We investigated intraepithelial T cells from the small intestine, SI (jejunum, ileum) and the large intestine, LI (colon) of euthymic (BALB/c, H-2d; C.B-17+/+, H-2d; C57BL/6, H-2b) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. From individual euthymic and athymic mice, 7 x 10(6) intraepithelial lymphocytes (IEL) per mouse were isolated from the SI. Ten-fold lower numbers of IEL were obtained from the LI epithelium (4 x 10(5) IEL per mouse). Thymus-dependent and -independent T cells represented > 80% of SI-IEL but the fraction of T cells was reduced from 20% to 40% in LI-IEL. In euthymic mice, alpha beta T cells predominated in SI-IEL and in particular in LI-IEL populations, while SI-IEL and LI-IEL populations of athymic mice contained predominantly gamma delta T cells. The intraepithelial T cell subset distribution was different in SI versus LI: mainly CD8+ T cells were present in the SI, but a large CD4+ T cell subset was present in the LI. 'Double positive' CD4+ CD8 alpha+ T cells were present mainly in the SI epithelium but were rare in the LI epithelium. In euthymic as well as athymic mice, T cells expressing the homodimeric CD8 alpha alpha isoform predominated in the SI epithelium, while T cells expressing the heterodimeric CD8 alpha beta isoform predominated in the LI epithelium. LI-derived TCR alpha beta+ IEL displayed the CD2+ CD28+ LPAM-1/2- M290+ phenotype, and a fraction of them expressed the L-selectin LECAM-1. In contrast, a large fraction of TCR alpha beta+ SI-IEL was CD2- CD28- LPAM-1/2- M290+ and LECAM-1-. RAG-1/2 expression was detectable by RT-PCR in IEL from the SI but not the LI. Striking differences in phenotype were thus apparent between thymus-dependent and thymus-independent T cells in the epithelial layer of the jejunum/ileum and the colon of the mouse.

The initial reactions in anaerobic metabolism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o- or p-cresol as sole organic substrate with a generation time of 11 h; o- or p-cresol was completely oxidized to CO2 with nitrate being reduced to N2. A denitrifying Pseudomonas-like strain oxidized m- or -p-cresol as the sole organic growth substrate completely to CO2 with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps: (1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol----4-OH-benzaldehyde----4-OH-benzoate----4-OH-benzoyl-CoA----be nzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating). (2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH3-benzoyl-CoA as central intermediate as follows: o-cresol----4-OH-3-CH3-benzoate----4-OH-3-CH3-benzoyl-CoA----3-CH3-benzo yl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between 14CO2 and the carboxyl of 4-OH-3-CH3-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH3-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH3-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH3-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH3-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH3-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH3-benzoate and further to 3-CH3-benzoyl-CoA. (3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4- and 3,4-dimethylphenol were oxidized to 4-OH-3-CH3- and 4-OH-2-CH3-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.

SUMMARYTransfer of 2 2 10 5 congenic or semiallogenic purified TCR®¯ CD4 T cells to SCID mice leads to an infiltration of the recipient gut lamina propria and epithelium with a donor-derived CD4T cell subset which induces a lethal inflammatory bowel disease (IBD) in the recipients. In contrast, IBD was not observed in SCID mice transplanted with unfractionated splenic cells. The earliest detectable pathological changes after CD4T cell transfer were proliferation and hypertrophy of the entire colonic epithelial layer, including increased mitotic activity, increased expression of epithelial nuclear proliferation antigen, and elongation of the crypts. Later on, massive mononuclear cell infiltration, hypertrophy of all layers of the colon and occasional epithelial ulcerations were observed. At this stage, accumulations of IgA, IgM and small numbers of IgG1-, IgG2-and IgG3-secreting plasma cells were present in the lamina propria of both the small and large intestine. We conclude that low numbers of intraveneously transferred CD4T cells induce IBD in SCID mice. In the late stages of CD4 T cellinduced IBD, the colonic lamina propria becomes infiltrated with macrophages, neutrophils and plasma cells secreting IgA, IgM, and to a lesser degree IgG antibodies which might play an accessory role in the pathogenesis of IBD.

We studied which T cell subsets from the gut-associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H-2d (BALB/c, BALB/cdm2, C.B-17+/+) mice. No antigen receptor-expressing lymphoid cells were found in GALT of congenic C.B-17 scid/scid (scid) mice. The heterotopic transplantation of a full-thickness gut wall graft from the ileum or colon of immunocompetent (C.B-17+/+, BALB/cdm2) donor mice onto immunodeficient scid mice selectively reconstituted a CD3+ T cell receptor alpha beta+ CD4+ T cell subset. CD4+ cells of this subset expressed the surface phenotype of mucosa-seeking, memory T cells. In the immunodeficient scid host, this gut-derived CD4+ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial layer and lamina propria of the small and large intestine, but not in peripheral LN. Scid mice heterotopically transplanted with gut from a congenic, immunocompetent donor developed clinical and histological signs of inflammatory bowel disease (IBD). Hence, the selective repopulation of GALT compartments with CD4+ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.

Polyclonal, mucosa-seeking memory/effector CD4+ T cells containing a large fraction of blasts activated in situ accumulate in the gut lamina propria of severe-combined immunodeficient (SCID) mice developing colitis after CD4+ T cell transplantation. CD4+ T cells isolated from different repopulated lymphoid tissues of transplanted SCID mice proliferate in vitro in the presence of interleukin (IL)-2 + IL-7. CD3 ligation enhances this cytokine-supported proliferation in CD4+ T cells from the spleen and the mesenteric lymph node of transplanted SCID mice; CD3 ligation suppresses the cytokine-supported proliferation in CD4+ T cells from the gut lamina propria in a cell density- and dose-dependent manner. Almost all CD4+ T cells from repopulated lymphoid tissues of transplanted SCID mice express CD95 (Fas) on the cell surface, and a large fraction of CD4+ T cells from the gut lamina propria of transplanted SCID mice express the Fas ligand on the surface. Gut lamina propria CD4+ T cells show Fas-dependent cytotoxicity. A large fraction of gut lamina propria CD4+ T cells that infiltrate the inflamed colon in transplanted SCID mice are activated in situ and many CD4+ T cells are apoptotic. Hence, a large fraction of colitis-inducing CD4+ T cells undergo activation-induced cell death in situ and can damage other cells through Fas-dependent cytotoxicity.