Early bacterial dependent induction of inducible nitric oxide synthase (iNOS) in epithelial cells upon transfer of CD45RB(high) CD4(+) T cells in a model for experimental colitis.

BACKGROUND: Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear. METHODS: In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB(high) CD4(+) T cells to SCID mice. CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter muridarum, act A(-) mutant Listeria monocytogenes, segmented filamentous bacteria (SFB), or Ochrobactrum anthropi. RESULTS: Within 2 weeks CNV SCID mice injected with CD45RB(high) CD4(+) T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4(+) T cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H. muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice monoassociated with SFB did not show any iNOS expression, whereas SCID mice monoassociated with act A(-) mutant L. monocytogenes and O. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis. CONCLUSIONS: These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD.

Recent immune status determines the source of antigens that drive homeostatic T cell expansion.

Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host's previous state of T cell immunocompetency.

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts.

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Commensal bacteria influence innate status within gingival tissues: a pilot study.

BACKGROUND: The objective of this study was to determine the contribution of commensal bacteria to the innate defense status of gingival tissue by examining the expression of innate host defense mediators in germ-free and conventionally reared groups in both BALBc/ByJ and SCID C.B17 mice. METHODS: Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was utilized to determine the constitutive levels within each gingival tissue set (N = 5) for: E-selectin, P-selectin, interleukin-(IL)-8 homologue, tumor necrosis factor-alpha, IL-1beta, intercellular adhesion molecule-(ICAM)-1, ICAM-2, and vascular adhesion molecule-(VCAM)-1. In addition, IL-1beta protein content was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Gingival samples revealed that only IL-1beta mRNA expression among all mediators examined was significantly reduced in conventionally reared mice (P<0.01) compared to germ-free mice. In contrast, IL-1beta protein levels were significantly (P <0.001) higher in conventionally reared mice compared to germ-free animals. Conventionally reared and germ-free SCID C.B17 mice revealed a similar pattern in regard to reduced IL-1beta mRNA and significantly increased IL-1beta protein (P<0.0001). CONCLUSION: Commensal microbial colonization influences innate host defense mediator expression of IL-1beta at both the mRNA and protein levels in healthy periodontal tissue in mice.

Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2, a novel goblet cell-specific protein.

BACKGROUND & AIMS: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules. These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation. A limited number of intestinal goblet cell-specific proteins have been identified. In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene. METHODS: The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide. Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments. RESULTS: Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon. High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions. Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice. Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours. CONCLUSIONS: These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria.

B1 cells contribute to serum IgM, but not to intestinal IgA, production in gnotobiotic Ig allotype chimeric mice.

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.

Experimentally induced gluten enteropathy and protective effect of epidermal growth factor in artificially fed neonatal rats.

BACKGROUND: A protective effect of breast-feeding against the development of celiac disease has been described, but the nature and effects of the actual milk components have not been established. Epidermal growth factor (EGF), a milk cytokine affecting the proliferation and differentiation of mucosal epithelial cells, was studied as to its potential protective effect on the damage of intestinal mucosa by gliadin in a model system. METHODS: Enteropathy was induced by gliadin in inbred AVN strain rat pups delivered by cesarean section, breast-fed, or hand-fed a milk formula. All experimental groups were treated with interferon-gamma (1,000 U per animal, administered intraperitoneally) after birth. Gliadin (0.5 and 3 mg) was intragastrically administered to the pups on days 0 and 3, and a 30-mg challenge dose was given on day 20 (24 hours before the termination of the experiment). One group of artificially fed pups received EGF (100 ng/ml) continuously in the diet. RESULTS: Gliadin- and interferon-gamma-treated formula-fed rat pups showed villus atrophy, increase of inflammatory cells, including CD4+ T lymphocytes in the lamina propria, and damage to epithelial tight junctions and the enterocyte brush border. Morphometrically, the villus height was significantly less than in other groups. Recombinant EGF was markedly increased in the epithelial cells of injured jejunum. The intestinal mucosa of gliadin- and interferon-gamma-treated pups kept on a EGF-supplemented artificial diet resembled that of breast-fed pups. CONCLUSION: Pathologic changes in jejunal mucosa (villus atrophy and inflammation) resembling gliadin-induced atrophy appeared on administration of interferon-gamma and gliadin to rat pups fed an artificial milk diet immediately after birth. Addition of EGF to the diet protected the rats against pathologic mucosal changes.

Distinct mechanisms for cross-protection of the upper versus lower respiratory tract through intestinal priming.

A main feature of the common mucosal immune system is that lymphocytes primed in one mucosal inductive site may home to distant mucosal effector sites. However, the mechanisms responsible for such cross-protection remain elusive. To address these we have used a model of local mucosal infection of mice with reovirus. In immunocompetent mice local duodenal priming protected against subsequent respiratory challenge. In the upper respiratory tract this protection appeared to be mainly mediated by specific IgA- and IgG2a-producing B cells, whereas ex vivo active effector memory CTL were found in the lower respiratory tract. In accordance with these findings, clearance of reovirus from the lower respiratory tract, but not from the upper respiratory tract, of infected SCID mice upon transfer of gut-primed lymphocytes depended on the presence of T cells. Taken together this study reveals that intestinal priming leads to protection of both the upper and lower respiratory tracts, however through distinct mechanisms. We suggest that cross-protection in the common mucosal immune system is mediated by trafficking of B cells and effector memory CTL.

Monoassociation of SCID mice with Helicobacter muridarum, but not four other enterics, provokes IBD upon receipt of T cells.

BACKGROUND & AIMS: Recently, a number of animal models for different aspects of inflammatory bowel disease (IBD) have been developed. The aim of this study was to use one of these to determine whether particular, ostensibly innocuous, intestinal bacteria could provoke or exacerbate IBD. METHODS: Conventionally reared C.B17 SCID mice were compared with germ-free and gnotobiotic mice, monoassociated with 1 of 5 intestinal bacteria, after transfer of CD45RB(high) CD4(+) T cells from conventionally reared congenic BALB/c mice. Recipient mice were monitored over 7-12 weeks for clinical signs of IBD, and tissues were analyzed by histology/flow cytometry for abnormal inflammation and CD4(+) T cell outgrowth. RESULTS: Neither germ-free mice nor mice monoassociated with segmented filamentous bacteria, Ochrobactrum anthropi, a nonpathogenic mutant of Listeria monocytogenes, or Morganella morganii developed any signs of IBD. In contrast, mice monoassociated with Helicobacter muridarum displayed an accelerated development of IBD in 5-6 weeks compared with 8-12 weeks observed in conventionally reared mice. The outgrowth of CD4(+) T cells in spleen and large intestine of H. muridarum monoassociated mice, as well as in conventionally reared mice was significantly higher than that in the other monoassociated mice. CONCLUSIONS: Among the intestinal bacteria tested, H. muridarum can serve as a provocateur of IBD in this model.

Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses.

Peyer's patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of IgA(+) and IgG2a(+) B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract.

Structural and functional differences between putative mucosal inductive sites of the rat.

Nasal-associated lymphoid tissue (NALT), bronchus-associated lymphoid tissue (BALT) and Peyer's patches (PP) were compared structurally and functionally using a model of local mucosal infection of rats with reovirus. Histological analyses showed that BALT lacks the typical lymphoid organization found in NALT and PP. After local reovirus infection, germinal centers developed in NALT with appearance of IgA+ cells, whereas no germinal centers or isotype-switched cells were found in BALT. Production of reovirus-specific IgA was observed in NALT and PP, but only small amounts of specific IgA were secreted by BALT. Both NALT and BALT showed considerable production of IgG2b, whereas this isotype was poorly produced by PP. These data reveal profound qualitative differences between these three mucosal sites, and strongly suggest that BALT is not a mucosal inductive site for reovirus-specific immune responses in the rat.