Stimulation of lamina propria lymphocytes by intestinal epithelial cells: evidence for recognition of nonclassical restriction elements.

We have addressed the restriction elements involved in the interaction of lamina propria lymphocytes (LPL) and intestinal epithelial cells using the model of primary mixed cell culture reaction. Whereas peripheral blood T cells proliferate in response to both allogeneic non T cells and class II antigen-bearing intestinal epithelial cells (non T cells >> epithelial cells), LP T cells appear to proliferate preferentially in response to intestinal epithelial cells. The interaction between these cells does not appear to be restricted by conventional products of the major histocompatibility complex as neither monoclonal antibodies to class I nor to class II antigens inhibit the mixed cell cultures, whereas they are inhibitory in conventional mixed lymphocyte reactions. Furthermore, treatment of epithelial cells with interferon gamma fails to augment the cells' ability to induce proliferation of LPL while successfully enhancing proliferation of peripheral blood T cells in parallel cultures. Taken together, these data suggest that alternate restriction elements or mucosa-specific accessory molecules may exist on intestinal epithelial cells that are preferentially recognized by LPLs. Such a distinct regulatory network may be critical to the maintenance of immunologic homeostasis in the gut.

CD1d is involved in T cell-intestinal epithelial cell interactions.

We assessed the role of the nonclassical class I molecule, CD1d, in the interaction between intestinal epithelial cells and T cells. In a mixed lymphocyte reaction (MLR) system where the stimulator cells were irradiated normal intestinal cells, the anti-CD1d monoclonal antibody (mAb) 3C11 inhibited T cell proliferation. In contrast, no inhibition was seen when mAb 3C11 was added to conventional MLR cultures (non T cell stimulators). Furthermore, no inhibition was seen when either airway epithelial cells were used as stimulator cells or lamina propria lymphocytes were used as responder cells. These latter two conditions along with a conventional MLR favor CD4+ T cell proliferation. However, we have previously shown that normal intestinal epithelial cells stimulate CD8+ T cells under similar culture conditions. Thus, CD1d expressed on intestinal epithelial cells may be an important ligand in CD8+ T cell-epithelial cell interactions.

Chemical Composition of Aerial Parts Essential Oils from Six Endemic Malagasy Helichrysum Species.

The essential oils of six endemic Malagasy Helichrysum species were investigated by GC (RI), GC-MS and 13C NMR spectrometry. In total, 153 compounds were identified accounting for 90.8% to 99.9% of the total composition. The main constituents were α-pinene for H. benthamii, 1,8-cineole for H. dubardii, (E)-ß-caryophyllene for H. indutum, and H. bojerianum. H. diotoides essential oil was characterized by the presence of two lilac alcohols and four lilac acetates whereas H. hirtum essential oil exhibited an atypical composition with 7ß-H-silphiperfol-5-ene, 7-epi-subergorgiol, and 7-epi-silphiperfol-5-en-13-oic acid as major components.

Patients with inflammatory bowel disease may have a transforming growth factor-beta-, interleukin (IL)-2- or IL-10-deficient state induced by intrinsic neutralizing antibodies.

Ulcerative colitis (UC) and Crohn's disease (CD) are considered to be immunologically mediated disorders that share certain features with murine models of colitis. Whether any of these models are physiologically relevant to the human condition remains controversial. The hypothesis is that increased amounts of antibodies neutralizing transforming growth factor (TGF)-beta, interleukin (IL)-2 or IL-10 create a relative immunodeficient state in inflammatory bowel disease (IBD) that predisposes to disease. To evaluate this, serum samples from patients with UC or CD and from normal healthy individuals were studied by enzyme-linked immunosorbent assays. Antibodies recognizing TGF-beta were most prevalent in UC (P<0.01); anti-IL-10 antibodies were elevated in CD (P<0.05), while anti-IL-2 antibodies were the same for all three groups. Importantly, the percentage of IBD patients with at least one of the antibody levels greater than any control value was 30% for UC and 33% for CD. To verify the presence of these antibodies, immobilized TGF-beta was exposed to UC sera and the attached proteins identified by Western blot assay. The proteins proved to be exclusively immunoglobulin (Ig) G. To evaluate the neutralizing activity of these antibodies, cytokine-specific IgG from subjects in each group of patients was incubated with TGF-beta, IL-2 or IL-10 before addition to a bioassay with changes in viability determined by a colorimetric analysis. Antibodies from most individuals in all three groups neutralized the action of each cytokine. This study shows that about one-third of IBD patients may have a relative deficiency of TGF-beta, IL-2 or IL-10 due to an increase in neutralizing antibodies in their sera.

Chemical composition of the essential oils from the aerial parts of two Malagasy endemic species (Apiaceae): Billburttia capensoides Sales & Hedge and Billburttia vaginoides Sales & Hedge.

The chemical composition of twenty-five essential oil samples from the aerial parts of two Malagasy endemic species Billburttia capensoides Sales & Hedge and B. vaginoides Sales & Hedge, were investigated for the first time. Based on chromatographic profiles, three selected samples were investigated using GC(RI), GC-MS and 13C NMR. The content of the main components varied drastically from sample to sample: p-mentha-1,3,8-triene (0.2-52.7%), terpinolene (2.8-40.7%) and dill apiole (0.0-22.2%). Statistical analysis of the 25 oil compositions allowed the distinction of two well-differentiated groups. Samples of group I contained mainly p-mentha-1,3,8-triene while the Group II was dominated by terpinolene and dill apiole.

A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.

Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion.

Chemical composition of Melicope belahe (Baill.) T. G. Hartley (Rutaceae) leaf essential oil from Madagascar.

Melicope belahe (Baill.) T.G. Hartley (Rutaceae) is an endemic species to Madagascar. The chemical composition of leaf essential oil is reported for the first time. A sample was extracted by hydrodistillation and analysis was carried out by combination of chromatographic (GC), spectroscopic and spectrometric (MS, 13C NMR) techniques. In total, 56 compounds have been identified. The chemical composition was dominated by α-pinene (42.6%) followed by linalool (6.2%) and (E)-ß-caryophyllene (5.2%).

The expression and regulation of class II antigens in normal and inflammatory bowel disease peripheral blood monocytes and intestinal epithelium.

Elevated constitutive expression of major histocompatibility (MHC) class II antigens occurs in the enterocytes of patients with IBD. It has been suggested that this aberrant expression of class II molecules may play a role in the pathogenesis of IBD. We examined two possible reasons for such a finding. 1) Heightened sensitivity of IBD enterocytes to endogenous gamma interferon (gamma IFN) and 2) enhanced endogenous secretion of gamma interferon by intestinal cells in close proximity to the enterocytes (lamina propria lymphocytes). Constitutive and gamma interferon stimulated HLA-DR and DP density on intestinal epithelial cells (IEC) and peripheral blood monocytes (PBM) from UC patients (IEC n = 13; PBM n = 20), CD patients (IEC n = 14; PBM n = 18) and non-IBD controls (IEC n = 12; PBM n = 20) were measured via flow cytometry (mean channel fluorescence). gamma IFN production by PHA stimulated and unstimulated lamina propria lymphocyte (LPL) cultures of UC patients (n = 11) CD patients (n = 8) and non-IBD controls (n = 11) was measured using a vesicular stomatitis virus/WISH cell bioassay. We found significantly greater gamma IFN secretion by IBD-derived PHA stimulated LPL than from non-IBD stimulated controls (CD = 39.4 +/- 12.4u; UC41.5 +/- 6.8u; NL = 22.4 +/- 8.3u, p less than 0.05) while gamma IFN induced HLA-DR and DP upregulation was no greater in IBD-derived IEC and PBM than in non-IBD controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an altered T-cell receptor repertoire in Crohn's disease.

We have compared the frequencies of T cells expressing each of four different T cell receptor (TCR) V beta segments in lamina propria and peripheral blood lymphocytes of 12 Crohn's disease (CD), six ulcerative colitis (UC), and 10 control patients in an attempt to identify disease-specific changes. The frequencies of CD4+ and CD8+ cells reacting with each of four fluoresceinated TCR-specific monoclonal antibodies directed against V beta 5, V beta 6.7a, V beta 8, and V beta 12 were determined by flow cytometry. There was no difference among the groups in the average frequency of any single V beta segment in either the CD4+ or CD8+ subpopulations. However, when the sum of the differences in V beta frequencies (delta score) between peripheral blood lymphocytes (PBL) and lamina propria lymphocytes (LPL) were determined for each individual, significant differences were observed between the CD4+ and CD8+ populations and among the patient groups. In all three patient groups, there were significant individual differences between LPL and PBL in the frequencies of CD8+ and CD4+ cells reacting with the four V beta-specific mAb. In Controls and UC, this difference was, on average, two-fold greater in CD8+ cells than in CD4+. In CD, however, this difference was, on average, the same for CD8+ and CD4+ cells. These observations suggest that (1) the human colonic LPL TCR repertoire is normally different from that of PBL, especially in the CD8+ population and (2) there is an alteration in the LPL TCR repertoire in CD which is not observed in Controls or UC.

Aspects on the role of tachykinins and vasoactive intestinal polypeptide in control of secretion, motility and blood flow in the gut.

Both intrinsic and extrinsic neurons of the gut respond to mechanical and chemical stimuli by the release of neurotransmitters. We summarize here some of our recent work on the role of vasoactive intestinal polypeptide (VIP), substance P (SP) and neurokinin A (NKA) in the secretory, motor and vascular effects of hydrochloric acid stimulation in the isolated rat duodenal loop and electrical nerve stimulation and mechanical stimulation of the cat colon. Isolated duodenal loops of conscious rats were perfused with isotonic saline, and challenged at hourly intervals with brief exposures to increasing concentrations of HCL. The concentrations of bicarbonate and prostaglandin E2 (PGE2) released from the duodenal mucosa were significantly augmented already by pH 5.0 whereas VIP was significantly augmented at pH 3.0 and the tachykinins SP and NKA at pH 2.0. Continuous electric stimulation of the pelvic nerve in cats at 4 Hz during 1 s with 10 s rest produced a marked release of NKA-LI and SP-LI from the colon to blood. Reflex activation of the pelvic nervae by mechanical stimulation of the anus or rectal distension produced a less pronounced release of NKA-LI and SP-LI from the colon to blood. There was a simultaneous colonic contraction and vasodilation during each nerve stimulation. Close intraarterial infusions of NKA, neurokinin B, SP, neuropeptide K (NPK), eledoisin and physalemin at doses of 0.1-100 pmol/min induced dose-dependent proximal and distal colonic contractions and vasodilation, NKA being the most potent. The effects of the tachykinins were reduced after tetrodotoxin and atropine, but unchanged after treatment with hexamethonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical composition of the essential oil from Croton kimosorum, an endemic species to Madagascar.

Croton kimosorum Leandri is an endemic species to Madagascar. The chemical composition of aerial parts, leaf and stem oils is reported for the first time. Analysis was carried out by combination of chromatographic (CC, GC), spectroscopic and spectrometric (MS, 13C NMR) techniques. In total, 76 compounds have been identified. Essential oil isolated from aerial parts contained mainly linalool (21.6%), sabinene (10.4%), 1,8-cineole (6.3%), beta-pinene (6.2%), (E)-beta-caryophyllene (5.9%), terpinen-4-ol (4.8%), geraniol (4,5%) and germacrene D (2.3%). In comparison with the first sample, the composition of leaf and stem oils varied slightly, while essential oil isolated by vapor distillation from a semi-industrial still exhibited similar composition.

Leaf oil from Vepris madagascarica (Rutaceae), source of (E)-anethole.

The volatile components isolated from leaves of Vepris madagascarica (Baillon) H. Perier (Rutaceae), an endemic species of north-eastern, sub-humid forests of Madagascar, were investigated by GC (Retention Indices), GC-MS and 13C NMR spectroscopy. Oil samples obtained on laboratory and industrial scales exhibited similar composition, dominated by phenylpropanoids. (E)-anethole (78.2% and 78.6%) was the major component followed by estragole (15.6% and 15.4%). In addition, trunk bark oil also contained (E)-anethole as its major component (84.6%), as well as various sesquiterpenes in low contents.

A novel method for the establishment of a pure population of nontransformed human intestinal primary epithelial cell (HIPEC) lines in long term culture.

A novel method for generating nontransformed human intestinal primary epithelial cell (HIPEC) lines in an in vitro culture system is reported here. Although several groups have reported the development of nontransformed intestinal epithelial cells (IEC) lines (Deveney et al, 1996; Latella et al, 1996; Pang et al, 1996; Perreault and Beaulieu, 1998), it still had been difficult to find an optimal condition to generate a pure population of nontransformed IEC in long-term cultures. It was hypothesized that an appropriate growth factor/cytokine milieu that would mimic the physiological microenvironment might favor the survival of the isolated cells and might play a critical role in epithelial cell growth. To test this hypothesis, isolated progenitor/crypt cells were cultured in collagen-coated petri dishes in the presence of mucosal tissue-derived growth factor containing culture supernatants (14-18 hours) and a combination of hormonal supplements. Cell attachment and growth was observed within 24 hours and confluent monolayers were seen between 7 and 12 days. Immunofluorescence staining and flow cytometric analysis of the cells demonstrated positive staining with anti-cytokeratin-18 antibody confirming their epithelial origin. The reproducibility of the method has been confirmed by establishing a number of HIPEC lines from various segments of the gastrointestinal tract. This novel method of HIPEC line generation, which maximizes the similarity of the ex vivo culture system to in vivo conditions, will serve as a valuable tool for the establishment of a large number of HIPEC lines (intestinal epithelial cell bank) and for subsequent use in studies of the immunological/physiological epithelial function in the intestine.

Antigen presentation in the intestine.

The intestinal mucosa is the largest surface area in the body which is continually exposed to an enormous amount of food antigens, viruses, bacteria, parasites or the by-products of these organisms. In such an antigen-loaded environment, specialized defence mechanisms must exist. There is clear evidence that the function of lymphocytes in the intestinal mucosa (IELs or LPLs) is different from that of lymphocytes of the peripheral blood, lymph node or spleen (these are antigen-free organs). The basic processes of these reactions are not completely understood. The role of differential antigen handling and presentation, and the non-random distribution of responsibilities between the professional and non-professional APC in this regard, have not been characterized. Thus, much remains to be learned about the basic mechanisms of antigen uptake, processing and presentation in the intestine which are necessary to induce an immune response. Diversity in APC function is a natural requirement for the maintenance of homeostasis in the intestine. Subpopulations of professional and non-professional APC may have been programmed to function in such a way that non-professional APCs may play a dominant role. It is anticipated that in vivo model systems will be developed and that eventually a clearer understanding will be gained in this rapidly evolving field.

Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells.

Intestinal epithelial cells (IEC) have been shown to act as antigen-presenting cells (APC) in vitro and may have this capacity in vivo. In order to determine whether IEC, like other APC, are able to produce accessory cytokines which may play a role in T cell activation, we assessed the accessory cytokine profile of IEC constitutively or after stimulation. We measured expression, production and regulation of accessory cytokines (IL-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) by the presence of mRNA as well as secreted protein. Freshly isolated IEC from surgical specimens were cultured in the presence or absence of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), IL-1 beta or TNF-alpha. mRNA was assessed by a specific RNAse protection assay which controlled for contaminating cell populations while protein secretion was measured by ELISA (IL-1) or bioassay (TNF and IL-6). Neither IL-1 beta nor TNF-alpha were detectable in cultured IEC supernatants, supporting the lack of macrophage contamination. All IEC spontaneously secreted IL-6 at levels comparable to those of macrophages. IEC IL-6 mRNA also increased approximately 200-fold during the first 24 h of culture. LPS, IFN-gamma or TNF-alpha had no effect on spontaneous IL-6 production, and neither resulted in the secretion of IL-1 beta or TNF-alpha. However, IL-1 beta up-regulated IL-6 synthesis by 6-7-fold. IEC express a profile of cytokine mRNAs distinct from conventional APC (low level constitutive IL-6 expression but no detectable IL-1 beta, TGF-beta or TNF-alpha), adding to their uniqueness as APC.

Expression of growth factor receptor-encoded mRNA by colonic epithelial cells is altered in inflammatory bowel disease.

A link between inflammation of the colon in inflammatory bowel disease (IBD) and the increased risk of colon cancer in ulcerative colitis (UC) may be provided by growth factor receptor genes. Their expression may be altered in response to growth factors present in the mucosa, and this, in turn, may induce further genetic changes, linked to carcinogenesis, in the cells of the colonic epithelium. To test this hypothesis, we assayed steady-state levels of eight growth factor receptor mRNAs in colonic epithelial cells of IBD patients and controls. Four of these genes (EGF-R, IGFI-R, CSF1-R, and PDGF-R-beta) were expressed in epithelial cells, whereas four (erbB-2, erbB-3, NGF-R, and met) were not. The level of the former in involved or uninvolved IBD was considerably lower than in normal epithelial cells from either sporadic colon cancer or diverticulitis patients. In contrast, expression was much higher in IBD patients with colon tumors than in active chronic IBD. The level of PDGF-R-beta mRNA was two- to fourfold higher in involved than in uninvolved areas of the colons of two UC patients, but not in one Crohn's disease patient. Message abundance of its ligand, PDGF-beta, however, was the same in paired UC samples. The pattern of expression of PDGF-beta and cripto was identical to that of EGF-R, whereas the level of mRNA of amphiregulin was the same in active chronic IBD and IBD patients with tumors. A fourth growth factor, Kfgf, was not expressed. Increased levels of PDGF-R-beta mRNA in involved UC relative to uninvolved UC may be related to the disease process in UC. Decreased expression of growth factor- and growth factor receptor-encoded mRNA in active chronic IBD may be related to the disease process, or it may be an effect of steroid therapy undergone by these patients. Enhanced expression of these genes in IBD patients with tumors compared to those without tumors suggests that this may be a marker for development of colon cancer in IBD.