Jirina Kolinska

Interaction of Mucosal Microbiota with the Innate Immune System

Organisms live in continuos interaction with their environment; this interaction is of vital importance but at the same time can be life threatening. The largest and most important interface between the organism and its environment is represented by surfaces covered with epithelial cells. Of these surfaces, mucosae comprise in humans approximately 300 m2, and the skin covers approximately 1.8 m2 surface of the human body. Mucosal tissues contain two effector arms of the immune system, innate and adaptive, which operate in synergy. Interaction with commensal bacteria, which outnumber the nucleated cells of our body, occurs physiologically on epithelial surfaces; this interaction could pose the risk of inflammation. The mucosal immune system has developed a complex network of regulatory signalling cascades that is a prerequisite for proper activation but also for a timely inactivation of the pathway. As demonstrated in gnotobiotic animal models of human diseases, impaired regulation of mucosal responses to commensal bacteria plays an important role in the development of several inflammatory and autoimmune diseases.

Bifidobacterium bifidum monoassociation of gnotobiotic mice: Effect on enterocyte brush-border enzymes

The effect of intestinal colonization withBifidobacterium bifidum (Gram-positive anaerobic bacterium colonizing the intestine of healthy new-born mammals, exhibiting a probiotic effect, protecting the intestinal mucosa against colonization by pathogenic microflora) on enterocyte brush-border enzymes was examined in weaned 23-d- and in 2-month-old gnotobiotic inbred mice and compared with that in corresponding germ-free (GF) and conventional (CV) controls. The two groups of GF mice were associated with humanB. bifidum 11 d before the end of the experiment. Specific activity of enterocyte brush-border enzymes—lactase, alkaline phosphatase and γ-glutamyltranspeptidase was significantly higher in both age groups of GF mice in comparison with CV ones; on the other hand, sucrase and glucoamylase activities were higher in CV mice. Monoassociation withB. bifidum accelerates biochemical maturation of enterocytes resulting in a shift of specific activities of brush-border enzymes between the values found for GF and CV mice. This effect ofB. bifidum supplementation was less pronounced for alkaline phosphatase, sucrase, glucoamylase and dipeptidyl peptidase IV in immature gut of weaned mice than of 2-month-old ones.

Constitutive expression of IL-18 and IL-18R in differentiated IEC-6 cells: Effect of TNF-α and IFN-γ treatment

The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, brush border enzyme activities, and gene expression of apoptotic markers. However, the addition of IL-18 stimulated production and release of the chemokine IL-8. These data suggest that IEC-6 cells may be not only a source of IL-18 but also a target for its action.

BRUSH BORDER ENZYME ACTIVITIES IN THE SMALL INTESTINE AFTER LONG-TERM GLIADIN FEEDING IN ANIMAL MODELS OF HUMAN COELIAC DISEASE

Cœliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.

Stimulation of enterocyte enzymatic activities, MHC class II expression and other immunological factors after oral treatment with Nocardia delipidated cell mitogen in germ-free rats

Nocardia delipidated cell mitogen (NDCM) was given intragastrically (200 micrograms/animal) to 2-month-old germ-free (GF) and conventionally (CV) reared AVN rats. On day 4, enzymatic activities of enterocyte brush border membrane vesicles (BBMV) isolated from jejunal scraping were measured. The results indicated that activities of sucrase, lactase and glucoamylase in BBMV were stimulated following NDCM treatment. The most pronounced increase of enzymatic activities was found in GF rats. In contrast to CV rats, GF rats do not express MHC class II on epithelial cells during their life. NDCM treatment induced MHC class II expression in enterocytes from GF rats. The levels of IgA and IgG in sera from NDCM-treated CV rats did not change significantly. The level of IgG in sera of GF rats was found to be enhanced after NDCM treatment. Markedly increased incorporation of 3H-thymidine by spleen lymphocytes stimulated with Con A in vitro was observed only in NDCM-treated GF rats. 3H-uridine incorporation into Con A-stimulated lymphocytes of GF rats was decreased after NDCM treatment.

Effect of nonpathogenic Escherichia coli monoassociation on small intestinal brush-border glycoconjugate moieties and cytokine production after colonization in ex-germ-free rats and pigs

We evaluated the contribution of nonpathogenic Escherichia coli O83 after coloniza- tion of germ-free (GF) rat pups and piglets on development of terminal α2,6- and α2,3-sialylated and broad range of terminal α1,2-, core α1,6-, and α1,3-, α1,4-fucosylated glycoconjugates in the suckling period relative to noncolonized GF and conventional (CV) counterparts. The ELISA-lectin approach was used to specify and quantify sialylated and fucosylated glycans in brush-border membrane vesicles (BBMV). Levels of pro- and anti-inflammatory cytokines in sera and splenocyte secretions were demonstrated using ELISA. Rat and pig intestinal responses to E. coli O83 monoassociation were different from those of GF and more similar to the CV animals in the decline of sialylated glycans, which is in agreement with the shorter life span of enterocytes in E. coli O83 monoassociated rats and pigs. No significant effect of E. coli O83 colonization on labeling fucosylated glycans was observed for immature fucosylation at the late suckling period. We demonstrate spontaneous secretion of pro-inflammatory cytokine IL-18 and anti-inflammatory IL-10 by GF rat splenocytes, and its suppression of IL-18 in E. coli O83 associated rat pups, and suggest that these cytokines serve as an immunomodulatory pool during the suckling period indicating the balance between T helper (Th) 1 and Th2 phenotypes.

Binding of the Galanthus nivalis Agglutinin to Thymocytes Reveals Alterations in Surface Glycosylation during T-Cell Development

Surface binding of the Galanthus nivalis agglutinin (GNA) to thymocyte subsets has been studied in pigs and rodents by multicolour flow cytometry. In all the species examined, analogous staining profiles have been recorded. Counter‐staining with anti‐CD3ε, anti‐CD4 and anti‐CD8 monoclonal antibodies (MoAb) revealed that a significant increase of the GNA targets on the cell surface occurred during early thymocyte differentiation and reached its maximum at the level of the CD3loCD4+CD8+ small cortical thymocyte. This was followed by a decrease in the GNA binding capacity upon terminal maturation to the single positive thymocytes. PAGE analysis has revealed a dominant GNA‐binding glycoprotein (molar mass approx. 90 kDa) present on thymocyte plasma membranes and absent on the surface of splenic lymphocytes, although both the whole cell lysates from both organs contained GNA ligands of the same size. Our findings are in agreement with previous data showing that immature thymocytes differ from their mature counterparts and peripheral T lymphocytes in the surface glycosylation pattern, and support the hypothesis that lectin–glycoprotein interaction plays a significant role in the cell‐to‐cell crosstalk in the thymic cortex.

Protective effects of nocardia delipidated cell mitogen on the mucosa of the small intestine after irradiation of germ‐free piglets.

The radioprotective effect of the bacterial immunomodulator Nocardia delipidated cell mitogen (NDCM) on intestinal mucosa and disaccharidase activities was studied in irradiated germ‐free piglets. Three‐week‐old germ‐free (GF) piglets were intragastrically pretreated with 1 mg NDCM per 1 kg body weight. The piglets were whole‐body irradiated with 2.5 Gray five days after the NDCM pretreatment and sacrificed eight days after irradiation. In the non‐irradiated group of GF piglets, NDCM application stimulated lactase activity and markedly increased sucrase activity. This stimulatory effect of NDCM disappeared after irradiation and the piglets exhibited a normal activity of lactase in the jejunal brush‐border membrane vesicles, while the sucrase activity decreased to the level found in irradiated controls. NDCM‐pretreated intestinal mucosa contained some infrequent lymphocytes which disappeared from the control irradiated tissue. It also exhibited less injury of the epithelium and stroma cells.

Differential effect of Bacillus firmus on immune response and enterocyte brush-border enzyme levels in BALB/c and B10.BR mice.

A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity,Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 μg every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, γ-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose α-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of γ-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-α-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.

Effects of Nocardia-delipidated cell mitogen on intestinal mucosa and spleen lymphocytes of germ-free rats.

Many compounds of bacterial origin can modulate basic physiological parameters of the mammalian organism including the immune system. One of them is Nocardia- delipidated cell mitogen (NDCM) which was isolated by delipidation from Nocardia opaca.1 NDCM stimulates proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells.2 The mucosa of small intestine, especially the enterocytes, cells with digestive and absorptive function, produce a number of glycohydrolases. The disaccharidase (sucrase, lactase, and glucoamylase) activities of brush border membrane vesicles (BBMV) of enterocytes after a short-term NDCM- treatment have not been studied. Measurement of lymphocyte proliferation is an established method of quantifying the immune response to foreign antigens. Antigenic stimulation of human peripheral blood lymphocytes by NDCM was measured by Barot-Ciorbaru.2 Neither 3H -TdR-nor 3H-UdR-uptake by T cells has been measured after NDCM stimulation.