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W Falk

Publications and source records attributed to W Falk.

At least 19 recordsLinked to original sources
PubMed2006

Adrenal insufficiency and colonic inflammation after a novel chronic psycho-social stress paradigm in mice: implications and mechanisms.

We investigated chronic psycho-social stress effects on stress-related parameters and on pathohistological changes in the murine colon. Moreover, we aimed to reveal the involvement of adrenal glucocorticoids in chronic stress effects. Chronic subordinate colony housing (CSC, 19 d) resulted in reduced body weight gain, thymus atrophy, adrenal hypertrophy, increased plasma norepinephrine, and increased anxiety. With respect to the time course of CSC effects, CRH mRNA in the hypothalamic paraventricular nucleus, light phase corticosterone and tyrosine hydroxylase expression in colonic tissue were found to be increased, whereas tyrosine hydroxylase expression in the locus coeruleus was found to be decreased on d 2 of CSC; these parameters returned to control levels thereafter. Nevertheless, after 19 d of CSC exposure, the adrenal corticosterone responses in vivo and in vitro, and glucocorticoid sensitivity of isolated splenic cells were found to be decreased. Importantly, in CSC mice a significant histological damage of the colon was found beginning on d 14 of CSC exposure. Additionally, pro- and antiinflammatory cytokine secretion by mesenteric lymph node cells was increased after CSC exposure. Adrenalectomy before CSC at least partially prevented these chronic stress effects as reflected by less increase in proinflammatory cytokine secretion and an equal histological damage score in adrenalectomized compared with sham-operated CSC mice. In conclusion, chronic exposure to CSC alters relevant neuronal, neuroendocrine and immune functions that could be directly or indirectly involved in the damage of the histological integrity of the colon comparable with that seen during the development of colitis.

Adrenal Glands↗
PubMed2006

Chronic intermittent psychosocial stress (social defeat/overcrowding) in mice increases the severity of an acute DSS-induced colitis and impairs regeneration.

Ulcerative colitis is a multifactorial disease, with immunological, genetic, and environmental factors playing an important role in its pathogenesis. Here we investigated the consequences of exposure to chronic psychosocial stress on the severity of a dextran sulfate sodium (DSS)-induced colitis in male C57BL/6 mice. Chronic stress was induced by repeated exposure to social defeat (SD, 2 h) and overcrowding (OC, 24 h) during 19 consecutive days. SD/OC mice showed a diminished body weight gain, thymus-atrophy, and adrenal hypertrophy, but similar light-phase plasma corticosterone concentrations, compared with unstressed mice. In contrast, the rise in dark-phase corticosterone concentration was significantly attenuated in SD/OC mice, whereas plasma ACTH concentrations and hypothalamic CRH mRNA expression did not differ between stressed and nonstressed groups. Additionally, adrenal cells from SD/OC mice showed a decreased in vitro response to ACTH stimulation. Subsequent treatment with 1% DSS for 7 d resulted in a more severe intestinal inflammation in SD/OC mice, as reflected by an increase in body weight loss, histological damage scores, and secretion of IL-6, TNFalpha, and interferon-gamma from mesenteric lymph node cells and by decreased colon length. The impaired health status of stressed mice was also reflected by a significantly lower survival rate after termination of the DSS treatment. In conclusion, the present findings demonstrate that chronic intermittent exposure to a psychosocial stressor before the induction of acute DSS-colitis results in adrenal insufficiency, increases in the severity of the acute inflammation, and impairs the healing phase.

Acute Disease↗
PubMed2006

Soluble galectin-3 is a strong, colonic epithelial-cell-derived, lamina propria fibroblast-stimulating factor.

BACKGROUND: Colonic lamina propria fibroblasts (CLPFs) play an important role in the pathogenesis of fibrosis and strictures in Crohn's disease. AIM: To identify colonic epithelial cell (CEC)-derived factors that activate CLPFs. METHODS: Primary human CECs and CLPFs were isolated from control mucosa and interleukin 8 (IL8) of CLPF cultures was quantified by ELISA. Activation of nuclear factor kappaB (NF-kappaB) was shown, and translocation of NF-kappaB was inhibited by a dominant-negative IkappaB-expressing adenovirus. The major CLPF-activating and IL8 inducing protein was purified using fast-performance liquid chromatography (HiPrep 16/60 Sephacryl S-200 High Resolution Column) and sodium dodecyl sulphate gel electrophoresis. RESULTS: A considerable increase in IL8 secretion by CLPFs cultured in CEC-conditioned media compared with that in unconditioned media (155.00 (10.00) pg/microg v 1.434 (0.695) pg/microg) was found. The effect of CEC-conditioned media on CLPF IL8 secretion was NF-kappaB dependent. A protein or DNA array confirmed the involvement of NF-kappaB and activator protein-1. Purification of a candidate band isolated with the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequent sequencing showed soluble galectin-3 to be a strong CLPF-activating factor. Depletion of galectin-3 from conditioned media by immunoprecipitation abolished the CLPF stimulatory effect. CONCLUSIONS: Using a classical biochemical approach, soluble galectin-3 was identified as a strong activator of CLPFs produced by CEC. Galectin-3 induced NF-kappaB activation and IL8 secretion in these cells and may be a target for future therapeutic approaches to reduce or avoid stricture formation.

Adult↗
PubMed2006

Monocyte chemoattractant protein-1 (MCP-1) inhibits the intestinal-like differentiation of monocytes.

Monocytes (MO) migrating into normal, non-inflamed intestinal mucosa undergo a specific differentiation resulting in a non-reactive, tolerogenic intestinal macrophage (IMAC). Recently we demonstrated the differentiation of MO into an intestinal-like macrophage (MAC) phenotype in vitro in a three-dimensional cell culture model (multi-cellular spheroid or MCS model). In the mucosa of patients with inflammatory bowel disease (IBD) in addition to normal IMAC, a reactive MAC population as well as increased levels of monocyte chemoattractant protein 1 (MCP-1) is found. The aim of this study was to investigate the influence of MCP-1 on the differentiation of MO into IMAC. MCS were generated from adenovirally transfected HT-29 cells overexpressing MCP-1, macrophage inflammatory protein 3 alpha (MIP-3alpha) or non-transfected controls and co-cultured with freshly elutriated blood MO. After 7 days of co-culture MCS were harvested, and expression of the surface antigens CD33 and CD14 as well as the intracellular MAC marker CD68 was determined by flow-cytometry or immunohistochemistry. MCP-1 and MIP-3alpha expression by HT-29 cells in the MCS was increased by transfection at the time of MCS formation. In contrast to MIP-3alpha, MCP-1 overexpression induced a massive migration of MO into the three-dimensional aggregates. Differentiation of IMAC was disturbed in MCP-1-transfected MCS compared to experiments with non-transfected control aggregates, or the MIP-3alpha-transfected MCS, as indicated by high CD14 expression of MO/IMAC cultured inside the MCP-1-transfected MCS, as shown by immunohistochemistry and FACS analysis. Neutralization of MCP-1 was followed by an almost complete absence of monocyte migration into the MCS. MCP-1 induced migration of MO into three-dimensional spheroids generated from HT-29 cells and inhibited intestinal-like differentiation of blood MO into IMAC. It may be speculated that MCP-1 could play a role in the disturbed IMAC differentiation in IBD mucosa.

Antigens, CD↗
PubMed2006

Cathepsins B, L and D in inflammatory bowel disease macrophages and potential therapeutic effects of cathepsin inhibition in vivo.

The cathepsins D (CTSD), B (CTSB) and L (CTSL) are important for the intracellular degradation of proteins. Increased cathepsin expression is associated with inflammatory diseases. We have shown previously an induction of CTSD expression in intestinal macrophages (IMAC) in inflamed mucosa of patients with inflammatory bowel disease (IBD). Here we investigated the regulation of CTSB and CTSL in IMAC during IBD and effects of CTSD and CTSB/CTSL inhibition in vivo. Human IMAC were isolated from normal and inflamed mucosa. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for CTSB and CTSL mRNA. Immunostaining was used to confirm PCR results. Cathepsin inhibition was investigated in the dextran-sulphate-sodium (DSS) colitis model in mice with application of pepstatin A (CTSD inhibitor), CA-074 (CTSB inhibitor) and Z-Phe-Tyr-aldehyde (CTSL inhibitor). CTSL mRNA was significantly up-regulated in IMAC isolated from IBD mucosa. Up-regulated protein expression was found mainly in areas of mucosal damage by immunostaining. Inhibition of CTSD in mouse DSS colitis was followed by an amelioration of the disease. Inhibitor-treated mice showed a significant lower histological score (HS) and less colon reduction in comparison to controls. Similarly, simultaneous inhibition of CTSB/CTSL was followed by a significant amelioration of colitis. Expression of tissue-degrading cathepsins is increased in IMAC in IBD. Inhibition of CTSD as well as CTSB/CTSL is followed by an amelioration of experimental colitis. The prevention of mucosal damage by cathepsin inhibition could represent a new approach for the therapy of IBD.

Animals↗
PubMed2005

Strange-quark contributions to parity-violating asymmetries in the forward g0 electron-proton scattering experiment.

We have measured parity-violating asymmetries in elastic electron-proton scattering over the range of momentum transfers 0.12 < or =Q2 < or =1.0 GeV2. These asymmetries, arising from interference of the electromagnetic and neutral weak interactions, are sensitive to strange-quark contributions to the currents of the proton. The measurements were made at Jefferson Laboratory using a toroidal spectrometer to detect the recoiling protons from a liquid hydrogen target. The results indicate nonzero, Q2 dependent, strange-quark contributions and provide new information beyond that obtained in previous experiments.

Journal Article↗
PubMed2005

TLR-4 surface display on human monocytes is increased in septic patients.

BACKGROUND: Sepsis is a serious condition, most often occurring as a complication of bacterial infections. The Toll-like receptors (TLR)-2 and TLR-4 have been identified as key molecules in response to Gram-positive and Gram-negative bacteria. This study aimed to assess possible alterations of the surface display of TLR-2 and TLR-4 on monocytes and granulocytes derived from patients with sepsis in comparison with healthy controls. - METHODS: We have utilized flow-cytometry to determine the presence of TLR-2 and TLR-4 on the cell surface at baseline and in response to LPS (40 ng/ml) in vitro. - RESULTS: We found no significant differences of TLR-2 display on monocytes and granulocytes from septic patients compared to controls. Surface display of TLR-4 on monocytes from septic patients at baseline was significantly higher than in healthy controls but there was no further response to LPS, whereas controls showed a significant increase of TLR-4 display on the cell surface after LPS stimulation. In contrast, TLR-4 baseline cell surface display on granulocytes was significantly lower in septic patients than in controls and there was no response to LPS in both groups. - CONCLUSION: Our data suggest a complex relationship between TLR-4 display and bacterial challenge in vivo and in vitro.

Adult↗
PubMed2005

In vivo CpG DNA/toll-like receptor 9 interaction induces regulatory properties in CD4+CD62L+ T cells which prevent intestinal inflammation in the SCID transfer model of colitis.

BACKGROUND AND METHODS: Cytosin-guanosin dinucleotide (CpG) motifs of bacterial DNA are known to be potent activators of innate immunity. We have shown previously that administration of CpG containing oligodeoxynucleotide (CpG-ODN) to mice before the onset of dextran sodium sulphate induced colitis ameliorated colitis and inhibited induction of proinflammatory cytokines. To investigate the possible involvement of CD4(+) T cells in the prophylactic CpG-ODN effects, we used the SCID transfer model of colitis. RESULTS: CD4(+)CD62L(+) T cells from CpG-ODN treated donors did not induce significant intestinal inflammation in SCID recipients, in contrast with control cells. Additionally, cotransfer of these cells with CD4(+)CD62L(+) cells from normal mice protected recipient animals from colitis, indicating regulatory activity. Also, CD4(+)CD62L(+) cells from toll-like receptor 9 deficient animals induced a significantly more severe colitis in SCID recipients than cells from wild-type littermate controls, suggesting a similar protective role of "endogenous" bacterial DNA leading to a less "aggressive" phenotype of these cells. There was no detectable difference in regulatory T cell surface markers between aggressive and attenuated cell pools but attenuated cell pools showed reduced proliferation in vitro and in vivo and produced less interferon gamma, interleukin (IL)-5, and IL-6 after anti-CD3 stimulation. CONCLUSIONS: Collectively, our data support the concept that both endogenous bacterial DNA and exogenously supplied CpG motifs of bacterial DNA induce regulatory properties in CD4(+) T cells. Therefore, bacterial DNA derived from the normal gut flora may contribute essentially to the homeostasis between effector and regulatory immune mechanisms in healthy individuals to protect them from chronic intestinal inflammation.

Animals↗
PubMed2005

Key role of the sympathetic microenvironment for the interplay of tumour necrosis factor and interleukin 6 in normal but not in inflamed mouse colon mucosa.

BACKGROUND: In the intestinal tract, the role of sympathetic neurotransmitters has been largely ignored in mucosal neuroimmunology. AIM: Our aim was to investigate the influence of the sympathetic microenvironment on the mucosal interplay of tumour necrosis factor (TNF) and interleukin 6 (IL-6). METHODS: Colon strips of normal and colitic BALB/c mice were superfused in vitro. Tissue was electrically stimulated to investigate the influence of endogenous norepinephrine (NE) on secretion of IL-6, with or without anti-TNF antibodies (anti-TNF) and adrenoceptor antagonists. IL-6 was secreted from macrophages. RESULTS: Superfusion with anti-TNF stimulated IL-6 secretion in normal but not in colitic colon (p<0.005). Parallel superfusion with a beta-adrenergic antagonist abrogated this phenomenon. Anti-TNF increased release of NE from normal colonic strips (p<0.05), which demonstrates TNF induced inhibition of preterminal NE release. In colitic mice, anti-TNF did not change NE release. In the presence of anti-TNF, exogenous and endogenous NE stimulated colonic IL-6 secretion via beta-adrenoceptors in normal (p<0.001) but not in colitic mice. In the absence of anti-TNF, endogenous and exogenous NE inhibited IL-6 secretion via the beta-adrenoceptor in normal but not in colitic mice (p<0.01). Colitic mice demonstrated loss of sympathetic nerve fibres. CONCLUSIONS: Modulation of mucosal IL-6 is largely dependent on the sympathetic microenvironment and availability of local TNF in normal but not in colitic mice. Anti-TNF strategies may lead to an increase in the proinflammatory cytokine depending on adrenergic tone. This would be relevant with normal sympathetic innervation, which is lost in colitic mice. We present a model of sympathetic regulation of colonic macrophage TNF and IL-6 secretion.

Adrenergic beta-Agonists↗
PubMed2005

Comparison of culture and real-time PCR for detection and quantification of five putative periodontopathogenic bacteria in subgingival plaque samples.

OBJECTIVES: Bacterial cultivation is a well-established method for analyzing plaque samples. Real-time polymerase chain reaction (PCR) is a novel rapid method for the identification and quantification of periodontopathogenic bacteria that has been recently introduced. In this study, we compared real-time PCR with conventional anaerobic cultivation. METHOD: A total of 78 subgingival plaque samples were harvested from pockets > or =5 mm in 22 patients with advanced chronic periodontitis and immediately transferred into transport medium. Aliquots were evaluated with species-specific probes by real-time PCR (meridol Perio Diagnostics, GABA) and anaerobic bacteria culture on selective media for the detection of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythensis. The analysis was performed by two separate, blinded examiners. RESULTS: When real-time PCR was compared with the culture method (golden standard) for the detection of putative periodontopathogenic bacteria, the sensitivity and specificity for A. actinomycetemcomitans were 67% and 100%, respectively (kappa: 0.79); for F. nucleatum 73% and 53%, respectively (kappa: 0.21); for P. gingivalis 94% and 84%, respectively (kappa: 0.77); for P. intermedia 33% and 94%, respectively (kappa: 0.26) and for T. forsythensis 92% and 56%, respectively (kappa: 0.51). Spearman's correlation coefficients for the quantitative results of both methods were 0.82 for A. actinomycetemcomitans, 0.33 for F. nucleatum, 0.83 for P. gingivalis, 0.38 for P. intermedia and 0.67 for T. forsythensis. CONCLUSION: Overall, the agreement between both test methods was excellent for A. actinomycetemcomitans and P. gingivalis, fair for T. forsythensis and poor for F. nucleatum and P. intermedia. The discrepancies in the results may be explained by the inability of cultivation methods to distinguish between close related taxa, and the problems of keeping periopathogenic bacteria viable, which is required for standard cultivation.

Aggregatibacter actinomycetemcomitans↗
PubMed2005

Glycoprotein (gp) 96 expression: induced during differentiation of intestinal macrophages but impaired in Crohn's disease.

BACKGROUND: The glycoprotein (gp) 96 links the adaptive with the innate immune system. It is a chaperone with a binding domain for peptides generated by proteasomal degradation. During cellular stress, peptide loaded gp96 can be released and presented to T cells by antigen presenting cells (APCs). METHODS: mRNAs from in vitro differentiated macrophages (iv mac) and normal intestinal macrophages (IMACs) were compared by subtractive hybridisation and Affymetrix GeneChip analysis. Differentiation induced expression of gp96 was investigated in the multicellular spheroid (MCS) model. In vivo gp96 protein expression was detected by double labelling immunohistochemistry of human colon and in the CD4+ CD62L+ T cell transfer mouse model. RESULTS: Five of 76 clones obtained by subtractive hybridisation revealed >99% sequence homology to gp96. Affymetrix GeneChip analysis confirmed induction of gp96 in IMACs. Gp96 mRNA was detected in IMACs from normal and intestinal bowel disease mucosa. Induction of gp96 protein was observed after seven days in the MCS model of IMAC differentiation. Immunohistochemistry confirmed the presence of gp96 protein in IMACs in normal mucosa as well as in mucosa from patients with ulcerative colitis and diverticulitis. In mucosa from Crohn's disease (CD) patients, gp96 protein was not detectable. In the CD4+ CD62L+ T cell transfer mouse model, gp96 was verifiable in non-activated IMACs. CONCLUSION: Gp96 is induced during differentiation of normal IMACs but is not detected in IMACs in CD mucosa. As gp96 has been described as having a role in tolerance induction, this may be relevant for loss of tolerance against luminal bacteria found in CD patients.

Animals↗
PubMed2004

Interferon gamma downregulates IL-8 production in primary human colonic epithelial cells without induction of apoptosis.

BACKGROUND: In acute or chronic inflammatory bowel disease (IBD) interferon gamma (IFNgamma) is still considered to be an important pro-inflammatory mediator. In the present study we investigated the impact of IFNgamma on interleukin-8 (IL-8) production as a read-out for cell activation in intestinal epithelial cell (IEC) lines and primary human colonic epithelial cells (CEC). METHODS: Primary cultures of human CEC were established from the mucosa of patients without inflammatory disease. CEC, HT-29 or Caco-2 cells were incubated with either IFNgamma, tumor necrosis factor (TNF)alpha or IL-10. IL-8 and IL-1Ra secretion was determined by ELISA. Competicon PCR was used for quantification of IL-8mRNA. Apoptosis was quantified by propidium iodine incorporation and fluorescence activated cell sorting (FACS) analysis. RESULTS: In contrast to HT-29 cells in primary human CEC 100 U/ml IFNgamma inhibited IL-8 secretion significantly to 70+/-15% of unstimulated primary CEC (p<0.005) more effectively than IL-10 (87+/-21% versus unstimulated cells, n.s.). In HT-29 cells, IL-8 secretion was induced to 405+/-101% of unstimulated cells. In Caco-2 cells, IFNgamma had no significant effect on IL-8 secretion. The effect in HT-29 and CEC was concentration dependent. In primary CEC, 200 U/ml IFNgamma further reduced IL-8 secretion to 48+/-18% of unstimulated CEC (p<0.05). Whereas IL-8 mRNA was strongly upregulated in HT-29 cells, no upregulation or even a downregulation was found in CEC. Pre-incubation with 100 U/ml IFNgamma did not increase the susceptibility to apoptosis mediated by anti-Fas antibody (CH-11) in primary CEC, whereas HT-29 cells showed increased rates of apoptosis after priming with IFNgamma. CONCLUSION: In contrast to HT-29, IFNgamma downregulated IL-8 secretion and did not induce IL-8 mRNA expression in primary human CEC. This effect was not due to induction of apoptosis.

Antineoplastic Agents↗
PubMed2004

Blocking lymphotoxin-beta receptor activation diminishes inflammation via reduced mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression and leucocyte margination in chronic DSS-induced colitis.

The lymphotoxin-beta receptor (LTbetaR) pathway is critical for maintenance of organized lymphoid structures and is involved in the development of colitis. To investigate the mechanisms by which LTbetaR activation contributes to the pathology of chronic inflammation we used a soluble LTbetaR-Ig fusion protein as a competitive inhibitor of LTbetaR activation in the mouse model of chronic colitis induced by oral administration of dextran sulphate sodium. Strong expression of LTbeta which constitutes part of the LTalpha(1)beta(2) ligand complex was detected in colonic tissue of mice with chronic colitis. Treatment with LTbetaR-Ig significantly attenuated the development and histological manifestations of the chronic inflammation and reduced the production of inflammatory cytokines such as TNF, IL-1beta, and IL-6. Moreover, LTbetaR-Ig treatment significantly down-regulated mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression, leading to reduced leucocyte rolling and sticking in postcapillary and collecting venules and reduced extravasation into the intestinal mucosa as quantified by in vivo fluorescence microscopy. Thus, LTbetaR pathway inhibition ameliorates DSS-induced experimental chronic colitis in mice by MAdCAM-1 down-regulation entailing reduced lymphocyte margination and extravasation into the inflamed mucosa. Therefore, a combined treatment with reagents blocking T cell-mediated perpetuation of chronic inflammation such as LTbetaR-Ig together with direct anti-inflammatory reagents such as TNF inhibitors could constitute a promising treatment strategy for chronic colitis.

Animals↗
PubMed2004

Cathepsin D is up-regulated in inflammatory bowel disease macrophages.

Down-regulation of receptors involved in the recognition or transmission of inflammatory signals and a reduced responsiveness support the concept that macrophages are 'desensitized' during their differentiation in the intestinal mucosa. During inflammatory bowel disease (IBD) intestinal macrophages (IMACs) change to a reactive or 'aggressive' type. After having established a method of isolation and purification of IMACs, message for cathepsin D was one of the mRNAs we found to be up-regulated in a subtractive hybridization of Crohn's disease (CD) macrophages versus IMACs from control mucosa. The expression of cathepsin D in intestinal mucosa was analysed by immunohistochemistry in biopsies from IBD and control patients and in a mouse model of dextran sulphate sodium (DSS)-induced acute and chronic colitis. IMACs were isolated and purified from normal and inflamed mucosa by immunomagnetic beads armed with a CD33 antibody. RT-PCR was performed for cathepsin D mRNA. Results were confirmed by Northern blot and flow cytometrical analysis. Immunohistochemistry revealed a significant increase in the cathepsin D protein expression in inflamed intestinal mucosa from IBD patients compared to non-inflamed mucosa. No cathepsin D polymerase chain reaction (PCR) product could be obtained with mRNA from CD33-positive IMACs from normal mucosa. Reverse transcription (RT)-PCR showed an induction of mRNA for cathepsin D in purified IMACs from IBD patients. Northern blot and flow cytometry analysis confirmed these results. Cathepsin D protein was also found in intestinal mucosa in acute and chronic DSS-colitis but was absent in normal mucosa. This study shows that expression of cathepsin D is induced in inflammation-associated IMACs. The presence of cathepsin D might contribute to the mucosal damage in IBD.

Acute Disease↗
PubMed2003

Contrasting activity of cytosin-guanosin dinucleotide oligonucleotides in mice with experimental colitis.

Intestinal inflammation in inflammatory bowel disease (IBD) and experimental models of colitis is characterized by a dysregulated intestinal immune response with elevated levels of Th1 cytokines. The luminal flora has been implicated as a major factor contributing to the initiation and perpetuation of inflammation in experimental colitis by mechanisms not known. Bacterial DNA contains unmethylated cytosin-guanosin dinucleotides (CpG) which strongly activate Th1-mediated immune responses. To test whether these CpG-motifs modulate intestinal inflammation we treated mice with dextran sulphate sodium (DSS)-induced colitis with CpG-containing oligodeoxynucleotides (CpG-ODN). CpG-ODN given after the onset of DSS colitis aggravated the disease, as indicated by a significantly increased loss of body weight and a 30% increase of the histological score. Further, we found a severe increase of proinflammatory cytokines (interleukin (IL)-6: 40-fold; interferon (IFN)-gamma: 11-fold). In a pretreatment setting CpG-ODN reduced weight loss significantly and reduced intestinal inflammation by 45%. Colonic IFN-gamma and IL-6 mRNA levels were reduced by 75%, and IL-10 was elevated by 400% compared to controls. The prophylactic CpG-effect was not imitated by IL-12 because IL-12 pretreatment was not protective. In time-course experiments, CpG-ODN pretreatment over 5 days resulted in a tolerance effect concerning its IFN-gamma-inducing quality, and during the following days of colitis induction IL-10 secretion from mesenterial lymph node cells was elevated compared to controls. Therefore, the prophylactic effect of CpG-ODN might be explained by its tolerizing effect and/or the increased ability for IL-10 production during the consecutive intestinal inflammation.

Acute Disease↗
PubMed2003

Inducible CD40 expression mediates NFkappaB activation and cytokine secretion in human colonic fibroblasts.

BACKGROUND: CD40 has been shown to be a functional activation antigen on a variety of cell types involved in immune responses. As intestinal fibroblasts and myofibroblasts may play a role during mucosal inflammation, we investigated the functional consequences of CD40 induction in primary cultures of human colonic fibroblasts. METHODS: Primary colonic lamina propria fibroblasts (PCLF) were isolated from endoscopic biopsies and surgical specimens. Cultures were used between passages 3 and 9. CD40 surface display was determined by FACS analysis and mRNA expression by reverse transcription-polymerase chain reaction. Secretion of cytokines was determined by ELISA. Nuclear factor kappaB (NFkappaB) activation was shown by electrophoretic mobility shift assay (EMSA). RESULTS: After priming with interferon gamma (IFN-gamma) (200 U/ml) for 72 hours, five of eight tested PCLF cultures showed induction of CD40 surface display (up to 10-fold). Induction of CD40 mRNA expression was demonstrated by semiquantitative polymerase chain reaction. In the responder-PCLF cultures, IFN-gamma alone caused a 1.5-5-fold increase in interleukin (IL)-8 secretion. Addition of 1 ng/ml CD40L was sufficient to achieve a further increase in IL-8, IL-6, or monocyte chemotactic protein 1 (MCP-1) secretion (2.5-18-fold of controls). Incubation with CD40L alone without priming with IFN-gamma had no effect. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN 100 microM) reduced IFN-gamma/CD40L mediated cytokine induction, suggesting participation of NFkappaB, which was directly demonstrated by EMSA. CD4+ T cells induced MCP-1 secretion by PCLF, which was prevented by addition of an excess of CD40-IgG fusion protein. CD40 expression on PCLF could also be demonstrated in vivo by immunohistochemistry. CONCLUSION: The CD40-CD40L pathway augments mucosal inflammatory responses via mucosal PCLF. CD40-CD40L mediated T cell/PCLF interactions could play an important role during intestinal mucosal inflammation.

Adult↗
PubMed2002

Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation.

BACKGROUND & AIMS: Bacterial wall products play an important role in the activation of immune and nonimmune cells of the intestinal mucosa. Toll-like receptors (TLRs) TLR2 and TLR4 have been identified as signaling receptors activated by bacterial wall components. METHODS: Expression of TLRs in human intestinal mucosa obtained by endoscopy and surgery was analyzed by immunohistochemistry. Intestinal macrophages were isolated by immunomagnetic beads armed with a CD33 antibody. Reverse-transcription polymerase chain reaction was performed for TLR1-5. Results were confirmed by Northern blot and flow cytometry. Interleukin-1beta messenger RNA (mRNA) was quantified by a polymerase chain reaction-enzyme-linked immunosorbent assay-kit. RESULTS: Immunohistochemistry revealed a significant increase in the TLR2 and TLR4 antigen expression on submucosal cells in inflamed intestinal mucosa compared with non-inflamed mucosa. TLR expression was localized in intestinal macrophages by double-labeling techniques. No TLR-polymerase chain reaction product could be obtained with mRNA from CD33-positive macrophages from normal mucosa. We observed an induction of mRNA for TLR2, TLR4, and TLR5 in inflammation-associated macrophages. TLR1 and TLR3 were only detectable in blood monocytes. Monocytes reacted to lipopolysaccharide stimulation with a 3-fold and in vitro differentiated macrophages with a 16-fold increase of cellular interleukin-1beta mRNA. Macrophages from normal mucosa did not respond to lipopolysaccharide showing the functional relevance of TLR expression. CONCLUSIONS: This study shows the inflammation-dependent induction of TLR2 and TLR4 expression in intestinal macrophages. The absence of TLRs abolishes the reactivity of mucosal macrophages to bacterial wall products. Presence of TLRs may thereby contribute to the inflammatory process.

Blood Cells↗