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Elimination of Colonic Patches with Lymphotoxin Receptor-Ig Prevents Th2 Cell-Type Colitis¹

Taeko Dohi^2,*,, Paul D. Rennert, Kohtaro Fujihashi^,, Hiroshi Kiyono^,¶, Yuko Shirai^*, Yuki I. Kawamura^*, Jeffrey L. Browning and Jerry R. McGhee

^* Department of Gastroenterology, Research Institute, International Medical Center of Japan, Tokyo, Japan; Immunobiology Vaccine Center, Departments of Microbiology and Oral Biology, University of Alabama, Birmingham, AL 35294; Biogen, Inc., Cambridge, MA 02142; and ^¶ Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin- receptor-Ig fusion protein (LTR-Ig) in utero, a treatment that blocked the formation of both Peyer’s and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LTR-Ig could be used to treat colitis in normal or Th2-dominant, IFN- gene knockout (IFN-^-/-) mice. Four weekly treatments with LTR-Ig resulted in deletion of Peyer’s and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-^-/- mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LTR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-. Thus, LTR-Ig may offer a possible treatment for the Th2-dominant form of colitis.

	Introduction

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Murine models for inflammatory bowel disease (IBD)³ are most conveniently studied in an Ag-specific manner by rectal exposure of colonic mucosa to the hapten, 2,4,6-trinitrobenzene sulfonic acid (TNBS) (1, 2, 3, 4). The TNBS-induced model has the important advantage of using normal mice with an intact immune system, thus allowing a systematic investigation of the exact roles of individual immunocompetent cells for the development of mucosal inflammation. Early evidence suggested that TNBS induced Th1-type responses that subsequently led to colitis. T cells from mice with TNBS colitis produced both IL-2 and IFN- (Th1-type), and in situ analysis of lesions revealed increased IFN- production (2). Treatment of mice with anti-IL-12 mAb markedly decreased the severity of TNBS colitis (2, 5). We previously used this model to show that mice that develop either predominant CD4⁺ Th1- or Th2-type responses exhibit distinct inflammatory lesions: Th1-dominant IL-4 gene knockout (IL-4^-/-) mice developed focal penetrating ulcers, whereas colitis induced in Th2-dominant IFN-^-/- mice was characterized by diffuse, atrophic changes in crypts accompanied by marked hypertrophy of colonic patches (6, 7). These characteristics of Th2-type hapten-induced colitis share the feature of ulcerative colitis in humans, which is considered to be Th2-mediated enteric inflammation (8, 9). Th2-type TNBS colitis was also accompanied by marked hypertrophy of colonic patches containing M cells and a follicle-associated epithelium covering the dome region. In addition, TNBS-specific Th2-type cytokine responses occurred in the colonic patches themselves (6).

Because there exists an association between colonic patch development and Th2-type responses, it is important to assess the role of colonic patches in colitis. It has been shown that lymphotoxin (LT) signaling is required for the genesis of Peyer’s patches and lymph nodes (10). For example, LT gene-deleted (LT^-/-) mice are deficient in both Peyer’s patches and lymph nodes (11). Furthermore, treatment of LT^-/- mice with an agonist mAb to LTR-induced lymph node development (12), whereas knockout of the LTR gene again resulted in failure of mice to develop Peyer’s patches and lymph nodes (13). Interestingly, treatment with soluble LTR-Ig fusion protein (LTR-Ig) in utero resulted in deletion of Peyer’s patches and peripheral lymph nodes; however, mesenteric and sacral lymph nodes (SLN), which drain the small and large intestines, remained intact (14, 15). Although the precise cellular and molecular mechanisms for the genesis of gut-associated lymphoreticular tissues (GALT) is not yet known, it is clear that membrane-associated LT12 on activated lymphocytes and LTR expressed by macrophages and dendritic cells partially account for the development of this mucosal inductive site (16).

Blockade of LTR in adult mice with LTR-Ig prevents germinal center formation in spleen and results in impaired IgG anti-sheep RBC Ab responses (17). Membrane LT12 expression by B cells and that of LTR by stromal cells play crucial roles in the formation of follicular dendritic cell networks (10, 18, 19, 20, 21). These studies have suggested that LT12/LTR interactions are necessary not only for ontogenesis of lymphoid tissue, but also for maintaining the normal architecture required for immune responses. The LT system is critical for the generation of chemokine gradients that are essential for cellular positioning (22) and for recruitment of NK and dendritic cells (23, 24). Likewise, disruption of the surface LT or LIGHT (another ligand for LTR)-LTR axis affects the generation of CD8⁺ T cells in anti-lymphochoriomeningitis virus immune responses (25, 26). Blocking of the LT/LIGHT axis with LTR-Ig was shown previously to be effective in preventing colitis in the CD3 and CD45RB^high T cell transfer models of colitis (27).

In this study we have generated Peyer’s patch-deficient mice by treatment with LTR-Ig fusion in utero to determine the effects of this treatment on the genesis of colonic patches as well as on the development of TNBS colitis. We also tested the idea that colonic patches are associated with Th2-type responses in three different aspects of murine colitis: 1) in mice deficient in Peyer’s patches, 2) in adult mice treated with LTR-Ig only, and 3) in adult mice treated with LTR-Ig with predominant Th2-type responses.

	Materials and Methods

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Fusion proteins

Fusion proteins were from the extracellular domain of either murine LTR or human TNFR p55 fused to the hinge, CH₂, and CH₃ domains of human IgG1 (LTR-Ig or TNFR p55-Ig, respectively) and were prepared as described previously (14, 28, 29).

Generation of Peyer’s patch-deficient mice

Timed pregnant BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in sterile cages under pathogen-free conditions at Biogen (Cambridge, MA) and under specific pathogen-free conditions in the immunocompromised mouse facility at the Immunobiology Vaccine Center, University of Alabama (Birmingham, AL). Mice were provided sterile food and water ad libitum and were free of microbial pathogens as determined by Ab screening and routine histologic analysis of organs and tissues. Because it has been shown that the genesis of lymph nodes and Peyer’s patches can be blocked in a sequential manner by varying the gestation day of LTR-Ig treatment (14, 15), we generated mice that had disrupted Peyer’s patches but that retained mesenteric and SLN for subsequent studies of colonic patch formation and TNBS colitis development. To do this, pregnant mice were injected i.v. with 200 µg LTR-Ig on gestational days 14 and 17 as described previously (14, 15). Mice born in the same facility and of the same age as those in the experimental group were used as controls.

Treatment of adult mice with fusion proteins and mAbs

IFN- gene-deficient (IFN-^-/-) mice and background/age-matched, normal BALB/c mice were purchased from The Jackson Laboratory and kept in our animal facility at the Immunobiology Vaccine Center or at the Research Institute, International Medical Center of Japan (Tokyo, Japan), under specific pathogen-free conditions as described above. Adult mice (8 wk of age) were given 200 µg LTR-Ig, TNFR p55-Ig, or control normal human IgG (control IgG) i.p. Young adult mice (8 wk of age) were given these fusion proteins on days -14, -7, 0 (the day of induction of colitis), and 7. In some experiments groups of mice were given 1 mg rat anti-IFN- mAb (clone XMG 1.2) by the i.p. route on days 0 and 7. Control groups of mice received i.p. doses of normal rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Induction of TNBS colitis

Mice were given rectally a solution of TNBS (Research Organics, Cleveland, OH) dissolved in a mixture of PBS (pH 7.2) and then mixed with an equal volume of ethanol at a final concentration of 2% TNBS in 50% ethanol. After being anesthetized with ketamine/xylazine, mice were given intrarectally 36 µg TNBS/g body weight on days 0 and 7 using a glass microsyringe equipped with a gastric intubation needle. Tissues and cells were assessed 3 days later (day 10).

Myeloperoxidase (MPO) assay

MPO activity in murine colonic tissues was determined by a method reported previously (30). Briefly, colonic tissues were homogenized in 5 mM phosphate buffer (pH 6.0) and centrifuged at 30,000 x g for 30 min at 4°C. The pellet was suspended in 50 mM phosphate buffer containing 0.5% hexadecyltrimethyl ammonium bromide. After centrifugation at 20,000 x g, the supernatant was subjected to MPO assay in a spectrophotometer with a rate assay system (U3200; Hitachi, Tokyo, Japan).

Histologic analysis and immunohistochemistry

The colon was removed from its mesentery to the pelvic brim by blunt dissection, opened longitudinally, and fixed in 5% glacial acetic acid in ethanol (v/v). After embedding in paraffin, 4-µm serial sections were prepared and stained with hematoxylin and eosin for histologic grading. Some specimens were snap-frozen, and 4-µm frozen sections were prepared. Histologic grading was performed on coded slides without knowledge of mouse group and according to the criteria described previously (6). For immunohistochemistry, frozen sections were fixed with cold acetone and incubated with 10% normal rabbit serum for blocking. Sections were then incubated with anti-mouse MAdCAM-1 mAb (x100; BD PharMingen, San Diego, CA), biotin-anti-mouse CD11c (x100, BD PharMingen), biotin-peanut agglutinin (biotin-PNA; x100, Vector Laboratories, Burlingame, CA), or FDC-M1 mAb (20 µg/ml; provided by Dr. K. Maeda, Yamagata University, Yamagata, Japan) for 1 h. Bound anti-MAdCAM-1 mAb or mAb FDC-M1 was detected with FITC-labeled anti-rat IgG (Southern Biotechnology Associates, Birmingham, AL). CD11c and PNA stainings were detected with FITC-streptavidin. For T and B cell analysis, each section was stained with FITC-anti-B220 and biotin-anti-CD3 mAb (BD PharMingen), followed by incubation with tetramethyl rhodamine-streptavidin (Southern Biotechnology Associates). Sections were examined with a fluorescence microscope (BX50/BXFLA, Olympus, Tokyo, Japan) equipped with a CCD camera and an image analyzer (ATTO densitograph; ATTO, Tokyo, Japan). Combination images of T and B cell staining were analyzed using identical settings in Photoshop 4.0 (Adobe Systems, San Jose, CA).

Isolation of lymphoid cells

Peyer’s and colonic patches were excised from the intestinal wall and washed once with RPMI 1640 (Cellgro; Mediatech, Washington, D.C.), and single cells were dissociated with collagenase (type V, Sigma, St. Louis., MO) at a concentration of 0.5 mg/ml in RPMI 1640 with 100 U/ml penicillin, 100 µg/ml streptomycin, and 40 µg/ml gentamicin for 20 min at 37°C (31). The cell dissociation step was performed twice more using fresh collagenase solution each time. Colonic lamina propria lymphocytes were prepared as described previously (32), with modification. Briefly, after excision of all visible lymphoid follicles, the colonic tissue was treated with 1 mM EDTA in PBS for 20 min to remove the epithelium. The tissue was then digested with collagenase (type V; Sigma) for 20 min, and this step was repeated one more time. The single-cell suspensions were then pooled and washed with RPMI 1640 two additional times. Mononuclear cells were further purified using a discontinuous Percoll gradient to avoid contamination with epithelial cells (32). The SLN were teased apart with forceps, and the resulting cell suspensions were washed twice more in RPMI 1640.

Culture conditions and proliferation assays

Cells from SLN were conjugated with TNBS by treatment of 1 x 10⁷ cells with 1 ml of 0.3 mg/ml TNBS in RPMI 1640 at room temperature for 15 min. Excess TNBS was removed by washing with RPMI 1640 and centrifugation of conjugated cells. For Ag-specific stimulation, conjugated cells were cultured in RPMI 1640 supplemented with 10% FCS, sodium pyruvate, L-glutamine, HEPES buffer, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentamicin, and 1 µg/ml amphotericin B (complete medium) at 37°C in an atmosphere of 5% CO₂ in air. For the proliferation assay (6), 2 x 10⁵ TNBS-conjugated cells were added to wells of 96-well plates and cultured for 3 days, and 0.5 µCi/well [³H]TdR was added 18 h before cell harvesting. The levels of [³H]TdR incorporation was determined by scintillation counting. Cells cultured without treatment with TNBS were used as controls. In some experiments TNBS-treated cells were cultured in complete medium for 24 h at a concentration of 3 x 10⁶ cells/ml. Culture supernatants were obtained and then assessed for cytokine levels as described below. Cells were stained with FITC-anti-CD4 mAb (BD PharMingen) and subjected to sorting with a FACStar^Plus (BD Biosciences, Mountain View, CA) to obtain purified CD4⁺ T cells (99% CD4⁺ T cells). Cytokine-specific RT-PCR was performed as described previously (33). In some experiments quantitative RT-PCR for mRNA expression of IFN- and IL-4 was performed using ABI PRISM 7700 with Taqman probes (PE Applied Biosystems, Foster City, CA). The PCR product for each cytokine was used as a standard for template DNA.

Cytokine assays

Culture supernatants were subjected to cytokine-specific ELISA as described previously (32, 34, 35). In brief, microtiter plates were coated with mAbs to individual cytokines and incubated overnight at 4°C. After blocking with 3% BSA in PBS at 37°C for 2 h, diluted samples were added to wells and incubated overnight at 4°C. The wells were then washed and incubated with detection mAbs, and bound Ab was detected using a peroxidase-labeled anti-biotin mAb (Vector Laboratories). The substrate used for peroxidase activity was 3,3',5,5'-tetramethylbenzidine. The following anti-cytokine mAbs were used for coating or in the biotinylated form for detection, respectively, in this ELISA: anti-IFN-, R4-6A2 and XMG 1.2 mAbs; anti-IL-2, JES6-1A12 and JES6-5H4 mAbs; anti-IL-4, BVD4-1D11 and BVD6-24G2 mAbs; anti-IL-5, TRFK-5 and TRFK-4 mAbs; anti-IL-6, MP5-20F3 and MP5-32C11 mAbs; and anti-IL-10, JES5-2A5 and JES5-16E3 mAbs. The ELISAs were capable of detecting 0.4 ng/ml IFN-, 0.05 U/ml IL-2, 3.0 pg/ml IL-4, 0.2 U/ml IL-5, 0.2 ng/ml IL-6, and 0.04 ng/ml IL-10. The levels of cytokine production by Ag-stimulated T cells were calculated by subtracting the results of control cultures that were not stimulated with TNBS.

Statistics

The data are expressed as the average ± 1 SD and were compared using the Mann-Whitney U test. The results were analyzed using the StatView-J 4.4 statistical program (Abacus Concepts, Berkeley, CA) for Macintosh computers and were considered statistically significant if p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Colonic patches are absent in mice treated in utero with LTR-Ig (generation of colonic patch-deficient mice)

In the initial experiments mice were treated in utero with LTR-Ig to determine whether loss of Peyer’s patches could be associated with a failure to form colonic patches. No visible Peyer’s patches were present in these mice; however, mesenteric and SLN developed normally as reported previously (14, 36). The mice treated in utero with LTR-Ig did not have visible colonic patches. To verify this, histologic sections of small and large intestines were assessed for the presence of follicles. No organized structures were seen in either small or large intestine, and only scattered aggregates of B cells were noted in the mucosal layer. These B cell aggregates were also observed in untreated normal BALB/c mice (data not shown).

TNBS colitis in mice treated in utero with LTR-Ig

We next induced TNBS colitis in 10-wk-old control and LTR-Ig-treated colonic patch-deficient mice by administering two doses TNBS-ethanol 1 wk apart. The histologic features of TNBS colitis observed on day 10 in untreated mice and colonic patch-deficient mice were remarkably different (Fig. 1). As noted previously (6), the inflammation induced by TNBS in normal BALB/c mice demonstrated crypt distortion, loss of goblet cells, and mononuclear cell infiltration in the mucosal layer with enlarged colonic patches (Fig. 1, A and C) and occasional ulcers. In contrast, colonic patch-deficient mice also developed colonic inflammation, which was clearly different from the control group. Open ulcers were found in all mice, whereas crypt lesions were less frequent (Fig. 1, B and D). Crypts were more hypertrophic and elongated, resulting in a thickened colonic wall. Transmural cell infiltration was seen with a predominant infiltration of polymorphonuclear leukocytes, even in the submucosa under the intact lamina muscularis mucosae (Fig. 1, B and D). An infiltration of polymorphonuclear leukocytes in colonic patch-deficient mice was verified by the finding of significant levels of MPO activity (Fig. 2A). This MPO activity was increased in the colon of mice after TNBS enema and was higher in colonic patch-deficient than in normal mice. No colonic patches were seen after induction of TNBS colitis. To control for vehicle-only effects, colonic patch-deficient mice were also given a 50% ethanol enema, This treatment resulted in only minimal superficial erosion and was identical with that induced in normal mice. We conclude that both mouse groups developed inflammation, but with distinct characteristics. Mice treated with LTR-Ig exhibited higher scores for acute ulcer-related lesions and lower scores for chronic-type inflammation and crypt changes (Fig. 2B).

View larger version (126K): [in this window] [in a new window]   FIGURE 1. Histologic features and cytokine responses in TNBS colitis induced in normal and colonic patch-deficient mice. Colonic lesions of control mice (A and C) and mice treated with LTR-Ig in utero (colonic patch deficient; B and D) are shown. Mononuclear cell infiltrates in the mucosal layer in control mice and polymorphonuclear leukocytes (PMN) cell infiltrates in the submucosa in colonic patch-deficient mice are shown in the lower panels. Top, Direction of the lumen; arrowheads indicate the layer of muscularis mucosae in C and D. The original magnification was x10 in the upper panels and x100 in the lower panels.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Disease severity in normal and colonic patch-deficient mice with TNBS colitis. A, MPO activity in the colon. All data are shown as the average ± 1 SD of four mice per group. B, Histologic scores are shown as the average ± 1 SD of mouse groups containing between four and eight mice. Open bars, normal BALB/c mice treated with ethanol; dotted bars, colonic patch-deficient mice treated with ethanol; hatched bars, normal mice treated with TNBS; shaded bars, colonic patch-deficient mice treated with TNBS; solid bars, colonic patch-deficient mice treated with TNBS and anti-IFN- mAb. Statistically significant differences are indicated by an asterisk.

The SLN of mice with TNBS colitis were enlarged and contained lymphoid cells undergoing proliferation. In a previous study (6) we extensively assessed TNBS-induced cytokine responses in lamina propria, colonic patches, and SLN and showed that the cytokine responses in the SLN accurately reflect the immunopathologic responses in colonic patches and lamina propria. Based on this analysis, we assessed cells from SLN of colonic patch-deficient mice for TNBS-specific proliferative and cytokine responses. Cultures of SLN cells exhibited brisk proliferative responses to TNBS-conjugated-self Ag (Fig. 2A). Cytokine production, which accompanied the TNBS-induced proliferative responses, was measured 24 h after stimulation (Fig. 3A). Following restimulation with TNBS, colonic patch-deficient mice did not produce IL-4, whereas control mice produced IFN-, IL-4, and IL-5. To confirm this, purified CD4⁺ T cells from cultures were subjected to cytokine-specific RT-PCR. The expression of mRNA in TNBS-specific CD4⁺ T cells was similar to the results obtained for secreted proteins (data not shown). Thus, in colonic patch-deficient mice, mucosal Th2-type responses were suppressed. Furthermore, to confirm this, lamina propria mononuclear cells were isolated from the colon. The total RNA was extracted, and cytokine-specific mRNA was quantified by real-time RT-PCR. The number of copies of mRNA for IL-4 was significantly lower in colonic patch-deficient mice. We conclude from these results that diffuse crypt-type inflammation is accompanied by Th2-type responses and is dependent upon the presence of colonic patches. In contrast, focal ulcer-type lesions occur independently of colonic patches.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. TNBS-specific proliferative and cytokine responses in normal and colonic patch-deficient mice with TNBS colitis. A, Cells were taken from SLN, and the stimulation index was determined as cpm of wells with TNBS/cpm of wells without TNBS. The level of [3H]TdR incorporation in control wells without TNBS was <600 cpm. B, Cytokine levels in culture supernatants in the same culture as that in A were measured by ELISA and are shown as the average ± 1 SD of three separate experiments using pooled cells from three to five mice per group. C, Expression of mRNA in colonic lamina propria cells. On day 10 after TNBS enema, lamina propria cells from three mice were pooled and subjected to quantitative RT-PCR in triplicate assays. mRNA levels were shown as relative values to the levels in mice treated with ethanol only. Dotted bars, colonic patch-deficient mice treated with ethanol; shaded bars, colonic patch-deficient mice treated with TNBS; hatched bars, normal mice treated with TNBS. Statistically significant differences are indicated by an asterisk.

TNBS colitis in colonic patch-deficient mice treated with anti-IFN- mAb

The results to this point suggest that inflammation in colonic patch-deficient mice is dependent upon IFN-, but not IL-4. We tested this assumption by treatment of TNBS colitis with anti-IFN- mAb. Treatment of murine TNBS colitis with anti-IFN- mAb was ineffective, and IFN-^-/- mice developed colitis, which was as severe as that in wild-type (WT) mice (6, 7). However, in colonic patch-deficient mice, blockade of IFN- markedly reduced the severity of inflammation (Fig. 2). We conclude that the inflammatory changes in colonic patch-deficient mice are largely mediated by IFN-, and that Th2-type inflammatory responses appear to be dependent upon the presence of colonic patches.

LTR-Ig treatment of adult mice decreases the cellularity of both Peyer’s and colonic patches

Our past studies in Th2-type TNBS-induced colitis revealed atrophic changes in the mucosal epithelium, with distorted crypts and fibrosis (6, 7). We proposed that this could be a model for the chronic stage of IBD, especially in ulcerative colitis. In this study we used colonic patch-deficient mice to provide further evidence that Th2-type inflammatory responses require the presence of colonic patches. Our model allowed us to treat Th2-type TNBS colitis by abrogating functional colonic patches. For this purpose we took advantage of the fact that signals through the LTR are also important in the maintenance of secondary lymphoid tissue in adult mice (10, 18, 19, 20, 21). Thus, we assessed the effects of blocking LTR signals by LTR-Ig on TNBS colitis induced in adult WT and IFN-^-/- mice (a model for Th2-dominant colitis). Because the beneficial effect of anti-TNF- mAb in experimental colitis (37, 38) and in treatment of Crohn’s disease has been documented (39, 40), TNFR p55-Ig was also tested for comparison. In our initial study we tested the effect of weekly i.p. administration of LTR-Ig on Peyer’s and colonic patches in adult mice without colitis. We found that weekly doses of LTR-Ig induced a flattened appearance in Peyer’s patches (Fig. 4A). Approximately three or four doses were required for maximum effects on Peyer’s patches, and this treatment also reduced by one-half the total number of macroscopically visible Peyer’s patches in LTR-Ig-treated adult mice. When treatment was discontinued, the Peyer’s patches recovered cellularity and increased in size (Fig. 4A, right panel). Interestingly, the cecal patches did not undergo visible changes in size or shape following administration of LTR-Ig (data not shown). Colonic patches, which are naturally flat, were assessed by histologic analysis. Two consecutive treatments with LTR-Ig resulted in diminished colonic patches with smaller germinal centers, which stained less intensely. The T and B cell zones were essentially intact (Fig. 4B, bottom row). After four treatments, colonic patches were further reduced in size, with only vestiges remaining, and were detected only in histologic sections (Fig. 4B). In untreated mice, CD11c-positive dendritic cells were present in the subepithelial dome of colonic patches and also scattered in the follicles (Fig. 4B, middle row). Two weekly doses of LTR-Ig markedly eliminated CD11c-positive dendritic cells from both areas of colonic patches (Fig. 4B). Similar effects were also observed in IFN-^-/- mice treated with LTR-Ig (data not shown). These effects of LTR-Ig suggested the possibility that this treatment abrogates the function of colonic patches by eliminating cells, including dendritic cells, and reducing cell-to-cell interactions in the colonic patches.

View larger version (86K): [in this window] [in a new window]   FIGURE 4. LTR-Ig treatment results in flattening of Peyer’s and colonic patches in normal adult mice. A, Mice were given 100 µg fusion proteins by the i.p. route. Representative Peyer’s patches from the proximal jejunum/duodenal region are shown. Mice were given control IgG (left) or LTR-Ig (middle) for 8 wk or were given LTR-Ig for 4 wk, followed by a 4-wk recovery period (right). The small intestine was soaked in 10% acetic acid for 1 day before photography. B, LTR-Ig treatment results in reduction of colonic patches with loss of germinal centers and dendritic cells. Mice were given 200 µg LTR-Ig i.p. once weekly, and frozen sections of the colon were prepared after 2 wk (middle) or 4 wk (right). The left column shows colonic patches without treatment. Sections were stained with hematoxylin and eosin, PNA, anti-CD11c and anti-CD3 (red), and anti-B220 (green). Stained areas indicated with an arrow are epithelial cells in an absence of germinal centers. CD11c+ dendritic cells are indicated with arrowheads.

Induction of TNBS colitis in LTR-Ig- and TNFR p55-Ig-treated mice

In the next study we induced TNBS colitis in mice pretreated with LTR-Ig. Adult mice were treated with either LTR-Ig or TNFR p55-Ig and were then given TNBS enemas following the third dose of fusion proteins. A second dose of TNBS was given at the same time as fusion proteins on day 7. These treated mice were then subsequently assessed for colitis and cellularity of patches on day 10 (Fig. 5). Pretreatment of both WT and IFN-^-/- mice with two doses of LTR-Ig markedly decreased the cellularity of Peyer’s and colonic patches (day 0, Fig. 5). In contrast, TNFR p55-Ig treatment did not affect the number of cells in either Peyer’s or colonic patches. After TNBS enema, both WT and IFN-^-/- mice treated with control IgG had >2-fold more colonic patch cells (day 10, Fig. 5B). In contrast, LTR-Ig-treated, IFN-^-/- mice exhibited markedly reduced numbers of colonic patch cells (Fig. 5B). In LTR-Ig-treated WT mice, most mice showed colonic patch hypertrophy after induction of colitis, and cell yields increased to levels comparable to those seen in mice treated with control IgG (day 10, Fig. 5B). Low numbers of cells were seen in Peyer’s patches of LTR-Ig-treated WT and IFN-^-/- mice even after induction of TNBS colitis. In mice treated with TNFR p55-Ig, the number of colonic patch cells increased after TNBS treatment, even though it remained lower than that in WT or IFN-^-/- mice treated with control-Ig.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. The effects of LTR-Ig and TNFR p55-Ig treatment and TNBS enema on the cellularity of Peyer’s and colonic patches. A and B, Cellularity of Peyer’s and colonic patches, respectively, in adult WT (IFN-+/+, upper panels) and IFN--/- mice (lower panels). LTR-Ig (squares), TNFR-Ig (diamonds), or control Ig (circles) were given on days -14, -7, 0, and 7, and TNBS in 50% ethanol was given rectally to induce colitis on days 0 and 7. Before administration of fusion protein (day -14), on day 0 (before TNBS enema), or on day 10, the numbers of cells in Peyer’s and colonic patches were determined. The values shown are the average ± 1 SD, and each experimental group included between 4 and 10 mice. The asterisk indicates a statistically significant difference from mice treated with control IgG.

In WT mice, LTR-Ig treatment decreased the disease severity only slightly; however, this was not significantly different from control mice (Fig. 6, left panel). LTR-Ig treatment eliminated colonic patches more efficiently in IFN-^-/- mice, and this group had the lowest histologic scores (Fig. 6, right panel). Treatment of normal mice with TNFR p55-Ig did not affect disease. To examine the effect of LTR-Ig in a Th2-type environment, we next treated WT adult mice with both LTR-Ig and anti-IFN- mAb. As noted previously (6), treatment with anti-IFN- mAb alone did not protect mice from TNBS colitis; however, coadministration of LTR-Ig with anti-IFN- mAb resulted in significant protection (Fig. 6, middle panel).

View larger version (34K): [in this window] [in a new window]   FIGURE 6. The effects of LTR-Ig and TNFR p55-Ig treatment on the severity of TNBS colitis. The histopathologic scores on day 10 in the experiment shown in Fig. 5 are shown for WT (IFN-+/+) and IFN--/- mice with TNBS colitis that had been treated with fusion proteins with or without anti-IFN- mAb. The hatched bars show mice treated with LTR-Ig; shaded bars show mice treated with TNFR p55-Ig; open bars indicate mice treated with control human IgG. Values shown are the average ± 1 SD. An asterisk indicates a statistically significant difference from mice treated with control IgG in each experiment.

View larger version (123K): [in this window] [in a new window]   FIGURE 7. Histologic features of the colon of WT adult and IFN--/- mice with TNBS colitis treated with LTR-Ig (hematoxylin and eosin staining). A and C, IFN--/- mice; B and D, WT mice. A and B, Paraffin-embedded sections; C and D, colonic patch vestiges in frozen sections. Original magnification, x10.

View larger version (100K): [in this window] [in a new window]   FIGURE 8. Immunohistochemical analysis of colonic patch vestiges after induction of TNBS colitis. Frozen sections were stained with anti-MAdCAM-1 and PNA to visualize germinal centers, anti-CD11c mAb to detect dendritic cells in the subepithelial dome, and FDC-M1 mAb for assessing follicular dendritic cells in the B cell zone. Original magnification, x30 for anti-MAdCAM-1 and PNA staining, x60 for CD11c and FDC-M1.

TNBS-specific responses were assessed in SLN cultures. No T cell proliferative responses were seen in IFN-^-/- mice treated with LTR-Ig or TNFR p55-Ig, whereas all groups of WT mice exhibited TNBS-specific T cell proliferative responses (Fig. 9A). Cytokine levels followed the degree of proliferative responses induced in culture. In IFN-^-/- mice treated with LTR-Ig or TNFR p55-Ig, a reduction in Th2-type cytokine synthesis was noted (Fig. 9B). The SLN cells from WT mice treated with LTR-Ig, TNFRp55-Ig, or control IgG all produced IFN-, IL-2, IL-4, and IL-5 (Fig. 9B). Reduction of these cytokines by CD4⁺ T cells in LTR-Ig-treated IFN-^-/- mice was confirmed by cytokine-specific RT-PCR (Fig. 9C, lane 4). Thus, although both LTR-Ig and TNFRp55-Ig tended to protect IFN-^-/- mice from TNBS-induced colitis, only LTR-Ig treatment resulted in elimination of colonic patches and prevention of colitis.

View larger version (31K): [in this window] [in a new window]   FIGURE 9. TNBS-specific proliferative responses in WT and IFN--/- mice with TNBS colitis treated with fusion proteins. A, SLN cells were taken, and the stimulation index was determined as cpm of wells with TNBS/cpm of wells without TNBS. The level of [3H]TdR incorporation for control wells without TNBS was 600 cpm. B, Cytokine production that accompanied proliferative responses was assessed in culture supernatants by ELISA. Values shown are the average ± 1 SD and are representative of three separate experiments using pooled cells from three to five mice per group. C, The expression of cytokine-specific mRNA in TNBS-stimulated CD4+ T cells was assessed. TNBS-stimulated total cell suspensions, which were prepared from SLN in mice treated with control IgG (lanes 1 and 3) or LTR-Ig (lanes 2 and 4) with colitis, were cultured for 24 h. CD4+ T cells were purified by sorting and subjected to cytokine-specific RT-PCR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Based upon these results, we assumed that abrogation of immune function of colonic patches would ameliorate TNBS colitis in a Th2-dominant state. We again used LTR-Ig for this purpose. In the initial study we found that pretreatment of adult mice with LTR-Ig resulted in loss of cellularity in both Peyer’s and colonic patches with disrupted follicular dendritic cell networks, and this response was most dramatic in IFN-^-/- mice. The precise mechanism for this observed effect is not yet known. Down-regulation of MAdCAM-1 was not seen after treatment with LTR-Ig, even in flattened colonic and Peyer’s patches. Blocking of the LTR signaling pathway in normal mice inhibited the formation of germinal centers in colonic patches, but these mice still exhibited colonic patch hypertrophy after induction of TNBS colitis. In contrast, colonic patches in IFN-^-/- mice were diminished even after induction of colitis and were accompanied by a lack of dendritic cells in both subepithelial dome and follicles in the vestiges of lymphoid follicles. Previous studies have shown that membrane-bound LT is essential for maintaining the architecture of follicular dendritic cell networks (11, 18, 19, 20, 21), and the use of mAb to block LT in adult mice causes decreased expression of chemokines such as B lymphocyte chemoattractant and secondary lymphoid tissue chemokine, a potent T cell attractant expressed by follicular stromal cells (22). Decreased numbers of dendritic cells in Peyer’s and colonic patches would result in decreased production of these chemokines, with subsequent lack of recruitment of mucosal lymphocytes. In our study the protective effect of LTR-Ig treatment was more obvious in the absence of IFN- and was closely associated with the loss of colonic patches. In contrast, in the presence of IFN- (normal mice) the colonic patches were initially eliminated efficiently by pretreatment with LTR-Ig; however, hypertrophy of patches was reinduced by TNBS enema treatment. It was evident that local inflammation induced the redevelopment of colonic patches in LTR-Ig-treated mice, because Peyer’s patches in the small intestine were absent in mice with TNBS colitis, whereas colonic patches became hypertrophic. Because dendritic cells reappeared in colonic patches after induction of colitis in LTR-Ig-treated WT mice, this clearly suggests the strong relation of colonic patch hypertrophy and recruitment of dendritic cells into colonic patches.

Our studies have clearly shown that the LT12/LTR signaling pathway maintains normal dendritic cell numbers and germinal centers in both Peyer’s and colonic patches. Our results further suggest the presence of inflammation-specific pathways for recruiting lymphocytes into colonic patches that are independent of the LT12/LTR pathway. In this regard, once tissue damage occurs, the production of IFN- and other inflammatory cytokines synergistically up-regulates other cytokines, inflammatory adhesion molecules, and chemokines, which may then accelerate the homing of lymphocytes into colonic patches to overcome the effects of LTR-Ig treatment. In IFN-^-/- mice, the effect of TNFRp55-Ig treatment was intermediate between those of control IgG and LTR-Ig treatments in preventing colitis and colonic patch hypertrophy. Because TNFR p55-Ig had no effect on the cellularity of colonic patches without colitis, this suggests that TNFR p55-Ig blocked only an inflammation-specific pathway. Thus, signaling via LTR may play a significant role in the constitutive maintenance of cellularity of colonic patches in WT mice, and additional inflammatory responses may cause colonic patch hypertrophy. In contrast, in IFN-^-/- mice colonic patch hypertrophy and inflammation itself were dependent on the LT12/LTR signaling pathway as well as maintenance of both Peyer’s and colonic patches in the uninflamed gut.

In contrast to the limited protective effect of LTR-Ig in WT mice in our study, previous reports of experimental colitis following T cell reconstitution of SCID mice demonstrated the efficacy of both LTR-Ig and anti-TNF- mAb treatment in preventing colonic inflammation (27). This latter study suggested that down-regulation of adhesion molecules by LTR-Ig was one of the mechanisms for the anti-inflammatory effects. In this T cell-adoptive transfer system, the induction of colitis primarily depended upon the homing of lymphocytes into the intestine. Because the tissue destruction occurred after infiltration and expansion of adoptively transferred T cells into the colon, the terminal inflammatory responses may be controlled by LTR-Ig in this experimental system by preventing the initial infiltration of lymphocytes into uninflamed mucosa. In contrast, in the case of TNBS colitis, rectal administration of TNBS with ethanol initially breaks the intestinal barrier, and haptenated Ags then enter the lamina propria. Therefore, this influx would trigger an initial inflammatory response, including the production of TNF- and IFN-. Indeed, these Th1-type cytokines are up-regulated in early phases of TNBS colitis, and this is followed by Th2-type cytokine production and hypertrophy of colonic patches occurring at a later stage. In IFN-^-/- and IL-12^-/- mice, the early burst of IFN- is absent, but these mice do develop a diffuse, mucosal type of colitis (7). This initial up-regulation of proinflammatory cytokines probably overcomes the effect of LTR-Ig in WT mice with TNBS colitis. LTR-Ig was effective for treatment of TNBS colitis in IFN-^-/- mice, most likely due to the lack of an early Th1 inflammatory response. Thus, LTR-Ig efficiently prevented the Th2-dominant type of inflammation in IFN-^-/- mice by disturbing the function of colonic patches. Indeed, when WT mice were treated with both anti-IFN- mAb and LTR-Ig, a significant reduction in disease was seen. Further investigation is required to clarify the precise role of IFN- in recruiting dendritic cells into colonic patches; however, our current findings in IFN-^-/- mice are of significance in terms of a model for the chronic type of colitis.

Although LTR-Ig provided limited benefits to WT mice, it does offer a new modality for treatment of human IBD, a new weapon in the current arsenal of proposed treatment that includes the neutralization of TNF- in humans (39, 40, 41) or IL-12 in the mouse model (2, 5). It is possible that disturbance of immunological events in GALT ameliorates inflammation mediated by Th2-type cytokine responses without induction of high levels of IFN- or TNF-. Ulcerative colitis is often suggested to consist of this type of inflammation (8, 9). Colonic patches are also very likely to be involved in the pathogenesis of IBD. Lymphoid follicles with an M cell-containing follicle-associated epithelium have been reported to be abundant in the human colon, although they are smaller in size than Peyer’s patches of the small intestine (42, 43). These findings suggest that colonic patches are a functional type of GALT for Ag uptake and presentation and for T and B cell interactions. Furthermore, evidence from animal models (44) and human studies (45, 46, 47) has shown that the indigenous flora is also important in the pathogenesis of IBD. Because the colon is the major colonization site for commensal bacteria, colonic GALT are likely to have specific functions in the maintenance of the homeostasis of mucosal immunity in the presence of bacterial Ags. In contrast to Crohn’s disease, which develops numerous secondary lymphoid follicles in all layers of the small and large intestinal walls, ulcerative colitis does not cause increased numbers of lymphoid follicles, but instead leads to an enlargement of colonic patches that line the mucosal layer (our unpublished observations). This suggests a primary role for colonic patches in the initiation of inflammation in ulcerative colitis. It is plausible that suppression of abnormal immunological events in these follicles by blocking the LT12/LTR signaling pathway, which may occur constitutionally in IBD patients, has beneficial effects in treating this disease.

In conclusion, our results have shown that anti-inflammatory effects of LTR-Ig are the result of a mechanism distinct from that involved in the neutralization of TNF- or the blocking of Th1-type cytokine pathways. By abrogating both Peyer’s and colonic patch development, this treatment effectively abrogates the Th2 type of TNBS colitis. Based upon our results, we envision that LTR-Ig would be beneficial in the treatment of ulcerative colitis and perhaps also of IBD cases refractory to other therapies.

	Acknowledgments

 
We thank Drs. Werner Meier, Paula Hochman, Konrad Miatowski, Joe Amatucci, Cathy Hession, Gerald Majeau, and Apinya Ngam-Ek for creating, characterizing, and producing receptor-Ig fusion proteins. We also thank Drs. Casey Weaver, Audrey J. Lazenby, and Kiyoshi Saito for important discussions concerning the histopathology of lesions, Dr. Atsushi Nakajima for MPO assay, and Annette M. Pitts for preparation of the tissues for histology.

	Footnotes

 
¹ This work was supported by U.S. Public Health Service National Institutes of Health Grants DK44240, AI18958, DE09837, DE12242, P30DK54781, AI43197, and AI35932 and by grants and contracts from International Health Cooperation Research (11A-1) from the Ministry of Health, Labor, and Welfare; Ministry of Education, Cultures, Sports, Science, and Technology; the Japan Health Sciences Foundation; and Organization for Pharmaceutical Safety and Research of Japan.

² Address correspondence and reprint requests to Dr. Taeko Dohi, Department of Gastroenterology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail address: dohi{at}ri.imcj.go.jp

³ Abbreviations used in this paper: IBD, inflammatory bowel disease; GALT, gut-associated lymphoreticular tissues; LT, lymphotoxin; LTR-Ig, LT receptor-Ig fusion protein; MPO, myeloperoxidase; PNA, peanut agglutinin; SLN, sacral lymph nodes; TNBS, 2,4,6-trinitrobenzene sulfonic acid; WT, wild type.

Received for publication November 17, 2000. Accepted for publication June 21, 2001.

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