The naturally occurring population of CD4+ CD25hi regulatory T cells (Tregs) is essential in suppressing potentially autoreactive T‐cells from inducing autoimmune diseases. Although Tregs are unresponsive to typical activation signals under in vitro conditions, these cells readily undergo proliferation upon adoptive transfer into syngeneic lymphopenic hosts. Recently, we have found that the source of antigens driving naïve T cell homeostatic expansion is determined by the recent immune status of the lymphopenic hosts. Consistent with this idea, the magnitude of Treg expansion differed considerably depending on the recent immune status of the host, i.e., in wild‐type hosts rendered acutely lymphopenic by irradiation, Tregs underwent moderate expansion, whereas a vastly stronger expansion was observed in congenic T cell‐deficient hosts, such as RAG– or SCID hosts. Experiments with various mutant mice raised under conventional and germ‐free conditions revealed that the moderate expansion of Tregs in acutely lymphopenic wild‐type hosts is largely driven by self‐MHC/peptide ligands with some contribution from IL‐2, but not from IL‐7 or IL‐15. In contrast, the bulk of the prodigious expansion of Tregs in immunodeficient mice is driven by MHC loaded with foreign peptides. These findings illustrate that Tregs resemble typical naïve T cells in their ability to respond to both self and foreign antigens.

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. In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB CD4 T cells to SCID mice. To this end, CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter.muridarum, act A (-) mutant L. monocytogenes, segmented filamentous bacteria (SFB), or O. anthropi. Within 2 weeks CNV SCID mice injected with CD45RB CD4 T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4 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 mono-associated with SFB did not show any iNOS expression, whereas SCID mice mono-associated with act A(-) mutant Listeria. monocytogenes and Ochrobactrum. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis. 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. Epithelial iNOS expression in the CD45RB transfer model of colitis 103 5.

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.

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 (VH) genes by H-CDR3 spectrotyping, cloning, and sequencing of VH genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of VH genes and multiple clonally related sequences. The restricted usage of VH 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 VH 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.

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.

BACKGROUND AND AIMS:The mechanisms by which commensal bacteria provoke intestinal inflammation in animal models of inflammatory bowel disease (IBD) remain incompletely defined, leading to increasing interest in the innate immune response of the colonic mucosa to bacterial colonisation.METHODS:Using gene expression profiling of colonic RNA from C.B17.SCID germ free mice and those colonised with altered Schaedler's flora, we investigated the innate immune response to bacterial colonisation in vivo. The two most consistently induced gene groups were RegIIIbeta and gamma as well as interferon gamma (IFN-gamma) response genes.RESULTS:Using quantitative reverse transcription-polymerase chain reaction, we showed that RegIIIbeta, RegIIIgamma, and IFN-gamma were constitutively expressed in the colon of conventionally housed SCID mice compared with either germ free SCID or conventionally housed BALB/c mice. Induction of these genes was reproduced by chronic monoassociation of germ free SCID mice with either of two separate gut commensal bacterial species-segmented filamentous bacteria and Schaedler's Escherichia coli. The cellular source for IFN-gamma on monoassociation of SCID mice with Schaedler's E coli was localised to a subset of intraepithelial natural killer (IENK) cells that express asialo-GM1. In vivo IFN-gamma immunoneutralisation studies failed to demonstrate any alteration in RegIIIbeta or gamma expression.CONCLUSIONS:Thus bacterial colonisation of the colon independently activates two distinct innate immune cell types at the mucosal interface with the colonic lumen, intestinal epithelial cells, and IENK cells, a response that may be regulated by the adaptive immune system. These innate immune responses may play a role in the pathogenesis of colitis in SCID adoptive transfer models in mice and possibly in patients with IBD.

This chapter discusses the role of mucosal microbiota in the development, maintenance, and pathologies of the mucosal immune system. Most mucosal sites of lymphoid tissue—respiratory tract, adenoids, salivary glands, and urogenital tract—in healthy mammals are in a quiescent state and generally resemble the status of lymphoid areas in spleen and most peripheral lymph nodes (PLN). The intestinal tract, palatine tonsils, and occasionally the nasal-associated lymphoid tissue (NALT) are the exceptions. These mucosal lymphoid tissues are in a “physiologically normal state of inflammation.” The chapter focuses on how the intestinal microbes drive the development of gut-associated lymphoid tissue (GALT) during neonatal life and act to maintain its physiologically normal steady state of inflammation. Specific and adaptive, “natural” and semi-specific, and aspecific elements of the mucosal immune systems may benefit and be activated by host interactions with environmental antigens (Ags). To provide the experimental rationale for implicating intestinal or oral/nasal microflora in the development of GALT, palatine tonsil, and sometimes NALT and their steady state of inflammation, the chapter briefly contrasts the status of systemic lymphoid tissue in healthy mammals—spleen, PLN—with GALT, palatine tonsils, and NALT.

Although mechanisms operative in the induction and maintenance of specific, adaptive immunity, including 'cognate' B/T interactions, have been extensively studied and defined, we still know little about the mechanisms operative in developing and maintaining B- and T-cell dependent 'natural' immunity. Particularly, we are still rather ignorant concerning gut microbial/gut or systemic APC, T cell and B cell interactions that lead to lymphoid cell mediated 'natural' immunity: specific or broadly reactive, activation via TCR and BCR and/or via other receptors such as the TLR series, and whether T/B interactions are operative at this level? Here we will address: (1) the general role of gut microbes in the development and maintenance of the intestinal, humoral immune system; (2) the general role of gut microbes in the development of B1 cell mediated, 'natural' gut IgA and the dependence of these B1 cells on bystander T cell help; (3) the relative contributions of B1 versus B2 cells to gut 'natural' and specific IgA responses; (4) the role for particular 'normal' gut microbes in the initiation of inflammatory bowel diseases (IBD) in mice with a dysregulated immune system; and (5) the possible roles of gut microbes in facilitating oral tolerance, a mechanism likely operative in forestalling or ameliorating IBD. A central theme of this paper is to attempt to define the specificities of activated, functional CD4+ T cells in the gut for Ags of particular, usually benign gut microbes. We will also consider the still-unresolved issue of whether the contributions of B1-derived IgA in the gut to the 'natural' Ab pool are Ag-selected and driven to proliferation/differentiation or whether the main stimuli are not via BCRs but rather other receptors (TLRs, etc.). The main experimental approach has been to use antigen-free, germ-free, or gnotobiotic (mono- or oligo-associated with precisely known bacterial species) mice.

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.

Backgr Backgr Backgr Backgr Background: ound: ound: ound: ound: We have recently shown that the development of colitis in SCID mice injected with CD45RB high CD4 + T cells is dependent on their gut flora. The mechanisms of this T cell and bacteria dependent induction of colitis are unclear. Since iNOS is expressed in epithelial cells of the inflamed mucosa of patients with inflammatory bowel disease (IBD), nitric oxide could be involved in this model of colitis. Aim: Aim: Aim: Aim: Aim: To study the induction of iNOS in relation to T cell infiltration and cytokine production in conventionally reared and mono-associated SCID mice in the CDRB45 high CD4 + T cell transfer model of colitis. Methods: Methods: Methods: Methods: Methods: CB-17 SCID mice were reared with conventional flora (CNV) or mono-associated with H.muridarum, act A (mutant nt L. monocytogenes, Segmented Filamentous Bacteria (SFB), or O. anthropi under specific pathogen free conditions. and mono-associated mice 6 to 11 weeks after injection of the CD45RB high CD4 + T cells. The iNOS and CD4 proteins were detected by immunohistochemistry. RT-PCR was performed for iNOS, TNF-α, IL-1β, IFN-γ, Gro, mMIP mRNA detection. Results: Results: Results: Results: Results: In CNV SCID mice injected with CD45RB high CD4 + T cells an early and focal, epithelial iNOS expression on the apical site of villi was observed that preceded the infiltration of CD4 + cells and cytokine production. This epithelial iNOS expression extended to the entire surface along the whole crypt axis in the course of colitis. SCID mice monoassociated with H.muridarum developed an severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice mono-associated with SFB did not show any iNOS expression, whereas SCID mice mono-associated with act A(mutant nt 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. Conclusion: Conclusion: Conclusion: Conclusion: Conclusion: Epithelial iNOS expression is an early bacteria dependent event in the CDRB45 high CD4 + T cell transfer model of colitis. These findings suggest that bacteria specific epithelial cell activation and subsequent iNOS induction and NO production is important for the development of IBD.

The h u m a n receptor for C3b and C4b (CR1) was initially isolated from pooled donor erythrocyte membranes and characterized as an integral membrane glycoprotein of ~205,000 dahons on SDS-polyacrylamide gels (1-3). Polyelonal antibodies against this glycoprotein immunoprecipi ta ted a similar molecule from other CR1bearing cells such as polymorphonuclear cells (PMN), macrophages, and B lymphocytes (1). Recently, we have demonstrated that CR1 is polymorphic in erythrocytes (E) obtained from normal donors (4). Autoradiographs o f purified surface-labeled receptor demonst ra ted that ~70% of individuals had a single major band at 190,000 daltons, ~3% of individuals had a major band at 220,000 dahons and the remaining individuals had both major bands at 190,000 and 220,000 dahons. Family studies provided evidence for transmission of the 190,000and 220,000-dalton major bands of CR1 by two codominant alleles. We have now extended these investigations by analyzing the structure of this receptor on h u m a n leukocytes. These studies demonstrate that a l though leukocytes from individual donors express CR1 polymorphism similar to that on E, specific structural differences in CR1 exist among peripheral blood cells.

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 physio-logic 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 RUMP 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, germfree, 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.