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. 2012 Jan 17;14(1):R9.
doi: 10.1186/ar3683.

A 4-trifluoromethyl analogue of celecoxib inhibits arthritis by suppressing innate immune cell activation

Asako Chiba  1 , Miho Mizuno, Chiharu Tomi, Ryohsuke Tajima, Iraide Alloza, Alessandra di Penta, Takashi Yamamura, Koen Vandenbroeck, Sachiko Miyake
Affiliations

A 4-trifluoromethyl analogue of celecoxib inhibits arthritis by suppressing innate immune cell activation

Asako Chiba et al. Arthritis Res Ther. .

Abstract

Introduction: Celecoxib, a highly specific cyclooxygenase-2 (COX-2) inhibitor has been reported to have COX-2-independent immunomodulatory effects. However, celecoxib itself has only mild suppressive effects on arthritis. Recently, we reported that a 4-trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2-inhibitory activity inhibits secretion of IL-12 family cytokines through a COX-2-independent mechanism that involves Ca2+-mediated intracellular retention of the IL-12 polypeptide chains. In this study, we explored the capacity of TFM-C as a new therapeutic agent for arthritis.

Methods: To induce collagen-induced arthritis (CIA), DBA1/J mice were immunized with bovine type II collagen (CII) in Freund's adjuvant. Collagen antibody-induced arthritis (CAIA) was induced in C57BL/6 mice by injecting anti-CII antibodies. Mice received 10 μg/g of TFM-C or celecoxib every other day. The effects of TFM-C on clinical and histopathological severities were assessed. The serum levels of CII-specific antibodies were measured by ELISA. The effects of TFM-C on mast cell activation, cytokine producing capacity by macrophages, and neutrophil recruitment were also evaluated.

Results: TFM-C inhibited the severity of CIA and CAIA more strongly than celecoxib. TFM-C treatments had little effect on CII-specific antibody levels in serum. TFM-C suppressed the activation of mast cells in arthritic joints. TFM-C also suppressed the production of inflammatory cytokines by macrophages and leukocyte influx in thioglycollate-induced peritonitis.

Conclusion: These results indicate that TFM-C may serve as an effective new disease-modifying drug for treatment of arthritis, such as rheumatoid arthritis.

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Figures

Figure 1
Figure 1
Effect of TFM-C on cytokine production from activated U937 macrophages. A. Lay-out of cytokine-specific antibody spots in the 16-plex cytokine Stripwell array (upper image) and visualization of cytokine-specific chemiluminescence in culture medium of LPS/PMA-treated U937 cells in the absence or presence of TFM-C (lower images). The grey-shaded cytokines in the upper images are those showing the highest production in LPS/PMA-treated U937 cells at 24 h. B. Effect of TFM-C treatment (50 μM) on the viability of macrophages (PMA-stimulated U937 cells). Apoptotic cells were measured by DAPI staining, and the percentage of damaged DNA and condensed chromatin was calculated following 6, 12 and 24 h of TFM-C treatment (upper graph). Metabolic activity of cells, measured by AlarmBlue®, was expressed as growth inhibition percentage of untreated controls for 6, 12 and 24 h of TFM-C treatment (lower graph). Bars show average of three independent experiments with corresponding error bars. C. Quantification of the kinetics of cytokine secretion and mRNA production (IL-1β, IL-6, IL-8, IL-10 IL-12 and TNF-α) in differentiated macrophages treated with LPS/PMA in the absence (open squares) or presence (solid circles) of TFM-C. All values represent the averages of three independent experiments. For each cytokine, the upper graph represents amount of secreted cytokine quantified using Quansys 16-plex Stripwells, while the lower graph represents cytokine-specific mRNA quantified by QPCR. Asterisks indicate significant differences at * P < 0.05 between TFM-C-treated and -untreated cells at each time point using Student's t-test. D. Effect of 50 μM TFM-C on IL-23p19, HERP and GAPDH mRNAs (QPCR) in differentiated macrophages, stimulated by LPS and PMA. The levels of mRNA levels are shown as 2-Ct. Asterisks indicate significant differences at * P < 0.05 compared with baseline condition LPS/PMA-only using Student's t-test.
Figure 2
Figure 2
The effect of TFM-C on CIA. A. Clinical scores of CIA in DBA1/J mice treated with 10 μg/g TFM-C (closed circles), celecoxib (open triangles) or vehicle (open squares) every other day from 21 days after immunization. The data shown are pooled from two similar experiments. Error bars represent + SEM of 10 to 12 mice per group. * P < 0.05 compared with control group. * P < 0.05 compared with both control and celecoxib-treated groups. B. Quantification of histological assessment of joints 37 days after induction of CIA. Result shown is the mean + SEM of five mice per group. * P < 0.05, TFM-C-treated versus vehicle-treated group. * P < 0.05, celecoxib-treated versus TFM-C-treated group. C. Representative histological feature of joints in vehicle-treated (left), TFM-C-treated (right) and celecoxib-treated (middle) mice. (H&E stained; original magnification × 40). D. The effect of TFM-C on CII-specific response. CII-specific antibody responses in vehicle- (open bars), TFM-C- (filled bars) and celecoxib-treated (gray bars) group. Individual serum samples were obtained at Day 37 after the induction of arthritis and were analyzed as indicated in Materials and Methods. Data represent the mean + SEM of five mice per group.
Figure 3
Figure 3
The effect of TFM-C on CAIA. A. Clinical scores of CAIA in B6 mice treated with 10 μg/g TFM-C (closed circles), celecoxib (open triangles) or vehicle (open squares) every other day from two days before CAIA induction. * P < 0.05 compared with control group, * P < 0.05 compared with both control and celecoxib-treated groups. Results shown are the mean + SEM of five mice per group. The data shown are from a single experiment representative of two similar experiments. B. Quantification of histological assessment of joints 12 days after induction of AIA shown in A. Results shown are the mean + SEM of five mice per group. * P < 0.05 control versus TFM-C group, * P < 0.05 celecoxib versus TFM-C -treated group. C. Representative histological feature of joints in vehicle-treated (left), TFM-C-treated (middle) and celecoxib-treated (right) mice. (H&E stained; original magnification × 40).
Figure 4
Figure 4
TFM-C inhibits the mast cell activation in CAIA. CAIA was induced in B6 mice and the mice were then treated with 10 μg/g TFM-C, celecoxib or vehicle as described in Figure 2. A. Quantification of degranulated mast cells in synovium of joints 12 days after induction of CAIA. * P < 0.05, compared with vehicle-treated group. * P < 0.05, compared with celecoxib-treated group. Results shown are the mean + SEM of six mice per group and were pooled from two experiments. B. Hisopathologic features of degranulated or intact mast cells in joints of representative vehicle-, celecoxib- and TFM-C- treated mice (toluidine blue stained; original magnification, × 100). White arrows indicate intact mast cells and black arrows indicate degranulated mast cells.
Figure 5
Figure 5
TFM-C supresses the activation of macrophages. B6 mice recieved 10 μg/g TFM-C, celecoxib or vehicle on Day 0 and Day 2, and on Day 3, splenic macrophages were collected and were stimulated by LPS in vitro in the presence of TFM-C, celecoxib or vehicle. Cytokines were detected by ELISA. IL-1β and IL-6 were measured 24 h after stimulation. TNF-α was measured six hours after stimulation. The data shown are from a single experiment representative of three similar experiments. * P < 0.05 compared with control group, * P < 0.05 compared with celecoxib-treated group.
Figure 6
Figure 6
TFM-C supresses leukocyte influx in thioglycollate-induced peritonitis. B6 mice recieved 10 μg/g TFM-C, celecoxib or vehicle at two days and one hour before peritoneal injection of thioglycollate. At four hours after thioglycollate injection, peritoneal lavage fluid was collected and the infiltrating cells were counted. Cell numbers are shown from three separate experiments. * P < 0.05, TFM-C-treated versus vehicle-treated group. * P < 0.05, celecoxib-treated versus TFM-C-treated group.
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