Defective mucosal T cell death is sustainably reverted by infliximab in a caspase dependent pathway in Crohn's disease

Abstract

Background and aims: To verify whether targeting defective mucosal T cell death underlies the sustained therapeutic benefit of infliximab in Crohn's disease, we explored its in vivo proapoptotic effect after 10 weeks of treatment, and its in vitro killing activity on lamina propria T cells (LPT) and peripheral blood T cells (PBT), both isolated from Crohn's disease patients.

Methods: Endoscopic intestinal biopsies were collected from 10 Crohn's disease patients (six steroid refractory and four fistulising) before and after three consecutive infusions of infliximab, administered at week 0, 2, and 6 in a single intravenous dose (5 mg/kg), and from 10 subjects who proved to have functional diarrhoea. Apoptosis was determined in vivo by TUNEL assay, and in vitro by fluorescein isothiocyanate-annexin V/propidium iodide staining on LPT and PBT from Crohn's disease patients cultured with infliximab. The effect of the broad caspase inhibitor Z-VAD-FMK and the neutralising anti-Fas antibody ZB4 was tested in vitro on LPT and PBT treated with infliximab. Caspase-3 activity was determined by immunoblotting.

Results: In Crohn's disease patients, infliximab treatment induced a sustained LPT apoptosis, still evident four weeks after the last infusion. In vitro infliximab induced death of LPT from Crohn's disease patients occurred via apoptosis rather than necrosis. LPT showed a higher susceptibility to infliximab induced apoptosis than PBT in Crohn's disease patients. The signalling pathway underlying the restoration of infliximab induced LPT apoptosis occurred via the caspase pathway but not Fas-Fas ligand interaction in Crohn's disease.

Conclusions: These findings demonstrate that apoptosis is the major mechanism by which infliximab exerts its killing activity on LPT in Crohn's disease. The sustained LPT proapoptotic action of infliximab, which extends far beyond its circulating half life, may be responsible for the sustained remission induced in Crohn's disease patients by infliximab retreatment.

Figure 1

Detection of apoptotic cells by terminal deoxynucleotidyl transferase mediated digoxigenin-deoxyuridine triphosphate nick end labelling (TUNEL) staining in Crohn’s disease patients, before (A) and after 10 weeks of treatment with infliximab (B), and in normal intestinal mucosa (C). In Crohn’s disease patients before treatment, TUNEL positivity is evident at the level of epithelial (arrows) but not lamina propria mononuclear cells while in Crohn’s disease patients after treatment and in controls, a considerable proportion of lamina propria mononuclear cells are TUNEL positive (arrows) (original magnification, ×400). (D) Percentage of TUNEL positive lamina propria mononuclear cells in 10 patients with Crohn’s disease before and after 10 weeks of treatment with infliximab, and in 10 biopsied controls. The four patients with a complete response to treatment and the one non-responder are indicated with different symbols (filled squares and triangles, respectively). Horizontal bars represent median values.

Figure 2

Flow cytometric analysis of fluorescein isothiocyanate (FITC)-annexin V binding and propidium iodide (PI) uptake of Crohn’s disease lamina propria T cells (LPT) (A, B) and peripheral blood T cells (PBT) (C, D) cultured with infliximab. LPT and PBT were incubated with IgG1 as control antibody (A and C, respectively) and with infliximab at a concentration of 5 μg/ml (B and D, respectively). The lower left quadrant of each panel shows live cells (FITC-annexin V negative/PI negative), the lower right quadrant represents apoptotic cells (FITC-annexin V positive/PI negative), and the upper right quadrant contains the non-viable dead cells (FITC-annexin V positive/PI positive). Numbers within the dot plots represent the percentages of apoptotic and dead cells. The data, which are representative of experiments performed in all patients with Crohn’s disease, show the higher percentage of apoptotic LPT treated with infliximab (B) in comparison with those cultured with the IgG1 isotype control (A), and the higher susceptibility of LPT (B) to undergo infliximab induced apoptosis compared with PBT (D). The low percentages of double stained cells allowed us to exclude necrotic membrane damage as a consequence of infliximab treatment.

Figure 3

Percentages of apoptotic and necrotic lamina propria T cells (LPT) from Crohn’s disease patients, assessed by flow cytometric analysis of fluorescein isothiocyanate-annexin V/propidium iodide binding. LPT were incubated with IgG1 as a control antibody and with infliximab at concentrations of 1 and 5 μg/ml. Results are mean (SD).

Figure 4

Effect of infliximab on lamina propria T cells (LPT) and peripheral blood T cells (PBT) obtained from 10 Crohn’s disease patients and 10 control subjects. Cells were cultured with infliximab added to the culture medium at different concentrations (1 and 5 μg/ml) or its isotype matched control (human IgG1). The percentage of apoptotic cells was assayed by flow cytometric analysis of fluorescein isothiocyanate-annexin V/propidium iodide binding. Results are mean (SD).

Figure 5

Involvement of caspase signalling pathway but not Fas-Fas ligand interaction in the transduction of infliximab induced apoptosis in Crohn’s disease lamina propria T cells (LPT). (A) LPT from intestinal lesions of 10 Crohn’s disease patients were treated with infliximab at a concentration of 5 μg/ml or IgG1 isotype control, after preincubation with ZB4 anti-Fas blocking antibody. Jurkat T cells cultured in RPMI 1640 medium and stimulated to undergo apoptosis by incubation with the mouse monoclonal antihuman Fas antibody (MoAb), an inducer of apoptosis in Fas expressing cells, were used as a positive control of ZB4 anti-Fas blocking action. (B) LPT isolated from intestinal lesions of 10 Crohn’s disease patients and treated with infliximab at a concentration of 5 μg/ml or IgG1 isotype control were preincubated with Z-Val-Ala-Asp(OMe)-monofluoroketone (Z-VAD-FMK) caspase inhibitor. Quantitation of apoptotic cells in cell cultures was performed by flow cytometry with fluorescein isothiocyanate-annexin V and propidium iodide staining. Results are mean (SD). ***p<0.0001 versus Jurkat T cells cultured in the absence of ZB4 anti-Fas blocking antibody; †††p<0.0001 versus cells cultured in the absence of Z-VAD-FMK.

Figure 6

Involvement of caspase-3 in infliximab induced apoptosis in Crohn’s disease lamina propria T cells (LPT). LPT from intestinal lesions of Crohn’s disease patients were treated with infliximab at different concentrations (1 and 5 μg/ml) or IgG1. Jurkat T cells, cultured for seven hours in the absence or presence of 2.5 μg/ml actinomycin D (act D), were used as controls. Caspase-3 activity was determined by immunoblot analysis. The blot shows two bands of 32 and 12 kDa, corresponding to the proform and active form of caspase-3, respectively. Data are representative of experiments performed in all patients with Crohn’s disease.