This was first reported in mice deficient in a4. T-cell receptor (TCR) T cells,' interleukin-l0 (IL-10) knock- out mice (_/_)2 andIL-2 -/- mice,3 but is also seen in mice.
Immunology 1997 91 73–80
Characterization of the mucosal cell-mediated immune response in IL-2 knockout mice before and after the onset of colitis S. A. C. MCDONALD, M. J. H. J. PALMEN,* E. P. VAN REES* & T. T. MACDONALD Department of Paediatric Gastroenterology, St Bartholomews and the Royal London School of Medicine and Dentistry, London, UK, and *Department of Cell Biology and Immunology, Faculty of Medicine, Vrije Universiteit, Amsterdam, the Netherlands
SUMMARY One of the major advances in the understanding of inflammatory bowel disease has been the observation that mice with immunoregulatory defects, such as interleukin-2 knockout ( IL-2 –/–) mice, develop spontaneous gut inflammation. Here we have characterized the immune response in the ileum, caecum and colon of these mice before and after the onset of colitis by examining the cellular infiltrate, the cytokines produced by these cells and the mucosal vascular addressin MAdCAM-1. IL-2 –/– mice developed colitis after 35 days of age and before this the mice were apparently healthy. IL-2 –/– mice aged over 35 days with colitis had large numbers of CD4+, CD8+, ab T-cell receptor (TCR)+ and cd TCR+ T cells, macrophages, dendritic cells and MAdCAM-1+ endothelial cells in the caecum and colon. This was associated with an increase in the number of interferon-c (IFN-c), IL-1 and tumour necrosis factor-a ( TNF-a) transcripts and a decrease in IL-4 and IL-10 transcripts. Treatment of IL-2 –/– mice with cyclosporin A significantly delayed mortality. Interestingly, IL-2 –/– mice under 35 days, although healthy, did show some subtle immunological signs of preclinical disease. There was a significant increase in the number of macrophages and dendritic cells in the colonic lamina propria and increased mRNA for IL-1 and TNF-a. There were also increased numbers of MAdCAM-1+ endothelial cells, but IFN-c transcripts were not elevated. These results suggest that T-cell-mediated colitis in IL-2 –/– mice may be secondary to an initial non-specific inflammation.
IL-2 –/– mice have been reported to develop normally for the first 3–4 weeks but then about 50% of the animals die between 4 and 6 weeks of a disease that is characterized by splenomegaly, lymphadenopathy, weight loss and anaemia.3 All remaining mice develop colitis. Colitis in IL-2 –/– mice is a chronic, continuous, non-granulomatous inflammation of the mucosa and submucosa and was initially reported as similar to ulcerative colitis ( UC ) in humans. It is characterized by a large increase in the number of B and T cells in the lamina propria.3 The T cells are activated, bearing high levels of membrane CD44 and CD69.8 When this model was first described it was considered that the colitis was antibodymediated; however, Ma et al.9 have now shown that disease develops in mice that are also immunoglobulin deficient, indicating a primary role for T cells in this disease. When IL-2 –/– mice are crossed with b -microglobulin –/– 2 mice, colitis still develops, suggesting that it is CD4+ cells and not CD8+ cells which are required for inflammation.8 When lamina propria T cells are isolated from these mice they exhibit increased cytolytic activity.8 Important questions, however, remain concerning IBD in IL-2 –/– mice. It is puzzling that disease develops in the colon and caecum while the ileum appears normal, but also has an
INTRODUCTION One of the major advances in the understanding of inflammatory bowel disease ( IBD) has been the observation that mice with immunoregulatory defects develop spontaneous gut inflammation. This was first reported in mice deficient in ab T-cell receptor ( TCR) T cells,1 interleukin-10 ( IL-10) knockout mice (–/–)2 and IL-2 –/– mice,3 but is also seen in mice that are transgenic for human CD3e when lethally irradiated and reconstituted with T-cell depleted bone marrow cells from normal (C57Bl/6×CBA/J ) mice4 and in G-protein knockouts.5 Severe combined immunodeficiency (SCID) recipients of small numbers of CD4+ T cells or CD45RBhi CD4+ T cells also develop an enteropathy.6,7 Germ-free or specific pathogen-free IL-2 –/– and IL-10 –/– mice do not develop IBD,2,3 indicating a crucial role for the gut bacterial flora.
Received 3 September 1996; revised 6 January 1997; accepted 8 January 1997. Correspondence: Professor T. T. MacDonald, Department of Paediatric Gastroenterology, Dominion House, 59 Bartholomews Close, St Bartholomew Hospital, London EC1A 7BE, UK. © 1997 Blackwell Science Ltd
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abundant bacterial flora.10 Although CD4+ T cells appear necessary for disease, whether these are of the T-helper type 0 (Th0 ), Th1 or Th2 phenotype has not been determined. The contribution of other inflammatory monokines is not known. Adhesion molecules such as mucosal addressin cellular adhesion molecule (MAdCAM ) are thought to play an important role in the homing of lymphocytes to sites of inflammation in the gut,11 and the expression of such molecules has not been defined in this model. The onset of inflammation has not been elucidated. The role of cd T cells is not clear, nor is the contribution of intraepithelial lymphocytes (IEL), and it is not known if the onset of this disease can be prevented by administering potent T-cell suppressor drugs such as cyclosporin A. In this study we have attempted to address all of these issues. MATERIALS AND METHODS Animals Adult IL-2 +/– C3H mice (a kind gift from Dr I. Horak, Department of Virology and Immunobiology, University of Wurzburg, Germany) were housed in the animal unit at St Bartholomew’s Hospital (London, UK). Mice were genotyped by amplification of the IL-2 gene by polymerase chain reaction ( PCR) with IL-2-specific primers ( Pharmacia Biotech, Uppsala, Sweden) as described elsewhere.12 Mice were classified as IL-2 –/– ( knockout), +/– (heterozygous) and +/+ (wild type). Antibodies The following antibodies were used for immunohistochemistry; hamster anti-mouse ab TCR ( H57; a kind gift from R. Kubo, National Jewish Hospital, Denver, CO), cd TCR (GL3; a kind gift from L. LeFrancois, University of Connecticut, CT ), rat anti-mouse CD4 (YTS.191) and CD8 (YTS.169) used as undiluted culture supernatants ( ECACC, Salisbury, UK ), rat anti-mouse dendritic cell ( NLDC.145; undiluted), macrophage ( MOMA-1; undiluted ) and class II major histocompatibility complex ( MHC) (M5/114; at 1510 dilution), all a kind gift from G. Kraal ( Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam, the Netherlands). Biotinylated goat anti-hamster IgG ( Vectra, Burlingame, CA) was used at a 1550 dilution, and biotinylated rabbit anti-rat IgG ( Dako, High Wycombe, UK ) was also used at a 1550 dilution. Hamster anti-mouse MAdCAM-1 was a kind gift from Jo Vivey (Genentech, South San Francisco, CA). Immunohistochemistry Cryostat sections of 6 mm were cut and air dried on glass slides. The tissue sections were then fixed in acetone for 10 min prior to either a two-step immunoperoxidase staining or a three-step avidin peroxidase staining as described previously.13 The number of positively stained cells in the lamina propria was quantified by a computer-assisted image analyser (Seescan, Cambridge, UK ). A stable light source and a fixed threshold and light intensity were used throughout the measurements. The image from the microscope was captured on a video monitor and areas of lamina propria were delineated and the number of positive cells marked by a mouse-controlled cursor. Results are expressed as number of positive cells per mm.2 Positively stained IEL were expressed as the number of positive
cells per 500 epithelial cells. MAdCAM-1+ blood vessels were expressed as the number of positively stained blood vessels per mm muscularis mucosa. Total RNA extraction Ileal, caecal and colonic tissue from IL-2 –/–, +/– and +/+ mice was snap-frozen in liquid nitrogen. The tissue was then homogenized in TRIzol ( Life Technologies, Paisley, UK ) and incubated at room temperature for 5 min. Molecular grade chloroform (Sigma Chemical Co., St Louis, MO) was then added and the sample was centrifuged for 15 min at 12 000 g. The aqueous phase containing the total RNA was removed and the RNA was percipitated out with isopropanol (Sigma) and pelleted by centrifugation at 12 000 g for 10 min. The pellet was then washed in 70% ethanol and resuspended in 50 ml water. The amount of total RNA was calculated by spectrophotometry. Reverse transcriptase (RT)-PCR Total RNA containing mRNA was reverse transcribed to cDNA by the addition of 500 ng of oligo(dt) 12–18 (Pharmacia Biotech, Uppsala, Sweden), 100 units of Superscript II reverse transcriptase ( Life Technologies) and 10 m dNTP mix (10 m each dATP, dCTP, dGTP and dTTP at neutral pH; Pharmacia Biotech) to 4 mg total RNA and incubated for 45 min at 42° and then 10 min at 70° to stop the reaction. For PCR, 1/40th of the cDNA product was added to a buered solution containing 2·5 m MgCl , 10 m dNTP mix, 40 ng of gene2 specific primers and 0·5 U of Taq polymerase (Pharmacia Biotech). The following gene-specific oligonucleotide primers were obtained from published sequences: IL-1b,14 IL-4,15 IL-10,16 IFN-c,17 TNF-a,18 IL-519 and b-actin.20 Samples were denatured at 94° for 5 min and then given 35 cycles ( 94° for 1 min, 58° for 1 min and 72° for 1·5 min) in a Cyclogene thermocycler (Techne, Cambridge, UK ). PCR products were analysed using a semi-quantitive method; IL1b, TNF-a, IL-4, IL-5 and IL-10 were standardized with bactin as a control, and IFN-c was standardized with TCR-b as a control. Each sample was electrophoresed in 1% agarose gel (Sigma), stained with ethidium bromide (Sigma) and the light intensity (measured in arbitary units) of each band was measured by an image analyser (Seescan UK, Cambridge, UK). Each sample was quantified as the number of arbitary units of cytokine to 1000 units of its control. Cyclosporin A Mice were injected intraperitoneally (i.p.) with 5 mg/kg cyclosporin A in olive oil (CsA; Sandoz, Geneva, Switzerland) and 200 ml of phosphate-buered saline (PBS ), pH 7·6. All mice were healthy before injections commenced and were given twice weekly injections for 12 weeks. Four groups of mice were studied: wild type mice given CsA; –/– mice given CsA; untreated –/– mice; and –/– mice given PBS with 1% olive oil. Results are expressed as percentage survival. Statistics The significance of the results was calculated using the Mann–Whitney U-test. © 1997 Blackwell Science Ltd, Immunology, 91, 73–80
Colitis in IL-2 –/– mice RESULTS Development of disease There were some dierences between our IL-2 –/– colony and that previously described.3 Generally there was very little overt bloody diarrhoea in our mice and they died from colitis at an earlier age, 6–14 weeks compared with 10–25 weeks as reported in earlier studies. There were also some histopathological dierences between the two colonies. IL-2 –/– mice were first reported as developing IBD similar to ulcerative colitis in humans with prominent crypt abscesses. IL-2 –/– mice in our colony only rarely showed overt ulceration and the lesions
consisted of mucosal thickening, crypt hyperplasia, goblet cell loss and general inflammation of the lamina propria of the colon and caecum. Immunohistochemistry All IL-2 –/– mice studied over 35 days of age had colitis and all less than 35 days were healthy. Mice dying aged less than 35 days had histologically normal mucosa and it is probable that they died of anaemia.3 IL-2 –/– mice with colitis over 35 days of age showed large increases in the number of CD4+, CD8+, abTCR+ and cdTCR+ T cells in the lamina propria of
Figure 1. Lamina propria cell counts for (a) CD4+, (b ) CD8+, (c ) ab TCR, (d ) cd TCR+ T cells, (e) NLDC.145+ dendritic cells, (f ) MOMA-1+ macrophages and ( g) MHC class II+ cells in IL-2 –/– mice under (n=5) and over (n=8 ) 35 days of age and their age-matched controls under (n=5) and over (n=8) 35 days of age. Counts were made in the ileum, caecum and colon. © 1997 Blackwell Science Ltd, Immunology, 91, 73–80
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the caecum and colon compared with wild-type controls ( Fig. 1a–d, Fig. 2). There were slight increases in CD8+ and cd TCR+ T cells in the epithelium ( Fig. 3a–d). There was no change in any of the above parameters in the ileum. Class II+ cells, MOMA-1+ macrophages and NLDC.145+ dendritic cells were also increased in the caecal and colonic lamina propria of IL-2 –/– mice aged over 35 days compared with wild-type controls ( Fig. 1e–g ). The mucosal addressin MAdCAM-1 showed a large increase in expression in the caecum and colon of IL-2 –/– mice compared with wild-type controls (Figs 4 and 5 ). IL-2 –/– mice aged under 35 days, although healthy,
showed some subtle immunohistological signs of preclinical disease. Figure 1e, f shows that there was an increase in the number of NLDC+ dendritic cells and MOMA-1+ macrophages in the colonic lamina propria of IL-2 –/– mice under 35 days of age compared with wild-type controls. There was also an increase in the number of MAdCAM-1+ endothelial cells ( Fig. 4). Cytokine RT-PCR There was a large increase in the amount of IFN-c and IL-1 mRNA in the caecum and colon of IL-2 –/– colitic mice
Figure 2. (a) ab TCR+ T cells in the colon of an IL-2 –/– mouse with colitis over 35 days of age. (b) ab TCR+ T cells in the colon of a wild-type mouse. Original magnification ×200.
Figure 3. Intraepithelial lymphocyte counts for cells staining for (a) CD4, (b) CD8, (c ) ab TCR, and (d ) cd TCR+ T cells in IL-2 –/– mice under (n=5) and over (n=8 ) 35 days of age and their age-matched controls under (n=5) and over (n=8) 35 days of age. Counts were made in the ileum, caecum and colon of each sample. © 1997 Blackwell Science Ltd, Immunology, 91, 73–80
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Figure 4. The expression of MAdCAM-1 in (a) the caecum and (b) the colon of IL-2 –/– mice over and under 35 days of age (n= 14) with age-matched controls (n=11).
Figure 5. The expression of MAdCAM-1+ blood vessels in the colon of (a) an IL-2 –/– mouse with colitis and (b) a wild-type mouse. Original magnification ×200.
aged over 35 days compared with wild-type controls ( Fig. 6a,b). TNF-a showed a similar pattern of expression to that of IL-1 with the exception of an increase in the amount of TNF-a mRNA in the ileum ( Fig. 6c). Th2 cytokines such as IL-4 and IL-10 were decreased in the colons of IL-2 –/– mice (Fig. 6d,e). There was also an increase in the amount of IL-1 and TNF-a mRNA in the colon of IL-2 –/– mice aged under 35 days and an increase in TNF-a mRNA was also seen in the ileum ( Fig. 6b, c ). Cyclosporin A Figure 7 shows that 37% of IL-2 –/– animals that received a twice weekly injection of 5 mg/kg body weight CsA survived for the duration of the experiment, whereas all untreated IL-2 –/– mice died by week 14. DISCUSSION This study has confirmed previous findings3,8 that there is an increase in the number of CD4+, CD8+ and ab TCR+ T cells in the caecal and colonic lamina propria of IL-2 –/– mice aged over 35 days compared with wild-type controls. The infiltrating T cells show a Th1 pattern of cytokine transcripts, with large amounts of IFN-c mRNA in the caecum and colon of IL-2 © 1997 Blackwell Science Ltd, Immunology, 91, 73–80
–/– mice aged over 35 days compared with wild-type mice. This is consistent with the cytokine profiles of other models of spontaneous inflammatory bowel disease.21,22 In adition, experimental models such as mouse graft-versus-host disease (GVHD) have shown that administration of anti-IFN-c antibodies abolishes virtually all the intestinal pathology.23 A human fetal gut model of mucosal damage has suggested that IFN-c may be involved in mucosal injury.24–26 Th2 cytokines such as IL-4 and IL-10 are decreased in the colons of IL-2 –/– mice aged over 35 days even before overt disease. IL-10 is a potent inhibitor of the Th1 response27 and a decrease in the local concentrations of IL-10 may help to explain why there is an uncontrolled immune response in these animals. Administration of a low dose CsA regimen to IL-2 –/– mice resulted in a 40% increase in survival rate and prolonged the lifespan of those that did develop the disease. This is further evidence that the colitis in these IL-2 –/– mice is T-cell mediated. An increase in the number of lamina propria dendritic cells and macrophages has been documented in hapten-induced colitis in rats28 and our present studies confirm that this is also the case in IL-2 knockout mice. Activated macrophages are able to secrete inflammatory cytokines such as IL-1, free radicals and enzymes,29–31 all capable of causing tissue injury. The increase in the number of dendritic cells may help to
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Figure 6. RT-PCR for (a) IFN-c, ( b) IL-1, (c ) TNF-a, (d ) IL-4 and (e) IL-10 in IL-2 –/– mice under (n=5) and over (n=8 ) 35 days of age and their age-matched controls under (n=5 ) and over (n=8 ) 35 days of age. Counts were made in the ileum, caecum and colon of each sample.
Figure 7. Percentage survival rate of IL-2 –/– mice treated with CsA (n=15 ) (+) or vehicle (n=15 ) (©) and wild-type mice treated with CsA (n=10) (#) or vehicle (n=10) ($).
perpeptuate inflammation by presenting antigen locally. In IL-2 –/– mice over 35 days, there was an increase in the number of macrophages and dendritic cells in the caecal and colonic lamina propria compared with wild-type controls. There was also a concurrent increase in the amount of IL-1 and TNF-a mRNA in these tissues and this can be inferred to originate from these cells. The increased production of IL-1 and TNF-a by macrophages and dendritic cells may either
have a direct eect on the mucosa of IL-2 –/– mice or induce surrounding mesenchymal cells to produce enzymes such as matrix metalloproteinases, which can degrade the lamina propria extracellular matrix.32 Only very minor changes were seen in the ileum of IL-2 –/– mice with colitis. The ileum contains an abundant bacterial flora10 and as colitis in IL-2 –/– mice is dependent on the presence of these bacteria3 it is puzzling why these animals do not develop an ileitis. There was, however, an increase in the amount of TNF-a mRNA in the ileum of IL-2 –/– mice both aged over and under 35 days. This suggests that there may be a subtle immune response occurring in the ileum but it is not sucient to result in tissue injury. Endothelial adhesion molecules are known to play an important role in the emigration of inflammatory cells from the blood to tissues.33,34 In the gut the a b integrin on 4 7 lymphocytes, and its ligand the mucosal vascular addressin MAdCAM-1 which is present on mucosal lymphoid organ high endothelial blood venules and gut lamina propria blood vessels, are responsible for the extravasation of lymphocytes to sites of inflammation in the gut.35,36 There is an increase in the number of MAdCAM-1+ blood vessels in the caecal and colonic lamina propria of IL-2 –/– mice aged over 35 days © 1997 Blackwell Science Ltd, Immunology, 91, 73–80
Colitis in IL-2 –/– mice compared with wild-type controls. There is some evidence to suggest that cytokines such as IL-1 and TNF-a are responsible for inducing MAdCAM-1 on endothelial cells.37 In hapteninduced colitis in mice the numbers of MAdCAM-1+ blood vessels are also increased.38 There are three important issues regarding the IBD seen in IL-2 knockout mice arising from this and previous studies. First, why does IL-2 deficiency result in colitis; second, what is the reason for the development of the Th1-dominated cytokine response seen in the mucosa; and third, how does one interpret the evidence that inflammation is an early event in these mice, before the appearance of overt disease? It is still not clear why the deficiency of IL-2 should result in hypersensitivity to a variety of self and non-self antigens. On a BALB/c background, IL-2 –/– mice show evidence of excessive CD4 T-cell activation by 10 days of age and develop inflammatory lesions in a variety of tissues.39 Based on this, it has been proposed that IL-2 is needed to maintain self-tolerance and, in the case of the gut, perhaps tolerance to the flora as well.40 Our studies were carried out in C3H IL-2 –/– mice and, although the disease presents later, they also show evidence of multi-organ disease with nephritis and insulitis (McDonald & McDonald unpublished observations). Perhaps some kind of regulatory cell is highly dependent on IL-2, more so than eector cells. Apparently healthy IL-2 –/– mice under 35 days of age show some subtle immunological signs of preclinical disease in the gut, with a significant increase in the number of macrophages and dendritic cells in the colonic lamina propria, increased expression of IL-1 and TNF-a mRNA and an increase in the number of MAdCAM-1+ blood vessels. These results suggest that the colitis in IL-2 –/– mice may be secondary following initial nonspecific inflammation caused by the gut bacteria. Ciacci et al. have shown that intestinal epithelial cells have IL-2 receptors and proliferate in the presence of IL-2.41 The absence of IL-2 may impair the mucosal barrier, allowing bacterial entry to the lamina propria. Intestinal bacteria contain agents capable of non-specifically activating macrophages, resulting in increased production of IL–12. When the mucosal barrier is intact, the response to soluble antigens is Th2 dominated,42 but nonspecific injury and penetration of bacterial products into the gut wall will favour a Th1 response. If this is directed against the peptides of the flora itself, chronic inflammation will ensue because of persistent antigen challenge. In trinitrobenzene sulfonic acid (TNBS ) colitis in mice, where a major component of the tissue injury is due to the response to the flora,43 antiIL-12 prevents disease.44 Importantly, in the same model, T cells become sensitized to antigens of the flora, and this sensitization is also inhibited by anti-IL-12.45 We would therefore envisage that a deficiency of IL-2 leads to a non-specific influx of bacterial products into the mucosa that, via IL-12, produces an aberrant Th1 response in the lamina propria. ACKNOWLEDGMENTS This work has been funded by the National Association of Crohn’s and Colitis, Crohn’s in Childhood Research Appeal, BUPA and The Netherlands Organization for Scientific Research.
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