Mechanism involved in the immunomodulatory effect of pamidronate: a stronger early immune response driven by T cells?

R Lopez-Posadas2, B Ocon1, C Mascaraque1, C Aranda3, MD Suarez3, A Zarzuelo1, O Martinez-Augustin3, F Sanchez de Medina1. 1University of Granada/CIBERehd, Pharmacology 18071, Spain, 2Universitätsklinikum Erlangen, Zentrum für Molekulare Entzündungs- und Tumorforschung, Germany, 3University of Granada/CIBERehd, Biochemistry and Molecular Biology II 18071, Spain

Introduction: We hypothesized that nitrogen-contaning bisphosphonates (NBP) may modulate intestinal inflammation. We have previously shown that pamidronate (unpublished data) has intestinal immunomodulatory activity in two preclinical models of inflammatory bowel disease (TNBS- and DSS-induced colitis). Here we used several in vitro models (cell lines and primary cultures) in order to identify the cellular target. Several NBPs, as well as specific inhibitors of prenylation, have been included for reference. We have focused on the main cell types participating in mucosal immune response, i.e. macrophages, T lymphocytes and intestinal epithelial cells.

Methods: NBPs were used at 3 different concentrations (1, 10 and 100 μM). HT-29 and Caco-2 cells were used as model intestinal epithelium. Confluent monolayers were exposed to short or long treatment with NBPs (24 hours or 5 days) together with 1 μg/ml of LPS (HT-29) or 10 ng/ml of IL-1β (Caco-2). Negative selection using magnetic bead based technology was performed for purification of macrophages and T lymphocytes from spleen cells. Coculture of T lymphocytes and intestinal epithelial cells was carried out in Transwells®. Results: pamidronate had no effect on intestinal epithelial cells, unlike other NBPs such as alendronate or ibandronate, which produce a prenylation-independent increase in IL-8 production in both HT-29 and Caco-2 cells after stimulation with LPS or IL-1β, respectively (Table 1). Activation of ERK and p38 MAPK could be involved in this effect. Ibandronate but not pamidronate increased IL-6, IL-10 and TNF-α production in purified macrophages stimulated with LPS (Table 2). Despite its poor effect on intestinal epithelial cells and macrophages, pamidronate exerts important effects on T cells. Pamidronate increased ConA-induced IFN-γ and IL-2 production by purified T lymphocytes (Table 3), without modifying cell proliferation. Pamidronate also activates T-lymphocytes when added to the apical compartment in the IEC18-T cell coculture system, increasing IFN-γ, IL-6 and IL-10 production, stimulating cell proliferation and activating the STAT4 signaling pathway.

Conclusion: T lymphocytes appear to be an important target of pamidronate in the intestinal mucosa. Increased IFN-γ production by T cells after pamidronate treatment may enhance innate immune related mechanisms leading to a decreased invasion of intestinal epithelial cells by luminal bacteria.

Table 1: IL-8 production by LPS or IL-1β stimulated intestinal epitelial cell lines, *P<0.005 and ** P<0.001 vs. Control group (stimulus without treatment).

Table 2: Cytokine production by purified macrophages after stimulation with LPS (1 μg/ml) and treatment with BP (100 μM). *P<0.005 and ** P<0.001 vs. Control group (stimulus without treatment).

Table 3: Cytokine production by purified T lymphocytes T. *P<0.005 and ** P<0.001 vs. Control group (stimulus without treatment).