Autoimmune, Cholestatic and Biliary Disease
Differential mechanisms in the pathogenesis of autoimmune cholangitis versus inflammatory bowel disease in interleukin-2Rα−/− mice†
Article first published online: 22 AUG 2008
Copyright © 2008 American Association for the Study of Liver Diseases
Volume 49, Issue 1, pages 133–140, January 2009
How to Cite
Hsu, W., Zhang, W., Tsuneyama, K., Moritoki, Y., Ridgway, W. M., Ansari, A. A., Coppel, R. L., Lian, Z.-X., Mackay, I. and Gershwin, M. E. (2009), Differential mechanisms in the pathogenesis of autoimmune cholangitis versus inflammatory bowel disease in interleukin-2Rα−/− mice. Hepatology, 49: 133–140. doi: 10.1002/hep.22591
Potential conflict of interest: Nothing to report.
- Issue published online: 28 DEC 2008
- Article first published online: 22 AUG 2008
- Accepted manuscript online: 22 AUG 2008 12:00AM EST
- Manuscript Accepted: 18 AUG 2008
- Manuscript Received: 8 JUL 2008
- National Institutes of Health. Grant Numbers: DK39588, DK074768, DK077961
- UCD Center for Health and Nutrition Research Pilot Grant
Interleukin-2 (IL-2) receptor α knockout (IL-2Rα−/−) mice have a deficiency of CD25 and a corresponding functional defect in T regulatory cells (Tregs). These mice spontaneously develop portal inflammation with biliary ductular damage and colitis with features similar to human inflammatory bowel disease with T cell infiltrates in both the liver and colon. In humans, inflammatory bowel disease may be accompanied by primary sclerosing cholangitis (PSC), but seldom primary biliary cirrhosis (PBC). We hypothesized that the effector mechanism responsible for T cell infiltrates would differ for colon versus liver. To address this thesis, we developed three colonies of double-knockout mice including IL-2Rα−/− CD4−/−, IL-2Rα−/− CD8−/−, and IL-2Rα−/− T cell receptor (TCR)-β−/−. Tissue immunopathology, body weight, and serum levels of cytokines, immunoglobulins, and anti-mitochondrial antibodies (AMA) were assayed at 3 months of age. Relative to IL-2Rα−/− mice, IL-2Rα−/− CD4−/− mice had increased biliary ductular damage but reduced inflammation in the colon. In contrast, IL-2Rα−/− CD8−/− mice had increased colon inflammation but markedly attenuated biliary ductular damage. Both IL-2Rα−/− CD4−/− and IL-2Rα−/− CD8−/− mice demonstrated elevated serum levels of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-12p40 (IL-12p40), and interleukin-2 (IL-2) compared with C57BL/6J controls, but only IL-2Rα−/− CD8−/− mice had increased serum levels of immunoglobulin A (IgA), AMA and interleukin-17 (IL-17). Finally, and of importance, IL-2Rα−/− TCR-β−/− mice had abrogation of liver and colon pathological conditions and lacked AMA. In conclusion, on loss of Treg function in mice, CD8 T cells mediate biliary ductular damage whereas CD4 T cells mediate induction of colon-specific autoimmunity. (HEPATOLOGY 2009;49:133-140.)
The role of the interleukin-2 receptor α (IL-2Rα, CD25) and its relationship with T regulatory cells (Tregs) and autoimmunity has been well demonstrated in murine and human inflammatory bowel disease and, more recently, in murine autoimmune cholangitis and human primary biliary cirrhosis (PBC).1–6 The IL-2Rα−/− murine model became of interest originally because of peripheral lymphadenopathy, impaired activation induced T cell death, hemolytic anemia, and rapid onset of colitis.7 Recent investigations have shown that if mice are maintained on antibiotics to prolong survival and minimize colitis, they express portal inflammation and intrahepatic bile duct damage, with a CD8 T cell bias associated with increased serum levels of Th1-dependent proinflammatory cytokines, namely interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and interleukin-12p40 (IL-12p40) and high levels of serum anti-mitochondrial antibodies (AMA).8 Because colitis seldom co-exists with the autoimmune biliary ductular destruction of PBC, we hypothesized that the selective targeting of autoimmune responsiveness against colon and cholangioles was attributable to distinct effector mechanisms. This view prompted us to assess the effects on severity of ulcerative colitis and autoimmune cholangitis by genetic depletion of one or the other of the major T cell subsets in IL-2Rα−/− mice.
Accordingly, we generated double-knockout mice, IL-2Rα−/− CD4−/−, IL-2Rα−/− CD8−/− and IL-2Rα−/− T cell receptor (TCR)-β−/−. We report herein that IL-2Rα−/− CD4−/− mice had attenuated colitis but increased intrahepatic biliary ductular destruction, whereas IL-2Rα−/− CD8−/− mice lacked biliary ductular destruction but had worse colitis, compared with single-knockout IL-2Rα−/− mice. The predominant role of T cells in these models was illustrated by the lack of pathological conditions in IL-2Rα−/− TCR-β−/− mice. In addition to the severe portal tract cellular infiltration in IL-2Rα−/− CD4−/− mice, both proinflammatory and T helper 1 (Th1) cytokines were elevated in serum, whereas IL-2Rα−/− CD8−/− mice not only had elevated serum levels of Th1 and inflammatory cytokines, but also increased levels of interleukin-17 (IL-17) and total immunoglobulin A (IgA) levels, as seen in IL-2Rα−/− mice. The data reported herein further argue for a key role of CD8 T cells in the pathogenesis of autoimmune cholangitis.
Material and Methods
IL-2Rα−/− (C57BL/6J.129S4-Il2ratm1dw), IL-2Rα+/−, TCR-β−/−, CD8−/− (Cd8tm1Mak), and CD4−/−(Cd4tm1Mak) on a C57BL/6J background were initially obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were then bred at the University of California Animal Vivarium using ventilated cages and specific pathogen-free conditions. Because IL-2Rα−/− mice are infertile, IL-2Rα+/− mice were crossed with TCR-β−/−, CD8−/− or CD4−/− mice and subsequently screened by genotyping as specified by Jackson Laboratory for IL-2Rα+/− TCR-β+/−, IL-2Rα+/− CD8+/− or IL-2Rα+/− CD4+/− mice. The resulting F1 generation were mated to generate F2 litters and subsequently screened by genotyping as specified by Jackson Laboratory for IL-2Rα−/− TCR-β−/−, IL-2Rα−/− CD8−/− or IL-2Rα−/− CD4−/− mice. Mice were studied from 4 through 12 weeks of age. At that time the experiment was terminated according to Animal Welfare protocol because of severe colitis in some animals. Each time and data point reflects data from four to eight mice. All studies were performed under approval from the University of California Animal Care and Use Committee.
Sections of the liver and colon were prepared and immediately fixed with 4% paraformaldehyde (Electron Microscopy Services, Hatfield, PA) for 48 hours. Each tissue was then embedded in paraffin and cut into 4-μm sections for routine hematoxylin (DakoCytomation, Carpinteria, CA) eosin (American Master Tech Scientific, Lodi, CA) (HE) staining. Liver disease staging was performed based on histological evaluation of HE-stained sections using a set of four indices by a pathologist who was blind to the protocol of these studies. Parameters that were evaluated include levels of portal tract inflammation and bile duct damage. Colon HE-stained sections were scored according to their level of colitis. The staging was based on four indices, including erosion, inflammation, epithelial change, and crypt abscesses.
Spleens were homogenized between two glass slides and suspended in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) (PBS/BSA) (EMD chemicals, Gibbstown, NJ). Livers were homogenized and passed through a 100-μm cell strainer (BD Biosciences, San Jose, CA) and suspended in PBS/BSA as described.9 Hepatocytes were removed as pellets by centrifugation at 75g for 1 minute. The small intestine was washed with growth media, Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum (Clonetech Laboratories, Mountain View, CA), using a 6-mL syringe and a blunt 18-gauge needle. Using a pair of scissors, the small intestine was cut into 3-cm sections and incubated in growth media while shaking at 300 rpm for 1 hour at 37°C. Intestinal tissues were removed as pellets by centrifugation at 75g for 1 minute. Lymphocytes were isolated from suspended liver, spleen, and intestinal cells using Histopaque-1077 (Sigma Chemical Co., St. Louis, MO). Cells were then washed with PBS/BSA and live cells were counted using a hemocytometer after trypan blue dye (Sigma Chemical Co.) staining. Surface Fc receptors (FcR) were blocked by incubation of lymphocytes with anti-mouse FcR blocking reagent for 5 minutes at 4°C. Lymphocytes were then stained for 30 minutes at 4°C with a combination of fluorochrome-conjugated antibodies, including anti-NK1.1 fluorescein isothiocyanate (BioLegend, San Diego, CA), anti-CD8β phycoerythrin (eBiosciences, San Diego, CA), anti-CD4 PerCP (BioLegend), anti-CD19 Alexa Fluor 647 (BD Biosciences), and anti-CD3e APC/Cy7 (eBiosciences). The stained cells were then washed with PBS/BSA and subjected to multiple-color analysis by a FACscan flow cytometer (BD Immunocytometry Systems, San Jose, CA) upgraded by Cytec Development (Fremont, CA) to permit five-color analysis. CellQuest software (BD Biosciences) was used to analyze the results. T cells were gated as CD3+ NK1.1−, and B cells were gated as CD19+.
Serum Immunoglobulin and AMA Levels.
Quantitation of serum immunoglobulin G, IgA, and immunoglobulin M (IgM) were carried out, using a mouse immunoglobulin enzyme-linked immunosorbent assay (ELISA) kit (Bethyl, Montgomery, TX). Known murine immunoglobulin myelomas were used in parallel as standards. Mouse serum AMAs were detected by ELISA using recombinant pyruvate dehydrogenase-E2 as previously described.10, 11 Briefly, 100 μL purified recombinant pyruvate dehydrogenase-E2 antigen at 10 μg/mL in carbonate buffer (pH 9.6) were coated onto each well on a 96-well ELISA plates at 4°C overnight. Plates were washed three times with PBS with 0.05% Tween 20 and blocked with 200 μL 1% BSA in PBS for 1 hour at room temperature. After removing the blocking solution, 100 μL sera to be tested, diluted (1:200) in PBS with 1% BSA, was added into each well and incubated for 1 hour at room temperature. The plates were then washed five times with PBS with 0.05% Tween 20, and 100 μL horseradish peroxidase–conjugated anti-mouse polyvalent immunoglobulin G, IgA, and IgM (Zymed, San Francisco, CA) diluted (1:3,000) in PBS with 1% BSA was added into each well and incubated for 1 hour at room temperature. The plates were then washed five times, and 100 μL tetramethyl benzidine peroxidase substrate (BD Biosciences) was added to each well. After 15 minutes' incubation at room temperature, the optical density at 450 nm was measured by ELISA plate reader (Perkin Elmer, Waltham, MA). Previously determined positive and negative samples were included with each assay.
Levels of serum TNF-α, IL-2, interleukin-4, interleukin-5, interleukin-6, interleukin-10, and IFN-γ from 3-month-old mice were measured using a mouse inflammatory cytometric bead array (CBA) kit and the mouse Th1/Th2 Cytokine CBA kit (BD Biosciences). Detection and analysis of serum cytokine levels were performed using a FACScan flow cytometer (BD Immunocytometry Systems). Quantikine mouse IL-17 and IL-12p40 ELISA kits (R&D Systems, Minneapolis, MN) were used to measure serum IL-17 and IL-12p40.
All results are expressed with mean ± standard error of mean. Mann-Whitney U tests and analysis of variance were used to compare the differences between all groups of mice. P values less than 0.05 were defined as significant.
All IL-2Rα−/− CD4−/− mice (8/8) display bile duct damage at 3 months of age with marked mononuclear cell infiltration surrounding most bile ducts corresponding to the foci of biliary epithelial cell destruction (Fig. 1A). In contrast, liver sections from IL-2Rα−/− CD8−/− mice appeared normal, lacking any detectable cellular infiltrates within either the portal tract or parenchymal tissues. Note that liver sections from IL-2Rα−/− were used as a positive control (Fig. 1B). The absolute number of T cells in the hepatic mononuclear cells (HMNC) population of IL-2Rα−/− CD4−/− mice was significantly increased compared with that of IL-2Rα−/− CD8−/− mice, 7.83 ± 1.14 (×106) and 0.63 ± 0.10 (×106) cells, respectively (Fig. 1C). The total number of T cells from HMNC of IL-2Rα−/− mice, 9.37 ± 3.21 × 106, was comparable to those of IL-2Rα−/− CD4−/− mice, being significantly higher than that of IL-2Rα−/− CD8−/− mice. As we have previously reported, there is a significant increase in the CD8/CD4 ratio in the IL-2Rα−/− mice (3.95 ± 0.684) in comparison to the C57BL/6 mice (1.01 ± 0.091). There were no significant differences in the infiltration of B cells within the HMNCs between the various groups of mice. Similarly, there was no statistically significant difference in the total B cell number isolated from liver of IL-2Rα−/−, IL-2Rα−/− CD4−/−, and IL-2Rα−/− CD8−/− mice, although an overall trend toward decreased numbers of B cells from IL-2Rα−/− CD8−/− mice compared with the other strains of mice was noted. However, the absolute number of B cells within the HMNC population of IL-2Rα−/− CD4−/− mice, 0.89 ± 0.18 × 106, were significantly increased compared with C57BL/6J mice (0.21 ± 0.03 × 106) (Fig. 1D). Finally, it should be noted that the liver histology of IL-2Rα−/− TCR-β−/− mice was completely normal and identical to C57BL/6J control mice (Fig. 1B).
IL-2Rα−/− CD4−/− (2/8) mice at 3 months of age had only minimal colon inflammation, whereas IL-2Rα−/− CD8−/− (4/8) mice at the same age had significant colon inflammation. Sections of the colon from IL-2Rα−/− CD8−/− mice demonstrated lymphoid hyperplasia, crypt abscesses, and subserosal inflammation, whereas IL-2Rα−/− CD4−/− mice exhibited only mild lymphoid hyperplasia (Fig. 2A). Colon sections from IL-2Rα−/− were used as a positive control (Fig. 2B). Interestingly, colon histopathology of IL-2Rα−/− TCR-β−/− mice was normal and similar to C57BL/6J mice.
Small Intestinal Intraepithelial Lymphocytes.
The small intestinal intraepithelial lymphocyte (IEL) population from IL-2Rα−/− CD4−/− mice exhibited decreased total T cell numbers compared with that of IL-2Rα−/− CD8−/− mice, 0.53 ± 0.09 (×106) and 3.93 ± 1.22 (×106) cells, respectively (Fig. 2C). The total number of T cells from IEL of IL-2Rα−/− mice, 6.33 ± 0.84 (×106), was significantly increased compared with that of both IL-2Rα−/− CD4−/− and IL-2Rα−/− CD8−/− mice. IL-2Rα−/− mice also exhibited increased T cell numbers compared with C57BL/6J mice, 1.02 ± 0.16 (×106). There is no significant difference in the number of CD8 T cells between IL-2Rα−/− CD4−/− (0.53 ± 0.09) and C57BL/6J mice (0.75 ± 0.14). The absolute number of B cells within the IEL populations of IL-2Rα−/− CD8−/− and IL-2Rα−/− CD4−/− mice was 0.06 ± 0.01 × 106 and 0.02 ± 0.01 × 106 cells, respectively (Fig. 2D). These absolute numbers were significantly less than within IELs of IL-2Rα−/− mice, 0.23 ± 0.04 × 106. In contrast, IL-2Rα−/− CD4−/− mice had significantly fewer B cells than C57BL/6J mice (0.16 ± 0.06 × 106).
Serum IgA levels of IL-2Rα−/− mice (53.5 ± 12.7 μg/mL) and IL-2Rα−/− CD8−/− mice (70.4 ± 9.7 μg/mL) were increased compared with those of IL-2Rα−/− CD4−/− (9.9 ± 2.9 μg/mL), and C57BL/6J mice, 9.8 ± 3.6 μg/mL (Fig. 3). Serum IgM levels of C57BL/6J (12.5 ± 0.6 μg/mL) and IL-2Rα−/− CD8−/− (14.3 ± 1.0 μg/mL) mice were significantly increased compared with those of IL-2Rα−/− CD4−/− mice (10.0 ± 0.8 μg/mL).
IL-2Rα−/− and IL-2Rα−/− CD8−/− mice had significantly increased levels of AMA (Fig. 4), greater than 3 standard deviations above the means of control C57BL/6J mice. There were no detectable levels of serum AMA in IL-2Rα−/− TCR-β−/− (data not shown).
Serum levels of TNF-α, IFN-γ, IL-2, and IL-12p40 in IL-2Rα−/− CD4−/−, IL-2Rα−/− CD8−/−, and IL-2Rα−/− were increased compared with C57BL/6J mice (Fig. 5). In addition, IL-2Rα−/− CD4−/− mice had increased serum levels of TNF-α compared with IL-2Rα−/− CD8−/− mice. The only pro-inflammatory cytokine not elevated in IL-2Rα−/− CD4−/− mice was IL-17. In contrast, IL-2Rα−/− CD8−/− and IL-2Rα−/− mice exhibited significantly elevated IL-17 levels compared with C57BL/6J mice.
Lack of the high-affinity binding subunit for IL-2, CD25, results in a decreased frequency of peripheral Tregs in IL-2Rα−/− mice, and although Foxp3+ Treg cells do still exist within these mice,12 the deficiency in CD25+ Tregs is sufficient to cause spontaneous lymphoproliferation accompanied by the subsequent features typical of autoimmunity. We succeeded in completely abrogating the onset of autoimmunity by creating TCR-β–deficient mice on an IL-2Rα−/− background, with such mice remaining free from colitis and biliary damage up to 3 months of age. Another method to inhibit disease is to reconstitute the CD25+ population, emphasizing the key role of T cells, in particular Tregs, in the disease development of these mice.13 The approach taken herein is a direct examination of double-knockout mice. This greatly facilitates interpretation of adoptive transfer systems. Although one may postulate that these data could also be obtained by transferring CD4 cells or CD8 cells into our double-knockout mice, to recapitulate these data, such experiments are prone to artefact. For example, the transfer of CD4 T cells would also require the use of IL-2, because such cells would not survive in the absence of IL-2. In addition, dose response experiments involving different numbers of cells, likely with the need for low-dose irradiation of the recipient, would also confound the data. Hence, the use of double-knockout mice, although expensive to generate, provides a cleaner environment for data interpretation.
A number of studies have been conducted on the mechanism for intestinal infiltration and biliary damage. The transfer of CD4+ CD45RBhigh T cells into recombination activating gene (RAG)-1−/− mice is sufficient to trigger the onset of colonic inflammation accompanied by increased serum levels of Th1 cytokines, proving that CD4 T cells in the absence of CD8 T cells can initiate and sustain expression of inflammatory bowel disease.14, 15 In the current study, we demonstrate that although CD4 T cells alone are sufficient for the development of colitis mediated by Th1 and Th17 cytokines in the IL-2Rα−/− mice, such mice do not demonstrate any detectable levels of bile duct destruction. CD8 T cells were not only necessary, but also sufficient for biliary damage even in the absence of CD4 T cell help. Thus, by crossing CD8−/− mice onto an IL-2Rα−/− background, we examined the role of CD4 T cells in disease development in the absence of CD8 T cells. The loss of CD8+CD28− Tregs likely contributes to the reduction of colonic inflammation by inhibition of CD4 T cell proliferation and secretion of IFN-γ.16 Like IL-2Rα−/− mice, IL-2Rα−/− CD8−/− mice exhibit splenomegaly and are infertile (data not shown) and, even though they have more T and B cells among their IEL population than do C57BL/6J mice, these cells are still significantly less than those in IL-2Rα−/− mice. The exacerbated colitis in IL-2Rα−/− CD8−/− mice is likely to increased levels of CD4 T cell-dependent inflammatory cytokines and chemokines such as IL-17 levels (Fig. 5), which have been associated with colon pathological conditions in patients and in animal models of colitis.17–20 An increase in B cell infiltration within the colonic epithelium of IL-2Rα−/− CD8−/− mice could account for elevated levels of total and mucosal IgA, corresponding with the degree of mucosal inflammation. Taken together, our data show that a functional CD8 T cell population is dispensable for initiation or persistence of colonic inflammation. We believe that the model of xenobiotic-induced autoimmune cholangitis also requires functional CD8 T cells to mediate autoimmune cholangitis.21–23
In the liver, a different situation was observed. The portal tract inflammation and biliary destruction appears to be mediated by Th1 and inflammatory cytokines produced by CD8 T cells that have infiltrated into the portal tracts. IL-2Rα−/− CD4−/− mice exhibit increased serum levels of IFN-γ, TNF-α, IL-2, and IL-12p40 comparable to levels found in IL-2Rα−/− mice. In agreement with our previous studies,8 these data suggest that CD8 T cells are responsible for the actual production of these Th1-dependent inflammatory cytokines. Comparably, CD8 T cells isolated from the peripheral blood mononuclear cells of PBC patients show increased intracellular staining levels for IFN-γ.24 This cytokine-facilitated destruction of biliary epithelium is presumably mediated by elevated levels of IFN-γ and TNF-α. These cytokines can compromise tight junction integrity in immortalized mouse cholangiocyte cultures and enhance priming of CD8 T cells by autoantigens of the biliary epithelium.25 Incubation of IFN-γ and TNF-α with biliary epithelial cells has been shown to increase not only intercellular adhesion molecule-1 expression but also antigen processing and presentation, specifically in PBC patients.26 There could also be priming of autoreactive CD8 T cells by cross presentation by activated dendritic cells or even biliary epithelial cells.24, 27 Once these autoreactive CD8 T cells are primed and activated, they could be localized to the biliary epithelium by local chemokine production28 or by the normal retention of activated CD8 T cells in the liver.29, 30 Although not addressed in this study, autoreactive CD8 T cells could also serve as effectors of biliary ductular injury by direct cytotoxicity via perforin or granzyme pathways.
The long-standing question of whether serum AMA participates in the pathogenesis of human PBC is also addressed herein. The data herein on IgA deficiency in the (IL-2Rα−/− CD4−/−) are expected, based on the need for CD4 cells for class switching. Unlike patients with PBC, there are no dramatic differences in sera IgM. These data as well as the immunohistology further highlight the critical role of lymphocytic responses in mediating disease, rather than a direct role for AMAs. Indeed, we have previously suggested that the anti-mitochondrial response in patients should be considered a multi-lineage response involving CD4 and CD8 T cells in addition to autoantibody.31 Our data indicate that CD4 T cell–deficient IL-2Rα−/− CD4−/− mice exhibit severe biliary ductular damage in the absence of significant levels of serum AMA, whereas CD8 T cell–deprived (IL-2Rα−/− CD8−/−) mice demonstrated lesson biliary damage yet had significantly higher levels of serum AMA (Fig. 4). Thus, in this model the participation of CD8 T cell in biliary destruction is dominant and CD8 T cell effector function can be developed in the absence of CD4 T cell help, whereas B cells that produce AMA require CD4 T cell helper effects. In humans with PBC, we have demonstrated a higher number of autoreactive CD8 T cells in PBC liver compared with blood and also a higher frequency in earlier stage disease. The control of activated CD8 T cells merits attention, as does the use of biotherapies that specifically target such effector T cells. Therapies directed at restoring the balance of Treg function also may prevent or alleviate features of autoimmunity, particularly in progressive autoimmune diseases in which CD8 T cell activity is suspected, including PBC. We should also note that the availability of murine models allow several additional key questions to be addressed, including the role of autophagy, apoptosis, and molecular mimicry, all issues that have been raised in the pathogenesis of PBC and the selective immune destruction of bile ducts.32–34 To this end, our future studies in mouse models of PBC will investigate attenuation of biliary damage by disabling the functions of effector cytotoxic T cells and their cytokines, as well as restoration of immunoregulatory mechanisms.
- 3The straw that stirs the drink: insight into the pathogenesis of inflammatory bowel disease revealed through the study of microflora-induced inflammation in genetically modified mice. Inflamm Bowel Dis 2007; 13: 490–500..
- 18Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD. Inflamm Bowel Dis 2008; 14: 437–445., , , , , , et al.