The bisphosphonate acute phase response: rapid and copious production of proinflammatory cytokines by peripheral blood gd T cells in response to aminobisphosphonates is inhibited by statins

Abstract

The bisphosphonates are a novel class of drug that have been registered for various clinical applications worldwide. Bisphosphonates, and in particular the aminobisphosphonates (nBPs), are known to have a number of side-effects including a rise in body temperature and accompanying flu-like symptoms that resemble a typical acute phase response. The mechanism for this response has been partially elucidated and appears to be associated with the release of tumour necrosis factor (TNF)alpha and interleukin (IL)6, although the effector cells that release these cytokines and the mechanism of action remain enigmatic. Here, we show that the nBP-induced acute phase response differs from the typical acute phase response in that CD14+ cells such as monocytes and macrophages are not the primary cytokine producing cells. We show that by inhibiting the mevalonate pathway, nBPs induce rapid and copious production of TNFalpha and IL6 by peripheral blood gammadelta T cells. Prior treatment with statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, blocks nBP-induced production of these proinflammatory cytokines by gammadelta T cells and may offer a means of avoiding the associated acute phase response. In addition, our findings provide a further mechanism for the anti-inflammatory effects attributed to inhibitors of HMG CoA reductase.

Fig. 1

nBPs induce rapid and copious production of TNFα by peripheral blood γδ T cells. (a) nBPs, but not nonaminoBPs, activate the Vγ9JPVδ2 T cell clone Bob in IFNγ ELISpot. The TCR of this clone has been sequenced and is published elsewhere [25]. ELISpots were performed with 1000 Vγ9JPVδ2 T cells and 25 000 spinner HeLa cells as antigen presenting cells per well and incubated for 6 h prior to development. Standard deviation from the mean of two replicate assays is shown, although in all cases these errors are smaller than the plot symbol. (b) nBPs stimulate TNFα production from direct ex vivo human PBMC. 10⁶ fresh human PBMC were incubated in 75 × 5mm FACS tubes at 37°C and 5% CO₂ in 1 ml of R10 (RPMI, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin), l ml of R10 + 10 µm risedronate or 1 ml of R10 + 100 µm pamidronate. 60 µl aliquots were removed at the specified times and added to TNFα ELISA assays. Error bars show standard deviation from the mean. (c) Activation of Vγ9JPVδ2 T cell clones by risedronate is extremely rapid. Standard deviation from the mean of two replicate TNFα ELISA assays is shown for three separate clones expressing a Vγ9JPVδ2 TCR. Clone P expresses CD8α; clones M and Bob are CD8^–. Clone P appears to make more TNFα and to produce it earlier. 10⁶ T cells were activated by 10 µm risedronate in 1 ml R10 in 75 × 5mm FACS tubes at 37°C and 5% CO₂. 60 µl aliquots were removed at the specified times and added to TNFα ELISA assays. Background TNFα production after a similar time period in the absence of risedronate was negligible (data not shown).

Fig. 2

The TNFα and IL6 produced by PBMC in response to nBPs is derived from γδ T cells. Magnetic depletion of γδ T cells from human PBMC removes their ability to manufacture TNFα in response to 100 µm pamidronate (a) and 10 µm risedronate (b). 5 × 10⁶ PBMC from a healthy donor ± magnetic depletion of γδ T cells were suspended in 1 ml of R10 ± antigen in 75 × 5mm FACS tubes at 37°C and 5% CO₂ for the times shown. 60 µl of cell supernatant was removed, applied to TNFα ELISA plates in duplicate and developed according to the manufacturers’ instructions. Standard deviation from the mean of the two replicate ELISAs is shown although in most cases this error is smaller than the plot symbol. (c) Exposure to nBPs activates only lymphocytes that express a Vγ9 receptor. 10⁶ fresh human PBMC were exposed to R10 (top panel) or R10 + 10 µm risedronate (bottom panel) for 6 h in an intracellular cytokine staining (ICS) assay. Plots show all the cells in the lymphocyte gate stained for PE-Vγ9 and APC-cytokines (TNFα, IL2, and IFNγ) as described previously [25]. Exposure to risedronate induces cytokine production only in lymphocytes that express a Vγ9 TCR. The percentage of total lymphocytes in the Vγ9⁺cytokine⁺ gate shown is indicated in the upper right of each panel. Almost 10% of the lymphocytes expressing a Vγ9 receptor are activated by exposure to nBPs. Similar results were observed with 100 µm pamidronate (data not shown). It is noticeable that exposure to risedronate lowers the expression of the Vγ9 TCR. (d) Intracellular cytokine staining (ICS) shows that nBPs induce direct ex vivo Vγ9-expressing T cells to make TNFα and IL6. Plots are gated to show only Vγ9-expressing lymphocytes. The left hand panels show IL6 production induced by 100 µm pamidronate and the right hand panels show TNFα production induced by 10 µm risedronate. The percentage of cytokine positive cells (fluorescence intensity > 20) is shown in the upper right corner of each panel.

Fig. 3

Incubation with nBPs does not activate cells of the monocytic lineage. Six hour exposure to 10 µm risedronate does not activate CD14⁺ cells. 10⁶ fresh human PBMC were exposed to R10 (a,c) or R10 + 10 µm risedronate (b,d) in an ICS assay. (a,b) show all live cells as determined by dead cell exclusion with 7-amino-actinomycin D (7-AAD; BD BioSciences). CD14⁺ cells such as monocytes and macrophages do not produce TNFα when exposed to nBP for this duration. The cells that produce TNFα in this assay do not express CD14 (compare bottom right quadrants in (a) and (b)). The cells that do respond in this assay express a Vδ2 TCR (c,d). The number of cells in the upper right quadrant of each plot as a percentage of total live cells is indicated in the upper right corner. There were approximately 30% fewer CD14⁺ cells after 6 h treatment with nBP compared to treatment with R10 alone as a fraction of the CD14⁺ cells that became 7-AAD⁺.

Fig. 4

The mevalonate biosynthetic pathway. nBPs inhibit farnesyl pyrophosphate (FPP) synthase and lead to a build up of IPP [23]. Statins inhibit HMG CoA reductase, a proximal enzyme in this pathway.

Fig. 5

Statins inhibit nBP-induced TNFα production by human PBMC. (a) Pretreatment of human PBMC with 1 µm pravastatin, 100 n m simvastatin or 100 n m fluvastatin for 2 h inhibits their ability to manufacture TNFα in response to 10 µm risedronate. Addition of IPP restores TNFα production and controls for any toxicity effects of these statins. 5 × 10⁵ statin-treated or untreated cells/well in 200 µl R10 were incubated with nBP for 12 h at 37°C and 5% CO₂ in 96 well U-bottomed tissue culture plates ± 1 µm IPP. 60 µl of supernatant was assayed for TNFα content by ELISA (Peprotech). Assays were performed in duplicate. Bars show standard deviation from the mean of two replicate assays. (b) ICS shows that pretreatment of PBMC with statins inhibits nBP-induced activation of Vγ9-expressing T cells. FACS plots show results with 1 µm pravastatin. Panels show results of incubation for 6 h in R10 only on the top row, R10 + 10 µm risedronate in row 2, R10 + 10 µm IPP in row 3, R10 + 10 µm risedronate + pravastatin in row 4 and R10 + 10 µm risedronate + pravastatin + 10 µm IPP on the bottom row. Pravastatin inhibits nBP-induced activation of Vγ9-expressing T cells (compare rows 2 & 4). Addition of IPP with pravastatin (bottom row) rescues activation and controls for any toxic effect of the statin. The Vγ9-expressing cell population (less than 3% of total lymphocytes) is shown in black. Vγ9 cells expressing cytokines appear in the upper right quadrant. The percentage of Vγ9 cells expressing cytokine is shown in the upper right corner of each plot. (c) Experiments performed as for (b) but with with 100 n m simvastatin or 100 n m fluvastatin instead of pravastatin. Experiments with pravastatin, simvastatin and fluvastatin were performed with different PBMC and show that between 2 and 8% of Vγ9-expressing T cells make cytokines when exposed to 10 µm risedronate. Bar charts show the percentage of Vγ9⁺cytokine⁺ cells from flow cytometry plots for PBMC exposed to R10, R10 + 10 µm risedronate, R10 + 10 µm IPP, R10 + 10 µm risedronate + statin and R10 + 10 µm risedronate + statin + 10 µm IPP. Simvastatin and fluvastatin inhibit nBP-induced activation of Vγ9-expressing T cells. Addition of IPP with the statin rescues activation and controls for any toxic effect of the statins. Flow cytometric analysis allowed confirmation that cells remained alive during the experiment and showed that non-Vγ9-expressing cells such as monocytes and macrophages did not make cytokines in response to nBP during the experiment (data not shown).