Gut 2001;48:489–495 489 Role of T in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after ãä depletion and no eVect of áâ T cell depletion

JCHoVmann, K Peters, S Henschke, B Herrmann, K Pfister, J Westermann, M Zeitz

Abstract oral tolerance induction.6 In addition, the per- Background and aim—Indirect evidence centage of ãä T cells in the intraepithelial lym- suggests that CD4+ T cells have a patho- phocyte compartment is several fold higher genic while ãä T cells have a protective role compared with peripheral lymphocytes, in the initiation and perpetuation of particularly in mice but also in rats and inflammatory bowel disease. To define the humans.7–9 In spite of these important aspects, role of T cell subsets in a rat colitis model little is known of the role of ãä T cells in (2,4,6-trinitrobenzene sulphonic acid inflammatory bowel disease (IBD). As ãä T (TNBS)) we analysed colitis severity after cells can downregulate contact sensitivity eVective depletion of T helper cells, áâ Ô responses to skin and prevent diabetes cells, or ãä T cells. in non-obese diabetic mice, a regulatory func- 10 11 Methods—T helper cells, áâ T cells, or ãä tion of ãä T cells in IBD seems likely. T cells were depleted using previously The 2,4,6 trinitrobenzene sulphonic described monoclonal directed acid (TNBS) in 50% ethanol induces in at the CD4 molecule (OX38), the CD2 susceptible rodents a subacute or even chronic 12 13 molecule (OX34, both depleting CD4+ T form of colitis (TNBS colitis). Histologi- cells), the áâ T cell receptor (R73), and the cally, the inflammatory response includes ãä T cell receptor (V65). Depletion was mucosal and submucosal infiltration of, ini- tially, , , and mast verified by flow cytometry and/or immuno- 12 histology. Colitis was induced using intra- cells, and later lymphocytes and fibroblasts. colonic application of TNBS. Granulomas and even Langhans’-type giant Results—Surprisingly, depletion of T cells can be detected. Because of these helper cells or áâ T cells had no influence similarities with IBD (particularly Crohn’s dis- on survival, macroscopic or microscopic ease) this model has been used by many inves- tigators since its first description in 1989. Indi- scores, or myeloperoxidase activity fol- + lowing colitis induction. In contrast, de- rect evidence suggests that CD4 T cells play a pletion of ãä T cells resulted in major role in the initiation and perpetuation of significantly increased mortality (V65: TNBS colitis, particularly a T helper 1 (Th1) 73%, n=15) compared with controls (30%, response as seen after CD3/CD28 stimulation of lamina propria T lymphocytes14 Innere Medizin II, n=13; p<0.03). In addition, colitis was his- and the attenuating eVects of T cell directed Medizinische Klinik, tologically more severe in the ãä T cell (anti-IL-12 monoclonal Universitätskliniken depleted group compared with controls (mAb), anti-CD40L mAb, anti- des Saarlandes, (p<0.05). D-66421 interferon-ã mAb, or OX40-Ig).14–16 However, Conclusions—T helper cells or áâ T cells Homburg/Saar, it has never conclusively been shown that CD4+ did not influence the initiation or perpetu- Germany T cells have a pathogenic role in TNBS colitis. JCHoVmann ation of rat TNBS colitis. In contrast, ãä T The role of other T cell subsets—for example, K Peters cells had a protective role in rat TNBS ãä T cells—has not been investigated in this S Henschke colitis as depletion caused increased mor- B Herrmann model. tality. K Pfister We therefore examined if depletion of ãä T (Gut 2001;48:489–495) M Zeitz cells, CD4+ T cells, or áâ T cells influenced the initiation and/or perpetuation of rat TNBS Zentrum Anatomie, Keywords: áâ T cells; ãä T cells; experimental colitis; Medizinische inflammatory bowel disease; rat colitis. Hochschule Hannover, D-30623 Hannover, Germany A subpopulation of T lymphocytes expresses a Materials and methods J Westermann T cell receptor (TCR) that is distinct from the ANIMALS conventional áâ TCR (that is, it is character- Inbred female Lewis rats were obtained from Correspondence to: DrJCHoVmann, ised by a ã and a ä chain). Several important Charles River, Germany, at the age of 10–12 Medizinische Klinik I, immune functions have been reported for weeks and weighing 160–180 g. The animals Universitätsklinikun intestinal ãä T cells, namely T cell help for Benjamin Franklin 1 Hindenburgdamm 30, immunoglobulin class switching, production 2 Abbreviations used in this paper: IBD, D12200 Berlin, Germany. of epithelial growth factors, first line defence [email protected] 34 inflammatory bowel disease; ko, knock out; PALS, against various pathogens, an immuno- periarteriolar sheath; TCR, T cell Accepted for publication downregulatory role during infection (for receptor; TNBS, 2,4,6-trinitrobenzene sulphonic acid; 14 November 2000 example, coccidiosis),5 and requirement for Th1, T helper 1; mAb, .

www.gutjnl.com 490 HoVmann, Peters, Henschke, et al

were kept in polycarbonate cages and had free INDUCTION OF TNBS COLITIS access to standard chow and tap water. Animals were randomised into treatment or control groups in a blinded fashion by BH, as stated above. After mAb pretreatment rats were mAb lightly anaesthetised with ether and a central The following depleting mAbs were used in venous catheter (OD 1.7 mm) was inserted vivo: OX34 (IgG2a), directed at CD2 (depletes 17 18 intrarectally such that the tip was 8 cm T helper cells), OX38 (IgG2a), directed at 12 19 proximal to the anus, as previously described. CD4 (depletes T helper cells), both kindly TNBS 40 mg (Fluka, Munich, Germany) in provided by AN Barclay (University of Oxford, 0.5 ml of 50% ethanol (vol/vol) was injected UK), R73 (IgG1), directed at the rat áâ TCR intrarectally. Thereafter, the catheter was 20 21 (depletes áâ T cells), and V65 (IgG1), slowly removed and the animals were kept in a 21 directed at the rat ãä TCR, both kindly vertical position for 30 seconds. donated by T Hünig (University of Würzburg, Clinical assessment included daily determi- Germany). Mouse mAbs directed at 1 nation of weight and general well being. of human CD58 AICD58.6 (epitope 1 of Animals were killed after a weight loss of domain 1, IgG2a) and TS2/9 (epitope 1 of greater than 20% of original weight (n=5) or domain 1, IgG1) served as controls.22 The other clinical signs of severe colitis (for AICD58.6 mAb was a gift from SC Meuer example, massive abdominal distention due to (University of Heidelberg, Germany); the megacolon (n=2) or severe lethargy (n=1)). hybridoma TS2/9 was obtained from ATCC. These animals were counted as fatal cases in mAb were purified from supernatants of hybri- the mortality analysis with maximal macro- doma cell lines using spinner flasks followed by scopic and microscopic colitis scores. aYnity chromatography employing protein A Sepharose beads (Pharmacia, Germany). Puri- ASSESSMENT OF COLITIS fied mAb was filtered (0.25 µm) and adminis- Macroscopic scoring tered intraperitoneally. All depleting mAb were At the end of the study animals were used in regimens that have previously been exsanguinated under ether anaesthesia and the demonstrated to strongly deplete indicated T small and large intestine as well as the spleen cell subsets.17 19 21 EVective depletion was con- were excised. Samples of the terminal ileum firmed by either flow cytometry or immunohis- and spleen were quickly frozen in liquid nitro- tology at the end of the study when depletion gen for immunohistology (see below) and sam- was least pronounced (see below). ples of the descending colon were obtained for paraYn section and measurement of myeloper- oxidase activity (see below). The remaining mAb TREATMENT descending, sigmoid colon, and rectum were Treatment with mAb was performed in two opened longitudinally, pinned out on a board, treatment blocks: and the macroscopic appearance was scored by (1) Anti-CD4 treatment: 37 animals were two blinded investigators on a scale of 0–4, blindly assigned to one of three mAb treat- similar to the original scoring system of Morris + ment groups: (a) CD4 T cell depleting and colleagues12: briefly, 0, no evidence of + anti-CD2 mAb (OX34, n=14), (b) CD4 inflammation; 1, erythema only; 2, erythema, T cell depleting anti-CD4 mAb (OX38, slight oedema, and small erosions; 3, two or n=10), or (c) matched control more bleeding ulcers and/or inflammation mAb (anti-LFA-3, AICD58.6, n=13). and/or moderate adhesions; 4, severe ulcera- mAbs were given over three consecutive tion and/or stenosis with prestenotic dilations days starting with 2 mg followed by 1 mg and/or severe adhesions. each. On day 3, TNBS was administered (see below). These studies were performed Microscopic scoring in three separate experiments. Tissues were fixed in phosphate buVered saline (2) Anti-TCR áâ or ãä treatment: 40 animals containing 4% formalin and embedded in par- were blindly assigned to one of three treat- aYn. Sections (5 µm) were stained with ment groups: (a) áâ T cell depleting mAb haematoxylin and eosin. The degree of inflam- (R73, n=12), (b) ãä T cell depleting mAb mation was blindly assessed by two investiga- (V65, n=15), or (c) isotype matched tors using the scoring system described by control mAb (anti-LFA-3, TS2/9, n=13). Neurath et al from 0–4 (0, no signs of TNBS was injected into the colon on day inflammation; 1, low level of leucocyte infiltra- 0. The áâ T cell depleting mAb (R73) and tion; 2, moderate level of leucocyte infiltration; the control mAb (TS2/9) were started two 3, high level of leucocyte infiltration, high vas- days prior to colitis induction at 200 µg, cular density, thickening of bowel wall; 4, followed by 100 µg on days −1, 0, 2, 4, 6, 8, transmural infiltrations, loss of goblet cells, and 10. The ãä T cell depleting mAb V65 high vascular density, strong bowel wall thick- was given seven days prior to colitis induc- ening).14 tion and at the same time as colitis induc- tion at a dose of 500 µg each. These previ- DETERMINATION OF MYELOPEROXIDASE ACTIVITY ously reported treatment regimens were Colonic myeloperoxidase activity was used to obtain depletion of either áâ or ãä measured as previously described by Grisham T cells over the whole observation period et al and Suzuki and colleagues.23 24 Briefly, tis- of 13 days.21 This treatment block was per- sue was homogenised in approximately 10 vol

formed in three separate experiments. of 50 mM KPO4, pH 7.4, and centrifuged for

www.gutjnl.com Role of T lymphocytes in TNBS induced colitis 491

20 minutes at 20000 g. The pellet was homog- Mann-Whitney U test. Values are expressed as

enised in 10 vol 50 mM KPO4, 10 mM EDTA, mean (95% confidence intervals) (colitis pH 6, containing 0.5% hexadecyltrimethylam- scores, myeloperoxidase activity) or mean (SD) monium bromide (wt/vol). After thaw-freezing (cell counts). once, the homogenate was sonicated and myeloperoxidase activity was determined by Results measuring H2O2 dependent oxidation of 3,3’,5,5’-tetramethylbenzidine and expressed DEPLETION OF T HELPER CELLS DOES NOT as units per gram of tissue. INFLUENCE THE INITIATION OR PERPETUATION OF RAT TNBS COLITIS Previously, we have shown that the anti-CD2 IMMUNOHISTOLOGY mAb OX34 ameliorates chronic adjuvant Cryostat sections were made (thickness 5 mm), arthritis and selectively depletes T helper air dried, wrapped in aluminum foil, and stored cells.17 18 To test the potential benefit of this ° at −20 C. To determine localisation and anti-CD2 mAb in hapten induced colitis and to phenotype of diVerent cell types a immuno- clarify the role of T helper cells in the initiation histochemical staining technique was used as of hapten induced colitis, we randomly treated previously described employing the APAAP 37 female Lewis rats over three days with either method17 using the following antibodies: R73 + anti-CD2 (OX34, CD4 depleting, n=14), anti- (áâ T cells), V65 (ãä T cells), and OX8 (CD8 CD4 (OX38, CD4 depleting, n=10), or an iso- T cells and NK cells) (kindly provided by AN type matched control mAb (AICD58.6, n=13). Barclay, Oxford, UK). The slides were counter- On the third treatment day animals received stained and mounted in glyceryl (Dakopatts, 40 mg of TNBS in 50% ethanol (day 0 after Hamburg, Germany). The number of stained 2 TNBS colitis induction). As anti-CD2 mAb cells/mm mucosa was determined in four treatment over three days was previously shown diVerent sections. Splenic lymphocytes were to result in long term depletion which is most counted as previously described on two sec- pronounced on day 1, depletion by anti-CD2 tions per animal and are expressed as number or anti-CD4 was only confirmed at the end of of lymphocytes within the periarteriolar lym- 2 17 the observation period—that is, day 13 after phocyte sheath (PALS)/mm of the spleen. colitis induction. As shown in table 1, áâ T Percentages of lamina propria lymphocytes or cells of the spleen and mucosa of the ileum lymphocytes expressing áâ or ãä T cells were were strongly depleted on day 13, as demon- calculated using these values. strated by immunohistology (mean reduction of 95% in the spleen and >80% in the lamina FLOW CYTOMETRY propria). Depletion of áâ T cells was due to Splenocytes and peripheral blood lymphocytes reduced numbers of T helper cells, as assessed were obtained by intracardial puncture or by two colour flow cytometry (CD8/TCR áâ) splenectomy and homogenisation as previously and immunohistology showing unchanged described.17 Single cell suspensions were ex- numbers of CD8 positive cells in the spleen posed to hypotonic solution to lyse erythro- and ileum (data not shown). However, no cytes. Cell viability was greater than 95% as diVerence was found with regard to mortality assessed by trypan blue exclusion. For flow for both anti-CD2 and anti-CD4 mAb com- cytometry staining R73 was biotinylated and pared with isotype matched control mAb OX8 was FITC labelled using standard treated animals. In addition, neither anti-CD2 techniques. T helper cells were defined as R73+ nor anti-CD4 mAb treatment resulted in OX8- cells using streptavidin PE. Only viable diVerences in macroscopic or microscopic cells, as determined by forward and side scatter scores, myeloperoxidase activity, or weight characteristics, were analysed using a FACS (data not shown). Vantage cell sorter (Becton/Dickonson, Hei- delberg, Germany). NO INFLUENCE OF áâ T CELL DEPLETION ON THE CLINICAL COURSE OF RAT TNBS COLITIS STATISTICAL ANALYSIS As depletion of T helper cells had no influence Statistical analysis was carried out using SPSS on the development of TNBS colitis, we next for Windows. Survival was analysed using examined if other T cell subsets (for example, Kaplan-Meier analysis. Comparison of macro- CD4−/CD8+ T cells) might be involved in the scopic and microscopic scores, and myeloper- initiation of TNBS colitis. After treatment with oxidase activity was carried out using the R73 on days −2, −1, 0, 2, 4, 6, 8, and 10

Table 1 Depletion of áâ T cells or ãä T cells in the spleen and the ileum after anti-CD2 monoclonal antibody (mAb), anti-CD4 mAb, anti-áâ T cell receptor (TCR), or anti-ãä TCR treatment. Values are mean (SD) of absolute numbers per PALS/mm2 of spleen or absolute numbers/mm2 mucosa of the ileum

No áâ T cells in No ãä T cells in No áâ T cells/mm2 No ãä T cells/mm2 mAb treatment PALS/mm2 spleen PALS/mm2 spleen ileum ileum Control (IgG2a) 829 (65) — 676 (32) — Anti-CD2 (IgG2a) 43 (11) 27.1 (3.5) 128 (22) 28.1 (3.1) Anti-CD4 (IgG2a) 47 (10) — 136 (25) — Control (IgG1) 774 (46) 29.5 (3.9) 661 (36) 26.7 (3) Anti-áâ TCR (IgG1) 48 (12) — 88.5 — Anti-ãä TCR (IgG1) — 2.7 (1.1) — 3.7 (1.3)

PALS, periarteriolar lymphocyte sheath.

www.gutjnl.com 492 HoVmann, Peters, Henschke, et al

4.0 A 4.0 B C 1.6 3.5 3.5

3.0 3.0 1.2

2.5 2.5 0.8 2.0 2.0

Microscopic score 0.4 Macroscopic score

1.5 1.5 MPO activity (U/mg)

1.0 1.0 0 n =n13 12 =n13 12 =11 10 Control αβ TCR Control αβ TCR Control αβ TCR

100 D

80 Control mAb

60

Survival (%) Anti-αβ TCR (R73)

40

20 024681012 Time after colitis induction (days) Figure 1 No significant eVect of áâ T cell depletion on colitis severity. Mean (95% confidence interval) values for (A) macroscopic score, (B) microscopic score, (C) myeloperoxidase (MPO) activity, and (D) survival (Kaplan-Meier analysis) comparing isotype matched control monoclonal antibody (mAb) (TS2/9, n=13) and the anti-áâ T cell receptor (TCR) mAb (R73, n=12). A dose of 200 µg followed by 100 µg of each mAb was started two days prior to colitis induction and continued on days −1, 0, 2, 4, 6, 8, and 10 after colitis induction.

(n=12), eVective depletion of áâ T cells was the anti-CD2 mAb treatment although ãä T confirmed by immunohistology in the spleen cells were shown to be CD2 positive (table 1). and mucosa of the ileum (table 1): 94% of áâ The most dramatic finding after depletion of T cells/mm2 PALS and greater than 85% of áâ ãä T cells was the markedly increased mortality T cells/mm2 lamina propria were depleted (anti-ãä TCR: 73% v control: 30%) as shown compared with controls 13 days after TNBS in fig 2D. Using Kaplan-Meier analysis this colitis induction. Although microscopic and diVerence was significant atapvalue of 0.026. macroscopic scores, and myeloperoxidase ac- The diVerences in microscopic scores were also tivity were higher in the áâ T cell depleted significant at p<0.05 (mean: 3.1 (anti-ãä TCR) group this diVerence was not significant, as v 2.1 (control)) while macroscopic scores were demonstrated in fig 1(A–C). In addition, áâ T not significantly increased (mean 3.0 (anti-ãä cell depleted animals showed only slightly TCR) v 2.3 (control); p=0.23) (fig 2A–C). No increased mortality compared with controls diVerence was seen with regard to myeloper- (anti-áâ TCR: 50% v control: 30%), which was oxidase activity. again not significant (fig 1D). Therefore, áâ T cell depletion does not aVect the initiation of Discussion TNBS colitis and has no influence on the per- The present study provides the first direct evi- petuation of this type of colitis. dence that depletion of ãä T cells aggravates rat TNBS colitis resulting in significant mortality. DEPLETION OF ãä T CELLS AGGRAVATES RAT TNBS In addition, no evidence was found that áâ T COLITIS cells or particularly T helper cells play a role in Conflicting evidence exists as to the pathogenic the initiation or perpetuation of rat TNBS or protective role of ãä T cells in colitis. We colitis as eVective depletion of these cell types therefore used a long lasting ãä T cell depletion had no eVect on the clinical course of this IBD protocol prior to TNBS colitis induction. animal model. EVective depletion of ãä T cells by anti-ãä There are conflicting data on the role of ãä TCR mAb treatment was confirmed by immu- T lymphocytes in IBD. This T cell subset is nohistology showing greater than 85% deple- involved in important regulatory immune tion of intestinal ãä T cells and greater than functions in the intestine, such as IgA produc- 90% depletion of splenic ãä T cells, even 13 tion, oral tolerance induction, and epithelial days after colitis induction (table 1). Interest- growth factor production.126 Also, ãä T cells ingly, no reduction in ãä T cells was found after have a role in first line defence against

www.gutjnl.com Role of T lymphocytes in TNBS induced colitis 493

p<0.05 4.0 A 4.0 B C 1.6 3.5 3.5

3.0 3.0 1.2

2.5 2.5 0.8 2.0 2.0

Microscopic score 0.4 Macroscopic score

1.5 1.5 MPO activity (U/mg)

1.0 1.0 0 n =n13 15 =n13 15 =11 10 Control γδ TCR Control γδ TCR Control γδ TCR

100 D

80 Control mAb

60

Survival (%) p<0.03

40

Anti-γδ TCR (V65) 20 024681012 Time after colitis induction (days) Figure 2 Increased mortality of ãä T cell depleted rats with TNBS colitis. Mean (95% confidence interval) values for (A) macroscopic score, (B) microscopic score, (C) myeloperoxidase (MPO) activity, and (D) survival (Kaplan-Meier analysis) comparing isotype matched control monoclonal antibody (mAb) (TS2/9, n=13) and the anti-ãä T cell receptor (TCR) mAb (V65, n=15).

pathogens34 and a immuno-downregulatory decreased numbers of intestinal ãä T cells. role during infection (for example, coccidi- However, the germfree environment also leads osis).5 Therefore, ãä T cell deficient mice to a reduction in áâ T cells which have been develop severe epithelial damage on infection shown to induce colitis in this model. There- with Eimeria vermiformis (coccidiosis) due to fore, the role of ãä T cells in both interleukin 2 failure to regulate the consequences of the áâ ko and TCR-á ko mice is presently unclear. In T cell response. Recently, ãä T cells at another contrast, ãä T cells in TNBS colitis seem to mucosal surface (the respiratory tract) were play a protective role which might be exerted shown to ensure clearance of damaged epithe- either by clearance of damaged epithelial cells, lial cells and to prevent airway inflammation/ secretion of epithelial growth factors, or by first hyperresponsiveness.25 26 Therefore, a protec- line defence against enteric pathogens. As the tive role for ãä T cells during bowel hapten TNBS primarily leads to epithelial inflammation seems likely. This notion is damage it is tempting to speculate that supported by a recent report in abstract form depletion of ãä T cells leads to defective clear- on ãä T cell deficient mice (truncated common ance of damaged epithelial cells and therefore cytokine receptor ã chain) who spontaneously to more severe inflammation in TNBS colitis. develop colitis at eight weeks of age.27 Further, On the other hand, the significance of epithelial three reports have described decreased num- growth factor production is exemplified by bers of ãä T cells in involved areas of Crohn’s attenuating eVects of keratinocyte growth colitis28–30 and one group reported increased factor for example, in rat TNBS colitis.35 numbers of ãä T cells.31 However, ãä T cells Finally, ãä T cells can function as regulatory T can also secrete proinflammatory cells in autoimmune diseases (for example, such as interferon ã. More importantly, T cells preventing murine dependent from áâ deficient mice can induce colitis after diabetes).11 Ultimately, studies using T cell transfer into immunocompromised tgå26 subset targeted and inducible ko mice of mice.32 Indirect evidence suggests that ãä T mediators involved in mucosal immunoregula- cells may be involved in genetic models of tion or epithelial integrity have to be done to ulcerative colitis, namely the TCR á knockout discriminate between these possibilities. (ko) and interleukin 2 ko mice.33 34 While Crohn’s disease is generally believed to be a TCR-á ko mice have only CD4+ á−/â+ T cells T cell dependent illness. To this end, most and ãä T cells left to induce colitis, interleukin patients with Crohn’s disease improve during 2 ko mice do not develop colitis in a germfree progression of human immunodeficiency virus environment and at the same time show infection due to progressive depletion of CD4+

www.gutjnl.com 494 HoVmann, Peters, Henschke, et al

T cells.36 As a model for Crohn’s disease, disease distribution (pancolitis in mice and TNBS colitis has also been proposed to be distal colitis in rats). controlled by CD4+ T cell driven illness, In conclusion, the present data demonstrate namely by T helper 1 (Th1) type cells.14 Our a protective role of ãä T cells in rat TNBS coli- data however suggest that CD4+ T cells are not tis. In addition, our results indicate that áâ T required for the induction and maintenance of cells, particularly CD4+ T cells, are not rat TNBS colitis as eVective depletion of CD4+ involved in the initiation or perpetuation of T cells or áâ T cells had no influence on the “classical” rat TNBS colitis. Future studies initiation or perpetuation of bowel inflamma- should investigate if ãä T cells are further tion. These results confirm previous observa- examples of regulatory T cells in other colitis tions that immunodeficient athymic rats de- models and how they mediate protection. velop even more severe TNBS colitis than immunocompetent (T cell bearing) controls.37 We thank AN Barclay, Oxford, UK, T Hünig, Würzburg, Ger- One might assume that production of Th1 type many, and SC Meuer, Heidelberg, Germany, for various hybri- cytokines (interferon ã and interleukin 2) in doma cell lines. This work was supported by DFG-grant Ho 1561/3-1 and by BMBF grant 01GI9986 (core facility IBD-in TNBS colitis might be a secondary phenom- vivo-models of the German competence network for IBD). The enon as TNBS colitis has recently been help of C Krick for statistical analysis and the technical assistance of S Lopez-Kostka are kindly acknowledged. The reported to occur in both interferon ã and critical comments of R Pabst and M HoVmann are greatly interferon ã receptor ko mice.38 39 These recent appreciated. data may also indicate that the beneficial eVect of anti-interleukin 12 mAb therapy is not nec- 1 Fujihashi K, Taguchi T, Aicher WK,etal. Immunoregula- essarily dependent on interferon ã producing T tory functions for murine intraepithelial lymphocytes: helper cells. Further, other supposedly T cell gamma/delta T cell receptor-positive (TCR+) T cells abro- gate oral tolerance, while alpha/beta TCR+ T cells provide directed immunotherapies could also target help. J Exp Med 1992;175:695–707. other cell types in TNBS colitis—for example, 2 Komano H, Fujiura Y, Kawaguchi M,etal. Homeostatic regulation of intestinal epithelia by intraepithelial gamma CD40L mast cells or anti-interleukin 12 natu- delta T cells. Proc Natl Acad Sci USA 1995;92:6147–51. ral killer cells. 3 Allison JP, Havran WL. The immunobiology of T cells with invariant gamma delta receptors. Annu Rev Immu- In favour of a role of T helper cells in TNBS nol 1991;9:679–705. colitis are two reports demonstrating beneficial 4 Kaufmann SH. Gamma/delta and other unconventional T lymphocytes: what do they see and what do they do? Proc eVects of CD4 T cell directed immunothera- Natl Acad Sci USA 1996;93:2272–9. pies.40 41 At first glance these data seem to be in 5 Roberts SJ, Smith AL, West BA,etal. T-cell áâ+ and ãä+ deficient mice display abnormal but distinct phenotypes contrast with our observation that depletion of toward a natural, widespread infection of the intestinal epi- T helper cells or even T cells have no ben- thelium. Proc Natl Acad Sci USA 1996;93:11774–9. áâ 6 Ke Y, Pearce K, Lake JP,etal. Gamma delta T lymphocytes eficial eVect on the course of TNBS colitis. regulate the induction and maintenance of oral tolerance. J Immunol 1997;158:3610–18. However, both studies induced colitis in a 7 Goodman T, Lefrancois L. Expression of the gamma-delta slightly diVerent way—that is, after systemic T-cell receptor on intestinal CD8+ intraepithelial lym- phocytes. Nature 1988;333:855–8. sensitisation employing adjuvant in rats and 8 Ullrich R, Schieferdecker HL, Ziegler K, et al. Gamma/delta coupling to BSA in mice. As TNBS is a hapten T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium. this may be important to induce a real Cell Immunol 1990;128:619–27. reaction rather than an im- 9 Helgeland L, Vaage JT, Rolstad B, et al. Regional phenotypic specialization of intraepithelial lymphocytes in mune reaction to a single colonic application of the rat intestine does not depend on microbial colonization. TNBS. Importantly, the first study was only Scand J Immunol 1997;46:349–57. 10 Szczepanik M, Anderson LR, Ushio H, et al. Gamma delta able to induce amelioration of colitis using a T cells from tolerized alpha beta T cell receptor synthetic mimetic of CD4 which binds to and (TCR)-deficient mice inhibit contact sensitivity-eVector T cells in vivo, and their interferon-gamma production in possibly modulates MHC class II positive cells. vitro. J Exp Med 1996;184:2129–39. However, a depleting anti-CD4 mAb had no 11 Harrison LC, Dempsey-Collier M, Kramer DR, et al. Aero- sol insulin induces regulatory CD8 gamma delta T cells eVect. Similarly, the anti-CD4 mAb therapy (a that prevent murine insulin-dependent diabetes. J Exp Med non-depleting mAb) in the second study might 1996;184:2167–74. + 12 Morris GP, Beck PL, Herridge MS, et al. Hapten-induced modulate CD4 T cells by induction of model of chronic inflammation and ulceration in the rat anti-inflammatory cytokines without showing colon. Gastroenterology 1989;96:795–803. + 13 Elson CO, Sartor RB, Tennyson GS, et al. Experimental the involvement of CD4 T cells in rat TNBS models of inflammatory bowel disease. Gastroenterology colitis. With regard to the “classical” rat TNBS 1995;109:1344–67. 14 Neurath MF, Fuss I, Kelsall BL, et al. Antibodies to colitis (without prior systemic sensitisation) we interleukin 12 abrogate established experimental colitis in can virtually exclude a role for CD45RC+CD4+ mice. J Exp Med 1995;182:1281–90. 15 Stuber E, Strober W, Neurath M. Blocking the CD40L- T cells in the initiation of colitis because the CD40 interaction in vivo specifically prevents the priming anti-CD2 mAb OX34 depletes this subset by of T helper 1 cells through the inhibition of interleukin 12 23 secretion. J Exp Med 1996;183:693–8. more than 95% in the whole body. It is pres- 16 Higgins LM, McDonald SA, Whittle N, et al. Regulation of ently unknown why OX34 depletes only this T cell activation in vitro and in vivo by targeting the OX40- OX40 ligand interaction: amelioration of ongoing inflam- subset while also binding to other T cell matory bowel disease with an OX40-IgG fusion protein, subsets—for example, ãä T cells or CD8+ T but not with an OX40 ligand-IgG fusion protein. J Immu- nol 1999;162:486–93. cells. As gene targeted models are not available 17 HoVmann JC, Herklotz C, Zeidler H, et al. Anti-CD2 in the rat there remains a theoretical possibility (OX34) MoAb treatment of adjuvant arthritic rats: attenu- + ation of established arthritis, selective depletion of CD4+ T that the remaining CD4 T cells might suYce cells, and CD2 down-modulation. Clin Exp Immunol 1997; to mediate colitis. Finally, it cannot be 110:63–71. 18 HoVmann JC, Herklotz C, Zeidler H, et al. Initiation and excluded that the same substance (TNBS) perpetuation of rat adjuvant arthritis is inhibited by the induces diVerent forms of disease in rats and anti-CD2 monoclonal antibody (mAb) OX34. Ann Rheum Dis 1997;56:716–22. mice. In favour of this hypothesis are certain 19 Shizuru JA, Seydel KB, Flavin TF, et al. Induction of donor- specific unresponsiveness to cardiac allografts in rats by diVerences—for example, the higher suscepti- pretransplant anti-CD4 monoclonal antibody therapy. bility of rats to TNBS colitis or the diVerent Transplantation 1990;50:366–73.

www.gutjnl.com Role of T lymphocytes in TNBS induced colitis 495

20 Hunig T, Wallny HJ, Hartley JK, et al. A monoclonal 31 McVay LD, Li B, Biancaniello R, et al. Changes in human antibody to a constant determinant of the rat T cell antigen mucosal gamma delta T cell repertoire and function asso- receptor that induces T cell activation. DiVerential reactiv- ciated with the disease process in inflammatory bowel dis- ity with subsets of immature and mature T lymphocytes. J ease. Mol Med 1997;3:183–203. Exp Med 1989;169:73–86. 32 Simpson SJ, Hollander GA, Mizoguchi E, et al. Expression 21 Pelegri C, Kuhnlein P, Buchner E, et al. Depletion of of pro-inflammatory cytokines by TCR alpha beta+ and gamma/delta T cells does not prevent or ameliorate, but TCR gamma delta+ T cells in an experimental model of rather aggravates, rat adjuvant arthritis. Arthritis Rheum colitis. Eur J Immunol 1997;27:17–25. 1996;39:204–15. 33 Mizoguchi A, Mizoguchi E, Chiba C, et al. Cytokine imbal- 22 Dengler TJ, HoVmann JC, Knolle P, et al. Structural and ance and production in T cell receptor-alpha functional of the human adhesion receptor CD58 mutant mice with inflammatory bowel disease. J Exp Med (LFA-3). Eur J Immunol 1992;22:2809–17. 1996;183:847–56. 23 Grisham MB, Benoit JN, Granger DN. Assessment of 34 Contractor NV, Bassiri H, Reya T, et al. Lymphoid leukocyte involvement during ischemia and reperfusion of hyperplasia, , and compromised intestinal intestine. Meth Enzymol 1990;186:729–42. intraepithelial lymphocyte development in colitis-free 24 Suzuki K, Ota H, Sasagawa S, Assay method for et al. gnotobiotic IL-2- deficient mice. J Immunol 1998;160:385– myeloperoxidase in human polymorphonuclear leukocytes. 94. Anal Biochem 1983;132:345–52. 35 Zeeh JM, Procaccino F, Ho mann P, . Keratinocyte 25 King DP, Hyde DM, Jackson KA . Cutting edge: V et al ,etal growth factor ameliorates mucosal injury in an experimen- protective response to pulmonary injury requires ãä T lym- phocytes. 1999; :5033–6. tal model of colitis in rats. Gastroenterology 1996;110:1077– J Immunol 162 83. 26 Lahn M, Kanehio A, Takeda K, et al. Negative regulation of airway responsiveness that is dependent on ãä T cells and 36 Pospai D, Rene E, Fiasse R, et al. Crohn’s disease stable independent of áâ T cells. Nat Med 1999;5:1150–6. remission after human immunodeficiency virus infection. 27 Kay Y, Takahashil I, Ito T, et al. Selected Vbeta subset of Dig Dis Sci 1998;43:412–19. Th2 type CD4+ T-cells mediate development of colitis in 37 Jacobson K, McHugh K, Vallance BA, et al. T lymphocyte mice with a truncated gamma chain. Immunol Lett reconstitution in athymic rats restores stress-induced reac- 1999;69(suppl 28):4. tivation of colitis. Gastroenterology 1996;110:A932. 28 Fukushima K, Masuda T, Ohtani H, et al. Immunohisto- 38 Dohi T, Fujihashi K, Rennert PD, et al. Hapten-induced chemical characterization, distribution, and ultrastructure colitis is associated with colonic patch hypertrophy and T of lymphocytes bearing T-cell receptor gamma/delta in helper cell 2-type responses. J Exp Med 1999;189:1169–80. inflammatory bowel disease. Gastroenterology 1991;101: 39 Camoglio L, Te Velde AA, de Boer A, et al. Interferon- 670–8. gamma receptor deficient mice are not protected against 29 Bucht A, Soderstrom K, Esin S, et al. Analysis of gamma TNBS induced colitis. Immunol Lett 1999;69(suppl 33):7. delta V region usage in normal and diseased human intes- 40 Watanabe M, Hosoda Y, Okamoto S, et al. tinal biopsies and peripheral blood by polymerase chain CD45RChighCD4+ intestinal mucosal lymphocytes infil- reaction (PCR) and flow cytometry. Clin Exp Immunol trating in the inflamed colonic mucosa of a novel rat colitis 1995;99:57–64. model induced by TNB immunization. Clin Immunol 30 Lee HB, Kim JH, Yim CY, et al. DiVerences in Immunopathol 1998;88:46–55. immunophenotyping of mucosal lymphocytes between 41 Okamoto S, Watanabe M, Yamazaki M, et al. A synthetic ulcerative colitis and Crohn’s disease. Korean J Intern Med mimetic of CD4 is able to suppress disease in a rodent 1997;12:7–15. model of immune colitis. Eur J Immunol 1999;29:355–66.

www.gutjnl.com