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1.
Sci Rep ; 7(1): 2805, 2017 06 05.
Article in English | MEDLINE | ID: mdl-28584241

ABSTRACT

γδT cells provide immune-surveillance and host defense against infection and cancer. Surprisingly, functional details of γδT cell antimicrobial immunity to infection remain largely unexplored. Limited data suggests that γδT cells can phagocytose particles and act as professional antigen-presenting cells (pAPC). These potential functions, however, remain controversial. To better understand γδT cell-bacterial interactions, an ex vivo co-culture model of human peripheral blood mononuclear cell (PBMC) responses to Escherichia coli was employed. Vγ9Vδ2 cells underwent rapid T cell receptor (TCR)-dependent proliferation and functional transition from cytotoxic, inflammatory cytokine immunity, to cell expansion with diminished cytokine but increased costimulatory molecule expression, and capacity for professional phagocytosis. Phagocytosis was augmented by IgG opsonization, and inhibited by TCR-blockade, suggesting a licensing interaction involving the TCR and FcγR. Vγ9Vδ2 cells displayed potent cytotoxicity through TCR-dependent and independent mechanisms. We conclude that γδT cells transition from early inflammatory cytotoxic killers to myeloid-like APC in response to infectious stimuli.


Subject(s)
Cytokines/metabolism , Escherichia coli/immunology , Phagocytes/microbiology , Phagocytes/physiology , Phagocytosis/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , B7-2 Antigen/metabolism , HLA-DR Antigens/metabolism , Humans , Immunoglobulin G/immunology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Phenotype , T-Lymphocytes/drug effects , Th1 Cells/immunology , Th1 Cells/metabolism , Zoledronic Acid/pharmacology
2.
Cell Microbiol ; 18(1): 66-79, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26153406

ABSTRACT

Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally.


Subject(s)
Activating Transcription Factor 2/metabolism , E-Selectin/metabolism , Endothelial Cells/microbiology , Endothelial Cells/physiology , Host-Pathogen Interactions , Neisseria meningitidis/physiology , Cells, Cultured , Endotoxins/metabolism , Humans
3.
Inflamm Bowel Dis ; 17(5): 1201-8, 2011 May.
Article in English | MEDLINE | ID: mdl-21484962

ABSTRACT

BACKGROUND: Human ex vivo evidence indicating that an inappropriate immune response(s) to nonpathogenic bacteria contributes to disease pathogenesis in pediatric Crohn's disease (CD) is limited. The aim of the present study was to compare and contrast the early innate immune response of pediatric "healthy" versus CD mucosa to pathogenic, probiotic, and commensal bacteria. METHODS: "Healthy control" and CD pediatric mucosal biopsies (terminal ileum and transverse colon) were cocultured for 8 hours with E. coli O42, Lactobacillus GG (LGG), Bacteroidesthetaiotaomicron (B. theta), or stimulated with interleukin (IL)-1ß (positive control). Matched nonstimulated biopsies served as experimental controls. IL-8 was the immune marker of choice. IL-8 mRNA and protein levels were quantified by quantitative polymerase chain reaction and sandwich enzyme-linked immunosorbent assay, respectively. RESULTS: IL-8 secretion was observed when control, ileal biopsies were exposed to pathogenic O42 and probiotic LGG, with no response noted to commensal B. theta. In comparison, Crohn's ileal biopsies showed impaired ability to induce IL-8 in response to O42 and LGG. Control colonic tissue showed a limited response to O42 or B. theta and LGG significantly reduced IL-8 secretion. Unlike control tissue, however, Crohn's ileal and colonic tissue did respond to B. theta, with more enhanced expression in the colon. CONCLUSIONS: We provide the first ex vivo data to support the notion that aberrant mucosal recognition of commensal bacteria may contribute to pediatric CD. While IL-8 responses to O42 and LGG varied with disease status and anatomical location, B. theta consistently induced significant IL-8 both in ileal and colonic CD tissue, which was not seen in control, healthy tissue.


Subject(s)
Bacteroides/immunology , Crohn Disease/immunology , Crohn Disease/microbiology , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Biopsy , Child , Colon/immunology , Colon/microbiology , Colon/pathology , Crohn Disease/pathology , Gene Expression/drug effects , Gene Expression/immunology , Humans , Interleukin-1beta/immunology , Interleukin-1beta/pharmacology , Interleukin-8/genetics , Interleukin-8/immunology , Intestinal Mucosa/pathology , Metagenome/immunology , Organ Culture Techniques , Probiotics
4.
J Clin Pathol ; 63(7): 592-4, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20501449

ABSTRACT

AIM: To review the gastrointestinal mucosal histological features of biopsies from children with Shwachman-Diamond syndrome (SDS) examined at a single specialist centre. METHODS: Search of a clinical database was performed to identify SDS cases and their gastrointestinal biopsies were reviewed for morphological parameters such as crypt:villous ratio, crypt hyperplasia and abnormal inflammatory infiltrates. Histological sections were also immunostained with CD4, CD20 and HLA-DR to determine the nature of the inflammatory infiltrate. RESULTS: 15 SDS cases were included, 7 (47%) of which showed morphologically normal duodenal villous architecture, whereas 8 (53%) showed varying degrees of enteropathic histological features ranging from villous blunting to partial villous atrophy and duodenitis. 11/15 (73%) showed some degree of duodenal inflammation, including increased lamina propria density of plasma cells, macrophages and eosinophils. CONCLUSION: Varying degrees of duodenal inflammatory enteropathic features are present in more than 50% of symptomatic children with SDS. This suggests that, in addition to pure pancreatic exocrine failure, an enteropathic component may contribute to symptoms in some cases, and be potentially responsive to appropriate therapy.


Subject(s)
Duodenum/pathology , Atrophy , Biopsy , Bone Marrow Diseases/immunology , Bone Marrow Diseases/pathology , CD4-Positive T-Lymphocytes/immunology , Child , Duodenitis/immunology , Duodenitis/pathology , Duodenoscopy , Duodenum/immunology , Exocrine Pancreatic Insufficiency/immunology , Exocrine Pancreatic Insufficiency/pathology , HLA-DR Antigens/metabolism , Humans , Immunity, Mucosal , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Lipomatosis , Shwachman-Diamond Syndrome
5.
Clin Exp Immunol ; 156(2): 263-70, 2009 May.
Article in English | MEDLINE | ID: mdl-19386083

ABSTRACT

In the small intestine members of both the alpha-defensin (DEFA5 and DEFA6) and beta-defensin (DEFB1 and DEFB2) family contribute to the anti-microbial barrier against infection. The aim of this study was to determine whether Staphylococcal enterotoxin B (SEB)-mediated immune activation and proinflammatory cytokines play a role in the regulation of intestinal defensin expression. Defensin mRNA and peptide secretion was studied after ex vivo tissue culture of duodenal biopsies over 24 h. Immune (T cell and macrophage) activation was induced by SEB, and in separate experiments exogenous proinflammatory cytokines were added individually. Defensin mRNA levels were quantified by reverse transcription-polymerase chain reaction, and peptide release into culture supernatants was quantified by immuno dot blot or enzyme-linked immunosorbent assay. Increasing concentrations of SEB down-regulated DEFA5, DEFA6 and DEFB1 mRNA in a dose-dependent manner but increased DEFB2 simultaneously. The down-regulation of alpha-defensins was reversed by dexamethasone. DEFA5 and DEFB2 peptide secretion levels were altered in parallel with mRNA. Interferon-gamma and interleukin (IL)-1beta exhibited a dose-dependent down-regulation of alpha-defensin mRNA, IL-6 significantly down-regulated only DEFA6; in contrast, tumour necrosis factor-alpha and IL-4 had no significant effect. Immune cell activation and proinflammatory cytokines down-regulated the constitutively expressed DEFA5, DEFA6 and DEFB1 defensins, and up-regulated DEFB2 in intact human intestinal tissue explants in short-term culture. The effect of local immune activation on innate defence may explain the reduced alpha-defensin expression noted in inflammatory T cell-mediated enteropathies.


Subject(s)
Defensins/metabolism , Duodenum , Enterotoxins/pharmacology , Intestinal Mucosa/metabolism , Staphylococcal Infections/metabolism , Superantigens/pharmacology , Adult , Defensins/genetics , Dexamethasone/pharmacology , Duodenitis/immunology , Duodenitis/microbiology , Enterotoxins/metabolism , Gene Expression/drug effects , Humans , Immunosuppressive Agents/pharmacology , RNA, Messenger/analysis , Staphylococcus aureus , Statistics, Nonparametric , Superantigens/metabolism , Tissue Culture Techniques , alpha-Defensins/genetics , alpha-Defensins/metabolism , beta-Defensins/genetics , beta-Defensins/metabolism
6.
Parasite Immunol ; 31(1): 2-9, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19121078

ABSTRACT

The microsporidian Encephalitozoon intestinalis develops within intestinal epithelial cells (enterocytes) and is an important opportunistic diarrhoeal pathogen associated with AIDS. Little is known about the protective immune response against the parasite although in mice IFN-gamma is involved and is required to prevent dissemination of the infection to other organs. The present study was designed to establish a suitable short-term in vitro culture technique for E. intestinalis that would enable studies of the role of cytokines such as IFN-gamma in the effector phase of immunity. Encephalitozoon intestinalis reproduced considerably better in the murine enterocyte cell line CMT-93 than in the three human enterocyte cell lines Caco-2, HT29 and HCT-8. Treatment of CMT-93 cells with IFN-gamma significantly reduced parasite reproduction in a dose- and time-dependent manner. IFN-gamma also inhibited development of the parasite in Caco-2 cells. Neither production of NO nor Fe deprivation appeared to be involved in IFN-gamma-mediated parasite killing. However studies suggested that tryptophan catabolism by indoleamine 2,3-dioxygenase played an important part in inactivation of E. intestinalis.


Subject(s)
Encephalitozoon/immunology , Enterocytes/immunology , Enterocytes/parasitology , Interferon-gamma/immunology , Animals , Cell Line , Cell Survival , Encephalitozoon/growth & development , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Iron/metabolism , Mice , Nitric Oxide/metabolism , Tryptophan/metabolism
7.
Clin Exp Immunol ; 145(3): 555-62, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16907926

ABSTRACT

Accumulating evidence suggests that intestinal epithelial cells (IECs) constitutively express the immunoregulatory cytokine interleukin (IL)-18. IECs also serve as the host cell for the intracellular parasitic protozoan Cryptosporidium parvum. In the present study, C. parvum infection of a human enterocyte cell-line HCT-8 resulted in increased expression of IL-18 mRNA as measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). IL-18 protein was detected in control uninfected cells and following infection there was increased expression as measured by enzyme-linked immunosorbent assay (ELISA). Gene expression revealed the presence of the IL-18 receptor subunits not only in cell-lines but also in freshly isolated IECs, suggesting that IL-18-mediated signalling events may contribute to epithelial host defence during infection. Recombinant IL-18 inhibited intracellular development of the parasite in HCT-8 and HT-29 cells. Increased expression of bactericidal antibiotic peptides LL-37 and alpha-defensin 2 by IL-18 in HCT-8 and HT-29 cells may represent one mode of action by which this pluripotent cytokine aids in limiting the development of intracellular pathogens such as C. parvum in the gastrointestinal tract.


Subject(s)
Cryptosporidiosis/immunology , Cryptosporidium parvum/physiology , Enterocytes/immunology , Enterocytes/microbiology , Interleukin-18/physiology , Animals , Anti-Bacterial Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Case-Control Studies , Cell Line , Cryptosporidiosis/metabolism , Cryptosporidium parvum/drug effects , HT29 Cells , Humans , Interleukin-18/genetics , Interleukin-18 Receptor alpha Subunit , RNA, Messenger/analysis , Receptors, Interleukin/metabolism , Receptors, Interleukin-18 , Recombinant Proteins/pharmacology , alpha-Defensins/metabolism , Cathelicidins
8.
Br J Haematol ; 129(6): 713-22, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15952996

ABSTRACT

Chemotherapy-induced neutropenia increases the risk of infection. There appears to be a wide variability in the severity and length of infective episodes. Susceptibility to infections is determined by the underlying malignant disease and its treatment, environmental factors (e.g. nutritional state of the patient and hygiene) and genetically determined variations of the immune system. The majority of primary immunodeficiencies are rare (c. frequency one in 10 000), whereas some genetic polymorphisms in the innate immune system, such as profound mannose-binding lectin deficiency, are much more common (c. frequency one in 10). Here, we review the potential role of the innate immune system in determining susceptibility to infections in patients with neutropenia.


Subject(s)
Neutropenia/immunology , Opportunistic Infections/immunology , Antineoplastic Agents/adverse effects , Genetic Predisposition to Disease , Humans , Mannose-Binding Lectins/immunology , Neutropenia/chemically induced , Receptors, Immunologic/immunology
9.
Gut ; 52(2): 166-7, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12524392
10.
Gut ; 51(3): 356-61, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12171956

ABSTRACT

BACKGROUND: beta-Defensins are a newly identified family of antimicrobial peptides that are expressed by epithelia on mucosal surfaces where their production is augmented by infection or inflammation. Helicobacter pylori colonises the gastric epithelium causing persistent gastric inflammation leading to antral and corpus gastritis, and peptic ulcer disease. AIMS: To evaluate the role of beta-defensins in the innate immune response of the gastric epithelium to infection and inflammation, we have assessed mRNA expression and regulation of human beta-defensins 1 and 2 (hBD1, hBD2) by H pylori and proinflammatory stimuli. We have also compared gene and peptide expression of these bactericidal agents in H pylori induced gastritis with that in normal gastric mucosa. METHODS: Modulation of expression of hBD1 and hBD2 by various stimuli was studied in three (AGS, MKN7, MKN45) gastric epithelial cell lines by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). Defensin mRNA expression was measured by semiquantitative RT-PCR in gastritis tissue and compared with controls. Peptide localisation was assessed by immunohistochemistry. RESULTS: Cytotoxic H pylori and interleukin 1 beta (IL-1 beta) markedly upregulated expression of hBD2 in a dose and time dependent manner in both AGS and MKN7 cell lines. A modest increase in hBD1 expression was also noted during infection. Interestingly, induction of hBD1 gene expression by IL-1 beta was only observed in MKN7 cells. The magnitude of this response was delayed and reduced compared with hBD2 expression. In gastric biopsies, hBD2 was undetectable in normal gastric antrum but a marked increase was observed in H pylori positive gastritis compared with control tissue (p<0.001). Constitutive expression of hBD1 was observed in normal gastric mucosa and there was a significant increase in gastritis (p<0.05). Immunohistochemistry revealed a parallel increase in hBD1 and hBD2 peptide expression in gastritis tissue with positive staining confined to the surface epithelium of the gastric glands. CONCLUSIONS: Modulation of beta-defensin expression by pathogenic and/or inflammatory stimuli and their cellular localisation places these antimicrobial peptides in the front line of innate host defence in the human stomach.


Subject(s)
Gastritis/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori , beta-Defensins/metabolism , Cell Line , Gastric Mucosa/immunology , Gastritis/genetics , Gastritis/immunology , Gene Expression Regulation/genetics , Helicobacter Infections/immunology , Humans , Interleukin-1/immunology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , beta-Defensins/genetics
11.
Gut ; 50(4): 451-9, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11889061

ABSTRACT

BACKGROUND AND AIMS: Approximately 10% of adults experience gastro-oesophageal reflux symptoms with a variable oesophageal response. A total of 60% have no endoscopic abnormality, 30% have oesophagitis, and 10% have Barrett's oesophagus. We investigated whether the inflammatory cell infiltrate and cytokine profiles of these clinical phenotypes merely vary in severity or are fundamentally different. METHODS: Patients with reflux symptoms and a normal oesophagus (n=18), oesophagitis (n=26), and Barrett's oesophagus (n=22 newly diagnosed, n=28 surveillance) were recruited. Endoscopic and histopathological degrees of inflammation were scored. Cytokine expression was determined by competitive reverse transcriptase-polymerase chain reaction and immunohistochemistry. RESULTS: In oesophagitis, endoscopic and histopathological grades of inflammation correlated highly. mRNA expression of proinflammatory interleukin (IL)-1beta, IL-8, and interferon gamma (IFN-gamma) were increased 3-10-fold compared with non-inflamed squamous or Barrett's oesophageal samples. There was a modest increase in anti-inflammatory IL-10 but no increase in IL-4. In Barrett's oesophagus, 29/50 had no endoscopic evidence of inflammation and histopathological inflammation was mild in 17/50 and moderate in 24/50, independent of acid suppressants. Expression of IL-1beta, IL-8, and IFN-gamma was similar to non-inflamed squamous mucosa. IL-10 was increased 1.6-fold similar to oesophagitis. IL-4 was increased fourfold, with 100-fold increase in IL-4/T cell receptor expression, compared with squamous oesophagus or oesophagitis. CONCLUSIONS: Barrett's oesophagus is characterised by a distinct Th-2 predominant cytokine profile compared with the proinflammatory nature of oesophagitis. The specific oesophageal immune responses may influence disease development and progression.


Subject(s)
Barrett Esophagus/immunology , Cytokines/metabolism , Esophagitis/immunology , Gastroesophageal Reflux/immunology , Th2 Cells/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry , Male , Middle Aged , Phenotype , Prospective Studies , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
12.
Gut ; 50(2): 186-90, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11788557

ABSTRACT

BACKGROUND: Coeliac disease (CD) is caused by a T helper cell type 1 (Th1) response in the small intestinal mucosa to dietary gluten. Paradoxically, interleukin (IL)-12, the major Th1 inducing factor, is undetectable in the mucosa of active CD. IL-18 is a recently described cytokine capable of promoting T cell interferon (IFN)-gamma production and facilitating Th1 cell polarisation. AIM: To examine expression of IL-18 and IL-18-associated Th1 proteins in CD. METHODS: IL-18 and IFN-gamma RNA transcripts were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). IL-18 and caspase-1 protein expression were assessed by western blotting. Caspase-1 activity was determined using a commercially available assay. RNA transcripts for the IL-18 receptor subunits, IL-1 receptor related protein (IL-1 Rrp) and accessory protein-like subunit (AcPL), and IL-18 induced Th1 specific T box transcription factor (T-bet) were measured by RT-PCR and Southern blotting. RESULTS: IL-18 RNA transcripts were found in all mucosal samples analysed, with no difference between CD patients and controls. By western blot analysis, a large protein of approximately 24 kDa, corresponding to the immature IL-18, was detected in all mucosal samples from CD patients and controls. In contrast, mature IL-18 was only seen in CD patients. Immunoreactivity corresponding to both immature and mature caspase-1 was present in both CD and control samples. Tissue homogenates from CD patients and controls expressed similar levels of caspase-1 activity. IL-1Rrp and AcPL were seen in all samples but were expressed at greater levels in the mucosa of CD patients. T-bet was also upregulated in CD. CONCLUSIONS: Active IL-18 is expressed in CD as well as other markers of Th1 polarisation.


Subject(s)
Celiac Disease/immunology , Interleukin-18/metabolism , Receptors, Interleukin , Th1 Cells/metabolism , Adolescent , Adult , Biomarkers/analysis , Caspase 1/metabolism , Celiac Disease/metabolism , Child , Child, Preschool , DNA, Complementary/metabolism , Humans , Infant , Interferon-gamma/metabolism , Interleukin-18 Receptor alpha Subunit , Interleukin-18 Receptor beta Subunit , Intestinal Mucosa/metabolism , Middle Aged , Proteins/metabolism , RNA/metabolism , Receptors, Interleukin-1/metabolism , Receptors, Interleukin-18 , T-Box Domain Proteins , Transcription Factors/metabolism , Up-Regulation
13.
Gut ; 49(2): 176-81, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11454791

ABSTRACT

BACKGROUND: Coeliac disease is characterised by increased epithelial renewal associated with a mucosal T cell response to gliadin. Keratinocyte growth factor (KGF) is produced by cytokine activated gut stromal cells and may be a link between mucosal T cell activation in untreated coeliac disease and epithelial hyperplasia. AIMS: To characterise expression of KGF in coeliac disease. METHODS: KGF transcripts in coeliac disease were measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and localised using in situ hybridisation. KGF production by gluten reactive CD4+ T cell clones was examined. In addition, KGF transcripts were measured following ex vivo challenge of coeliac biopsies with a peptic-tryptic digest of gliadin. RESULTS: KGF transcripts were elevated in coeliac biopsies compared with normal controls but were not different from non-coeliac disease controls. By in situ hybridisation, KGF mRNA containing cells were present in the upper half of the lamina propria, most abundantly just under the epithelium. There was no signal from cells within the epithelium. Gluten reactive T cell clones did not make KGF. In vitro challenge of coeliac biopsies generated a strong interferon gamma response but a specific KGF response could not be detected because of an extremely high number of KGF transcripts in all cultured biopsies. CONCLUSIONS: KGF is overexpressed in coeliac biopsies and in tissues with non-coeliac enteropathy. No evidence was found for KGF production by intraepithelial lymphocytes or lamina propria T cells.


Subject(s)
Celiac Disease/metabolism , Fibroblast Growth Factors/metabolism , Adolescent , Adult , Biopsy , CD4-Positive T-Lymphocytes/metabolism , Case-Control Studies , Celiac Disease/etiology , Child , Child, Preschool , Female , Fibroblast Growth Factor 7 , Fibroblast Growth Factors/analysis , Glutens/immunology , Humans , In Situ Hybridization , Infant , Interferon-gamma/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Middle Aged , RNA, Messenger/analysis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric
14.
Gastroenterology ; 120(1): 99-107, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11208718

ABSTRACT

BACKGROUND & AIMS: Interferon (IFN)-gamma plays an important role in the immunologic control of infection by the protozoan enteropathogen Cryptosporidium parvum. We tested the hypothesis that IFN-gamma may directly inhibit infection of enterocytes by this pathogen. METHODS: HT-29, Caco-2, and H4 human enterocyte cell lines were grown in monolayers and incubated with IFN-gamma before exposure with C. parvum. IFN-gamma receptor expression in the cell lines was determined by Western blot analysis. RESULTS: IFN-gamma inhibited C. parvum infection of both HT-29 and Caco-2 cells but not H4 cells. Response to IFN-gamma was related to the expression of the IFN-gamma receptor in the respective cell lines. The effect of IFN-gamma was partially reversed by inhibition of the JAK/STAT signaling pathway. IFN-gamma mediated its action by at least 2 mechanisms: (1) inhibition of parasite invasion and (2) by modification of intracellular Fe(2+) concentration. No role for tryptophan starvation or nitric oxide synthase activity was found. TNF-alpha and IL-1beta also had anti-C. parvum activity but had no synergistic effect with IFN-gamma. CONCLUSIONS: IFN-gamma directly induces enterocyte resistance against C. parvum infection; this observation may have important consequences for our understanding of the mucosal immune response to invasive pathogens.


Subject(s)
Antineoplastic Agents/pharmacology , Cryptosporidiosis/drug therapy , Cryptosporidium parvum/immunology , Interferon-gamma/pharmacology , Intestinal Mucosa/parasitology , Animals , Antineoplastic Agents/immunology , Caco-2 Cells , Cryptosporidiosis/immunology , Cryptosporidium parvum/growth & development , Drug Synergism , Enterocytes/cytology , Enterocytes/enzymology , Enterocytes/parasitology , Enzyme Inhibitors/pharmacology , HT29 Cells , Humans , Interferon-gamma/immunology , Interleukin-1/immunology , Interleukin-1/pharmacology , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Iron/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitroarginine/pharmacology , Receptors, Interferon/biosynthesis , Tryptophan/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/pharmacology , omega-N-Methylarginine/pharmacology , Interferon gamma Receptor
15.
Digestion ; 62(4): 240-8, 2000.
Article in English | MEDLINE | ID: mdl-11070407

ABSTRACT

BACKGROUND/AIMS: In our experience with the acute murine dextran sodium sulphate (DSS) model of experimental colitis, we noted both interstrain and interanimal variations in daily water consumption. One might critically question whether observed differences in injuries are just a dose dependency phenomenon reflecting variations in DSS intake. To clarify this important topic, we performed a dose and concentration dependency study of DSS in Balb/c mice. We also determined Th1 and Th2 cytokine levels to compare the cytokine profile to that from inflammatory bowel disease (IBD). METHODS: In four groups (14 animals each group) different concentrations of DSS (0, 2.5, 5 and 7.5%) were given for 7 days ad libitum. Mucosal injury of the entire colon was histologically assessed and graded. Cytokine levels were determined by competitive quantitative RT-PCR. RESULTS: A linear increase in the crypt damage score was noted with increasing concentrations (0, 4.9 +/- 0.7, 11.9 +/- 0.5 and 18.9 +/- 1.3, respectively), but the total dose of DSS intake did not correlate with mucosal damage. Progressive upregulation in the transcripts for Th1 cytokines (IL-12, IFN-gamma, IL-1, TNF-alpha) was observed with increasing dosage of DSS. Interestingly, an increase in IL-10, but not IL-4 mRNA transcripts was also noted. DISCUSSION: Acute DSS-induced mucosal injury is dependent on the administered DSS water concentration but not on the consumed DSS dose. The cytokine profile is a classic Th1 response and is similar to that of various inflammatory conditions in the colon. CONCLUSIONS: Minor variations in fluid consumption do not affect the severity of DSS-induced injury in mice. The acute murine DSS colitis model is useful for studying the pathophysiological aspects of colonic inflammatory diseases as IBD and for evaluating new potential therapeutic agents


Subject(s)
Antiviral Agents/adverse effects , Colitis/physiopathology , Cytokines/analysis , Dextran Sulfate/adverse effects , Animals , Antiviral Agents/administration & dosage , Colitis/veterinary , Dextran Sulfate/administration & dosage , Disease Models, Animal , Dose-Response Relationship, Drug , Drinking , Female , Mice , Mice, Inbred BALB C , Reverse Transcriptase Polymerase Chain Reaction , Severity of Illness Index
16.
J Immunol ; 165(9): 5315-21, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11046066

ABSTRACT

Animal studies have demonstrated that feeding Ags induces regulatory (Th2, Th3) cells in Peyer's patches (PP), which migrate to the periphery and produce immunomodulatory cytokines such as IL-4, IL-10, or TGF-beta. In this work we have attempted to extend this paradigm to man by analyzing the response of human PP T cells to in vitro challenge with the common dietary Ag beta-lactoglobulin (betalg) of cow's milk. PP T cells stimulated with betalg showed enhanced proliferation compared with blood T cells from the same patient. Increased expression of CD25 and the Th1-associated chemokine receptor CCR5 was also seen on CD4(+) and CD8(+) PP T cells, but not blood T cells, stimulated with betalg. By enzyme-linked immunospot assay and RT-PCR, the PP T cell recall response to betalg and casein was dominated by IFN-gamma, with negligible IL-4, IL-5, IL-10, or TGF-beta. To help explain the PP T cell response to betalg, we examined IL-12 expression. Both IL-12p40 and -p35 transcripts were abundantly expressed in PP, but not in adjacent normal ileal mucosa. Immunoreactive IL-12p40-containing cells were present below the PP dome epithelium. Furthermore, in culture, PP, but not paired PBMC, spontaneously released IL-12p70. These results suggest that the human response to oral Ags in the gut may be different from that in rodents.


Subject(s)
Antigens/administration & dosage , Cytokines/biosynthesis , Dietary Proteins/immunology , Peyer's Patches/immunology , T-Lymphocyte Subsets/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Adolescent , Adult , Antigens/immunology , Child , Child, Preschool , Cytokines/analysis , Dietary Proteins/administration & dosage , Enzyme-Linked Immunosorbent Assay , Female , Humans , Ileum/chemistry , Ileum/immunology , Interleukin-12/analysis , Intestinal Mucosa/chemistry , Intestinal Mucosa/immunology , Lactoglobulins/administration & dosage , Lactoglobulins/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/immunology , Male , Peyer's Patches/chemistry , Peyer's Patches/cytology , Peyer's Patches/metabolism , Prospective Studies , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocyte Subsets/metabolism
17.
Gut ; 47(1): 57-62, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10861265

ABSTRACT

BACKGROUND: Degradation of the extracellular matrix and ulceration of the mucosa are major features of inflammatory bowel disease (IBD). One of the most important enzymes in degrading the matrix and produced in excess by cytokine activated stromal cells, is stromelysin-1. The activity of stromelysin-1 is controlled by tissue inhibitor of metalloproteinase (TIMP-1), its natural inhibitor. In model systems excess stromelysin-1 produces mucosal degradation. METHODS: Quantitative competitive RT-PCR was used to analyse stromelysin-1 and TIMP-1 transcripts; western blotting was used to measure the amount of stromelysin-1 and TIMP-1 protein in biopsy samples from children with IBD. RESULTS: In biopsies from patients with active Crohn's disease (n=24), ulcerative colitis (n=23), and controls (n=16), TIMP-1 transcripts and protein were abundant and unchanged. Stromelysin-1 transcripts and protein were markedly elevated in mucosal biopsies obtained from inflamed sites of patients with active IBD but were not elevated in adjacent endoscopically normal mucosa (n=10). Elevated levels of stromelysin-1 transcripts in active Crohn's disease (n=5) returned to normal levels following treatment with enteral nutrition. CONCLUSIONS: Stromelysin-1 is markedly overexpressed at inflamed sites in patients with IBD whereas TIMP-1 remains unaltered. Excess stromelysin-1 is likely to be responsible for loss of mucosal integrity in IBD.


Subject(s)
Inflammatory Bowel Diseases/enzymology , Matrix Metalloproteinase 3/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Blotting, Western , Child , Colitis, Ulcerative/enzymology , Colitis, Ulcerative/metabolism , Crohn Disease/enzymology , Crohn Disease/metabolism , Enteral Nutrition , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/therapy , Intestinal Mucosa/enzymology , Intestinal Mucosa/metabolism , Matrix Metalloproteinase 3/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinase-1/genetics , Transcription, Genetic
19.
J Clin Invest ; 102(8): 1473-80, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9788959

ABSTRACT

Immune reactions in the gut are associated with increased epithelial cell proliferation. Here we have studied the role of keratinocyte growth factor (KGF; FGF7) and transforming growth factor-alpha (TGF-alpha) in the epithelial cell hyperplasia seen in explants of fetal human small intestine after activation of lamina propria T cells with the superantigen Staphylococcus aureus enterotoxin B (SEB). After the addition of SEB to the explants there is a 10-fold increase in KGF mRNA by 72 h of culture. KGF transcripts were abundant in the lamina propria using in situ hybridization and the culture supernatants contained elevated amounts of KGF protein. SEB had no direct effect on KGF mRNA and protein production by cultured lamina propria mesenchymal cells, but both were upregulated by TNF-alpha. Accompanying the increase in KGF there was also an increase in TGF-alpha precursor proteins in the culture supernatants and the phosphorylated form of the EGFR receptor was also detected in the tissue. Increased TGF-alpha precursor proteins were also detected in the supernatants of control explants stimulated with KGF alone. The direct addition of KGF and TGF-alpha enhanced epithelial cell proliferation and antibodies against KGF and TGF-alpha partially inhibited SEB-induced crypt hyperplasia. These results suggest molecular cross-talk between the KGF/KGFR and the TGF-alpha/EGFR in immune-mediated crypt cell hyperplasia.


Subject(s)
Epithelial Cells/pathology , Fibroblast Growth Factors , Growth Substances/metabolism , Intestine, Small/pathology , Th1 Cells/immunology , Transforming Growth Factor alpha/metabolism , Drug Interactions , Enterotoxins/immunology , Epithelial Cells/immunology , Fetus , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Humans , Hyperplasia/etiology , Immunosuppressive Agents/pharmacology , Intestine, Small/immunology , Lymphocyte Activation , Organ Culture Techniques , RNA, Messenger/analysis , Stromal Cells , Superantigens/immunology , Tacrolimus/pharmacology , Transforming Growth Factor alpha/genetics , Up-Regulation
20.
Cytokine ; 10(8): 627-34, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9722936

ABSTRACT

The cytokine profiles of mononuclear cells freshly isolated from Peyer's patch (PPMC), adjacent ileal lamina propria lymphocytes (LPMC) and peripheral blood (PBMC) in children without histological evidence of gastrointestinal disease has been investigated by single-cell enzyme-linked immunoabsorbent spot forming assay (ELISPOT) and reverse transcriptase (RT)-PCR. In the blood, interferon gamma and IL-4 ELISPOTs were regularly detected albeit at low frequency (< 50/10(5) cells). IL-5 and IL-10 ELISPOTs were not seen in most patients. In Peyer's patches and lamina propria there was a dramatic increase in cytokine secreting cells of all types compared to blood, reaching a very high frequency for interferon gamma in the lamina propria (1000-3000/10(5) cells). IL-4 and IL-5 ELISPOTs were 20-100-fold less common in both PP and LPL. At all sites, cytokine secretion depended on protein synthesis and enrichment for CD4+ cells in PP increased the frequency of all cytokine-secreting cells. Quantification of messenger RNA for cytokines using RT-PCR demonstrated that IL-4 and IL-10 transcripts were significantly greater than interferon gamma transcripts in PP and in lamina propria, IL-4, IL-10 and interferon gamma transcripts were equivalent. IL-5 transcripts were not detected in most samples of PP and lamina propria. These results clearly show that cells secreting interferon gamma predominate in human PP and LPL. However the high mRNA concentrations for IL-4 and IL-10 shows that although these cells are quantitatively few, they are highly transcriptionally active.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-4/biosynthesis , Interleukin-5/biosynthesis , Peyer's Patches/metabolism , Polymerase Chain Reaction/methods , Adolescent , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Child , Child, Preschool , Cycloheximide/pharmacology , Female , Humans , Infant , Interferon-gamma/genetics , Interleukin-10/genetics , Interleukin-4/genetics , Interleukin-5/genetics , Leukocytes, Mononuclear/drug effects , Male , Peyer's Patches/pathology , Protein Synthesis Inhibitors/pharmacology , RNA-Directed DNA Polymerase
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