Nina N. Pawlowski

Animal Models of Inflammatory Bowel Disease: An Overview

Inflammatory bowel disease (IBD) research has been performed in human in vitro studies and in in vivo studies using appropriate animal models. Such animal models allow both the examination of inflammatory processes (both early and late events) as well as the evaluation of new therapeutic modalities. Since the first description of the immune complex colitis in rabbits in 1961, overall 63 models have been described, most of which within the last decade. These IBD animal models can be divided into 5 different categories: (1) antigen-induced colitis and colitis induced by microbials; (2) other inducible forms of colitis (chemical, immunological, and physical); (3) genetic colitis models (transgenic and knock-out models); (4) adoptive transfer models, and (5) spontaneous colitis models. In spite of the high overall number of models, none of them is the ‘perfect’ model and therefore numerous aspects need to be considered when choosing one model for a particular study. Importantly, most clinical aspects (e.g. extraintestinal manifestations or fistula) have recently been described in one or the other model allowing further studies with relevance for almost all aspects of IBD. So far, IBD animal models have taught us important lessons, e.g. the requirement of T-helper cells in most models, the need of a particular genetic background, and the role of the flora in the initiation of IBD. It is expected that our understanding of IBD will further increase in a number of additional areas using animal models, e.g. exploring the role of the innate immune system in IBD.

Aggravation of intestinal inflammation by depletion/deficiency of γδ T cells in different types of IBD animal models

The role of γδ T cells in inflammatory bowel disease (IBD) is still controversial. Although γδ T cells induce IBD in immunodeficient animals, others suggest a protective role of γδ T cells. Therefore, this study was conducted in order to elucidate the effect of γδ T cell depletion/deficiency on different IBD animal models. Mice depleted of or deficient in γδ T cells were exposed to dextran sodium sulfate (DSS) in order to induce colitis. In addition, γδ T cells were depleted in mice with terminal ileitis (TNFΔARE) or colitis due to interleukin 2 deficiency (IL‐2 ko). Finally, DSS‐induced colitis was studied in mice deficient in interferon gamma (IFN‐γ ko) upon γδ T cell depletion. Depletion of γδ T cells aggravated DSS‐induced colitis and terminal ileitis of TNFΔARE mice. Exacerbated DSS‐induced colitis was also found in γδ T cell‐deficient mice. IL‐2 ko mice showed increased mortality upon early (starting at 4 wk of age) but not late depletion (starting at 8 wk of age). Early γδ T cell depletion or deficiency resulted in increased IFN‐γ production by both lamina propria lymphocytes and splenocytes in every model investigated herein. In IFN‐γ ko mice, γδ T cell depletion did not affect the development and course of DSS‐induced colitis. The protective effect of γδ T cells in IBD was confirmed in various IBD animal models. Particularly, during the early phase of intestinal inflammation, γδ T cells appear to be important. The mechanism seems to involve the control of IFN‐γ production and epithelial regeneration.

Human peripheral γδ T cells possess regulatory potential

Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.

Human peripheral gammadelta T cells possess regulatory potential.

Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.

γδ T lymphocytes: a new type of regulatory T cells suppressing murine 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis

BackgroundThe intestinal immune system is continuously challenged by antigen without becoming dysregulated. However, injury of the mucosa by, i.e. dextran sulphate sodium causes severe inflammation in γδ T-cell-deficient mice. We therefore asked whether γδ T cells have regulatory functions.Materials and methodsγδ T cells were isolated from spleens and mesenteric lymph nodes of C57BL/6 wild-type (wt) mice. Proliferation and cytokine secretion of γδ T cells were quantified by [3H] thymidine incorporation and ELISA. Additionally, proliferation of carboxyfluorescein diacetate succinimidylester-labelled CD4+ T cells cocultured with γδ T cells was analysed by flow cytometry. Finally, γδ T cells from wt or interleukin-10 transgenic (IL-10tg) mice were transferred into congenic mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis.Resultsγδ T cells were hyporesponsive to CD3/CD28 stimulation and suppressed CD4+ T-cell proliferation (up to 66 ± 7% suppression) in vitro. Further, the preventive transfer of wt or IL-10tg γδ T cells ameliorated TNBS-induced colitis resulting in prolonged survival and reduced histological damage (1.5 ± 0.4 and 1.3 ± 0.2, respectively vs. 3.8 ± 0.3 in untransferred mice, p < 0.05). This was accompanied by reduced TNF-α and increased IL-10 and TGF-β secretion from intestinal and splenic lymphocytes.ConclusionsMurine γδ T cells are a new type of regulatory T cells in vitro and act protective on mouse TNBS-induced colitis in vivo. Future studies have to define the underlying mechanism and to investigate whether γδ T cells can be used for immunotherapy of human inflammatory bowel disease.

αCD2 mAb treatment safely attenuates adoptive transfer colitis

Increased proliferation, defective apoptosis, and cytokine dysregulation of T lymphocytes are thought to be important for the pathogenesis of inflammatory bowel disease. Since these phenomena can be corrected by αCD2 mAb, we asked whether CD2 directed immunotherapy safely prevents and/or ameliorates adoptive transfer colitis. Colitis was induced by transfer of CD4+ T cell blasts to syngenic RAG1−/− mice or CD45RBhigh CD4+ T cells to SCID mice. The αCD2 mAb 12-15 or rat IgG was given, starting either initially or upon first signs of colitis. Disease activity was assessed by clinical monitoring, microscopic scoring, hemoccult, endoscopy, and blood count analysis. Cytokine production of stimulated LPL was measured by ELISA and cell proliferation by [3H]-thymidine incorporation. Parasite control was analyzed in a murine model of infection with Toxoplasma gondii. The αCD2 mAb significantly increased mean survival time when starting at transfer of blasts (survival >35 days: αCD2 69% vs 0% of controls, P<0.001). In the SCID colitis model hematochezia and macroscopic colitis were delayed. When used in established T-cell blast colitis, the benefit was less pronounced, even in combination with dexamethasone (mean survival±s.e.m.: αCD2+dexa: 13.5±2.9 vs dexa+IgG: 6.3±1.0, P<0.05). In the preventive experiment the αCD2 mAb markedly reduced IL-2 secretion and T-cell proliferation. The immune response towards Toxoplasma gondii was not impaired. These studies show for the first time that CD2 directed immunotherapy can attenuate or delay adoptive transfer colitis and ameliorate established colitis. Most likely inhibition of IL-2 secretion and T-cell proliferation are responsible for these effects. Still, immune defence towards T. gondii is maintained.

Targeting human CD2 by the monoclonal antibody CB.219 reduces intestinal inflammation in a humanized transfer colitis model

The cell adhesion molecule CD2 facilitates antigen-independent T-cell activation and CD2 deficiency or blockade reduces intestinal inflammation in murine models. We here aimed to evaluate the therapeutic potential of monoclonal antibodies (mAb) specific for human CD2 in colitis treatment. Transfer colitis induced by naïve CD4(+) T cells expressing human CD2 was treated with anti-human CD2 mAb. The mAb CB.219 protected from severe colitis in a preventive treatment regimen, while therapeutic treatment ameliorated intestinal inflammation. Diminished intestinal tissue damage was paralleled by a profound suppression of lamina propria lymphocytes to produce pro-inflammatory cytokines and tumor necrosis factor α as well as the neutrophil chemoattractant CXC motif ligand 1 and the CC chemokine ligand 3. Furthermore, infiltration with macrophages and T cells was low. Thus, reduced intestinal inflammation in our humanized colitis model by targeting CD2 on T cells with the mAb CB.219 suggests a novel approach for colitis treatment.

Lamina propria T cell activation: role of the costimulatory molecule CD2 and its cytoplasmic tail for the regulation of proliferation and apoptosis.

Background/aimsAccumulation of T lymphocytes in the gut is a hallmark of inflammatory bowel disease probably caused by insufficient T cell apoptosis. Activated peripheral T cells, or “resting” lamina propria T lymphocytes (LPLs), are highly susceptible to apoptosis induction, e.g., using the mitogenic anti-CD2 monoclonal antibody (mAb) pair T112+3. It is, however, unknown how CD2-mediated LPL apoptosis is related to proliferation and whether the whole CD2 molecule is required for apoptosis induction.Materials and methodsMapping of anti-CD2 mAb was performed using erythrocyte rosetting assays and cross-blocking enzyme-linked immunosorbent assay (ELISA). Lamina propria mononuclear cells (LPMNCs) or phytohemagglutinin (PHA) blasts were stimulated with a panel of 18 anti-CD2 mAbs followed by apoptosis analysis [Annexin V expression on propidium iodide (PI)-negative cells, 4c6-diamidino-2-phenylindole·2HCl (DAPI) staining]. Proliferation was measured by [3H]-thymidine incorporation. For structural analysis, EL4 cells were used which were transfected with human CD2 (wild type (WT), cytoplasmic-deficient, cytoplasmic CD28). Sorting was performed employing standard techniquesResultsAll three mitogenic anti-CD2 mAb pairs induced apoptosis of LPMNC and PHA blasts. Two out of four submitogenic anti-CD2 mAb, AICD2.M3, and ICRFCD2.3 lead to LPMNC proliferation but no apoptosis. Importantly, apoptosis was also detected in cytoplasmic-deficient CD2 tg or CD2/CD2/CD28 tg EL4 cells. Sorted CD45high huCD2 WT EL4 had higher apoptosis rates compared to WT huCD2tg EL4 cellsConclusionLPMNC apoptosis induction via CD2 is always associated with proliferation, although proliferation is not necessarily associated with apoptosis. The cytoplasmic tail of CD2 is not required, and CD45 appears to transmit apoptotic signals entering the T cell via CD2.

Human peripheral γδ T cells possess regulatory potential: Human γδ T cells have regulatory potential

Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.