Cutting edge: nitrogen bisphosphonate-induced inflammation is dependent upon mast cells and IL-1

Abstract

Nitrogen-containing bisphosphonates (NBPs) are taken by millions for bone disorders but may cause serious inflammatory reactions. In this study, we used a murine peritonitis model to characterize the inflammatory mechanisms of these agents. At dosages comparable to those used in humans, injection of NBPs into the peritoneum caused recruitment of neutrophils, followed by an influx of monocytes. These cellular changes corresponded to an initial increase in IL-1α, which preceded a rise in multiple other proinflammatory cytokines. IL-1R, IL-1α, and IL-1β were required for neutrophil recruitment, whereas other MyD88-dependent signaling pathways were needed for the monocyte influx. Mice deficient in mast cells, but not mice lacking lymphocytes, were resistant to NBP-induced inflammation, and reconstitution of these mice with mast cells restored sensitivity to NBPs. These results document the critical role of mast cells and IL-1 in NBP-mediated inflammatory reactions.

Figure 1. Characterization of the murine peritonitis model induced by nitrogen-bisphosphonates

A)C57BL/6 mice (n= 5–6) werei.p. injected with different doses of NBPs pamidronate (PMD), zoledronate (ZLD), and alendronate (ALD) as well as the non-nitrogen-bisphosphonate clodronate (CLD). Infiltrating cells were recovered 3 days after administration. B)Mice (n=5) were injected i.p. with 0.8mg/mL ALD and peritoneal infiltrating cells were recovered at different time points. C) The granulocytes populations were further differentiated into neutrophils, eosinophils, and mast cells based on morphology. Data are represented as means±SEMof pooled two independent experiments.

Figure 2. Alendronate causes a rapid increase and IL-1 α and KC

Peritoneal lavage fluid and serum were collected at indicated time points from mice (n=5) i.p. administered 0.8 mg ALD (black bars) or vehicle (white bars). The levels of chemokines and cytokines in lavage (A) and serum (B) were analyzed by Luminex bead assay. Data are represented as means+SEM. * p<0.05, and ** = p<0.01 compared to 0h or vehicle controls (at the time point of significance with ALD) by one-way ANOVA with Dunnett's test.

Figure 3. Alendronate requires mast cells to induce peritonitis

A) WT C57BL/6 (n=6/group), Rag1^−/−(n=7/group), beige (Bg^J, n=5–6/group) or Pretty2 (n=3/group) mice were injected with 0.8 mg ALD (black bar) or vehicle (white bar). The peritoneal lavage was collected after 3 days. B) W/Wv mice, littermate WT mice, and W/Wv mice reconstituted BMMC received ALD (black) or vehicle (white). The peritoneal lavage was collected and total cell number was determined. C) ALD was injected in WT mice (n=10–12/group) treated with cromolyn (Cromo, n=7–8/group) or compound 48/80 (48/80, n=5/group). Peritoneal infiltrating cells were collected 3 days after ALD (black) or vehicle (white) injection.D)Degranulation of BMMC treated with 100μM each compound for 1.5h measured by β-hexosaminidase released in supernatant (Å₄₀₅). For all panels data is the pooled means+SEM from 2–3 independent experiments, *p<0.05 by one-way ANOVA with Dunnett's test.

Figure 4. Mast cells and IL-1 were involved in NPB-induced peritonitis

A) WT C57BL/6 (n=10/group), Casp1^−/−(n=8 /group), Nlrp3^−/− (n=8/group), P2x7^−/−(n=6/group) or Il1r^−/− (n=7/group) mice wereinjected with 0.8mg ALD. The number of infiltrating cells (left) and neutrophil number (right) in the peritoneal lavage were determined 3 days after ALD administration.B) Five-6 week old WT C57BL/6 (n=6/group), Il1α^−/− (n=9/group), and Il1β^−/− (n=6/group) mice werei.p. injected with 0.8mg ALD and WT C57BL/6 (n=4/group) mice were injected with PBS. The number of infiltrating cells (left) and neutrophil number (right) in the peritoneal lavage were determined 24h after ALD administration. Data are pooled means+SEM from two independent experiments. C) W/Wvmice were reconstituted with mast cells derived from WT, and Myd88^−/−, Il1r^−/−, Il1α^−/−, and Il1β^−/− mice (n=8–9/group). The infiltrating cells were collected 3 days after administration of ALD. A–C) Data are pooled means+SEM from 2 independent experiments *p<0.05 by one-way ANOVA with Dunnett's test compared to WT ALD treated mice. D) IL-6 secretion in co-cultures of BMMC and peritoneal mesothelial cells (from WT or Il1r^−/− mice). BMMC derived from WT mice were cultured with mesothelial cells from WT or Il1r^−/− mice for 6 h in the presence and absence of ZLD (10 μM). IL-6 in the supernatant was quantified by ELISA. Data are mean+SEM of a representative experiment (n =3) performed in triplicate. *p<0.05 by one-way ANOVA with Bonferroni's post-hoc test. E) IL-1α and IL-6 release from BMMC 16h after treatment with ZLD or LPS. Data are mean+SEM of a representative experiment (n =2) performed in triplicate. *p<0.05 compared to vehicle by one-way ANOVA with Bonferroni's test.