resulted in a significantly increased pulmonary fungal burden with attenuated inflammatory cell infiltration. Corresponding to this finding, the expression of the macrophage‐ and neutrophil‐attracting chemokines CCL2 and CXCL1 was inhibited in the GM6001‐treated group and MMP12 levels were found to be correlated strongly with CCL2 mRNA expression. Thus, our data suggest that the induction of MMPs by C. neoformans infection potentiates inflammatory cell infiltration by modulating pulmonary chemokines, thereby promoting effective host immunity to pulmonary Cryptococcus infection.     

Intestinal oxidative damage in inflammatory bowel disease: semi-quantification,localization, and association with mucosal antioxidants

Intestinal inflammation is accompanied by excessive production of reactive oxygen and nitrogen metabolites. In order to counteract their harmful effects, the intestinal mucosa contains an extensive system of antioxidants. It has previously been shown that the levels of and the balance between the most important antioxidants are seriously impaired within the intestinal mucosa from inflammatory bowel disease (IBD) patients compared with normal mucosa. The present study investigated the consequences of this antioxidative imbalance by evaluating parameters of oxidative stress-related mucosal damage in the same tissue samples. The extent of apoptosis, peroxynitrite-mediated protein nitration (3-NT), and lipid peroxidation were assessed in relation to the expression of nitric oxide synthase (NOS) and the superoxide-producing enzyme xanthine oxidase (XO). In addition, bi- and multi-variate regression analyses were performed to associate these parameters with the levels of the antioxidants assessed previously. Apoptotic cell death was visualized by TUNEL staining in luminal epithelium of normal controls, and in IBD additionally in the inflammatory infiltrate and in deeper parts of the crypts, but its frequency was unrelated to the severity of inflammation. In Crohn's disease (CD), epithelial apoptosis levels were strongly associated with the expression of XO, implying a role for this enzyme in the regulation of epithelial cell homeostasis, although its levels were unaffected by intestinal inflammation and were comparable to those in normal control mucosa. 3-NT immunoreactivity was substantially increased in luminal crypt cells, neutrophils, and mononuclear cells in the inflamed mucosa of ulcerative colitis (UC) patients. The inflamed IBD luminal epithelium, but not the inflammatory cells, also contained increased amounts of NOS. The immunoreactivity of both 3-NT and NOS was significantly higher in UC than in CD. Unexpectedly, the increased 3-NT expression in UC was associated with neutrophilic myeloperoxidase and not with NOS, which suggests that 3-NT is formed in areas with a dense neutrophilic infiltrate via a peroxynitrite-independent oxidation pathway. Lipid peroxidation, as estimated by the malondialdehyde (MDA) concentration, was elevated in both the inflamed CD and the inflamed UC mucosa, and was identified in the luminal epithelium using a histochemical technique. In CD, lipid peroxidation was independently associated with the concentration of metallothionein and with Mn-superoxide dismutase activity, suggesting the involvement of hydroxyl radicals and superoxide anions. In UC, however, the amount of MDA was associated with epithelial catalase expression and neutrophilic myeloperoxidase activity, suggesting a hydrogen peroxide- and/or hypochlorous acid-mediated mechanism. The present study underlines the importance of oxidative stress in the pathogenesis of IBD and provides clues regarding the (anti)oxidants involved which indicate that this process evolves through diverging pathways in CD and UC.