Joint tissues amplify inflammation and alter their invasive behavior via leukotriene B4 in experimental inflammatory arthritis

Abstract

Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B(4) (LTB(4)) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB(4) synthesis and to respond to LTB(4) within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase(-/-) and leukotriene A(4) (LTA(4)) hydrolase(-/-) mice, we demonstrate that FLSs generate sufficient levels of LTB(4) production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs-which comprise the predominant lineage populating the synovial lining-are competent to metabolize exogenous LTA(4) into LTB(4) ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB(4) 3-fold without inducing the expression of LTA(4) hydrolase protein. Moreover, LTB(4) (acting via LTB(4) receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB(4) in joints, placing LTB(4) regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.

FIGURE 1

Transcellular biosynthesis of LTB₄ in K/BxN serum-induced arthritis. A, Clinical index of arthritis severity was monitored daily after arthritogenic K/BxN serum administration to irradiated mice reconstituted with BM from various genotypes. Symbols refer to the donors and to the recipients, respectively (donor genotype → iRAD recipient genotype). Values are the mean ± SEM of data pooled from three independent experiments. (n = 15 mice per group). Differences were significant at p < 0.001 for WT → iRAD WT, WT → iRAD LTA₄H^–/–, and LTA₄H^–/– → iRAD 5-LO^–/– versus 5-LO^–/– → iRAD WT ; p = NS for WT → iRAD WT and WT → iRAD LTA₄H^–/– versus LTA₄H^–/– → iRAD 5-LO^–/–. B, Quantification of LTB₄ production in arthritic joint tissues. Ankle tissues from recipient mice reconstituted with BM from specified genotypes were homogenized, and lipids were extracted at 14 d after K/BxN serum administration. Lipid extracts then were fractionated via RP-HPLC, and fractions corresponding to the retention time for LTB₄ were analyzed by ELISA as described in Materials and Methods. Values depicted are picograms per gram of joint tissue (mean ± SEM) of data pooled from three independent experiments. C, Histomorphometric arthritis quantification. Shown are quantitative measurements (mean ± SEM) of inflammation, bone erosion, and cartilage erosion in ankles from each irradiated recipient mice at 14 d after serum administration. Values are the mean ± SEM of data pooled from three independent experiments (n = 15 mice per group). D, Neutrophil enumeration in arthritic synovial tissue. Tissues from experimental mice in Awere stained with anti-Ly6G mAb NIMP-R14, and neutrophil density was quantified histomorphometrically. The data are the mean ± SEM of three independent experiments. The p values in B, C, and D, compared with those detected in 5-LO^–/– → iRAD WT, are indicated on the top of each bar. E, Representative histological sections of ankle tissues from mice examined in A. Scale bar, 100 μm. F, Immunohistochemical staining for NIMP R14⁺ neutrophils quantified in D. Scale bar, 25 μm.

FIGURE 2

Metabolism of exogenous LTA₄ in FLSs. A, The expression of LTA₄H in cellular lysates from WT, 5-LO^–/–, and LTA₄H^–/– FLSs was analyzed by Western blot. The expression of heat shock protein 90 was used as a control. Results shown are representative of three separate experiments. B, LTB₄ formation by FLSs is dependent on the concentration of exogeneous LTA₄. WT FLSs (8 × 10⁴ cells per milliliter) were cultured with LTA₄ at a concentration of 0.1 or 1 μM for 20 min at 37°C. The formation of LTB₄ from exogenous LTA₄ was determined by ELISA as described in Materials and Methods. The results represent the mean ± SEM of three experiments. C, FLSs from either WTor LTA₄H^–/– mice were incubated with 1 μM LTA₄ for 20 min at 37°C. LTB₄ production in the culture supernatants was measured by ELISA. Results represent the mean ± SEM of three experiments. The p values in B and C are indicated on the top of each bar. The assay limit of detection is denoted by the horizontal dotted line.

FIGURE 3

Transcellular biosynthesis of LTB₄ in FLS/neutrophil co-cultures. A, LTB₄ formation after stimulation of WT FLSs (8 × 10⁴ cells per milliliter) and WT neutrophils (8 × 10³ cells per milliliter) either alone or in coculture after stimulation with 1 μM A23187 for 30 min. B, LTB₄ formation after coculture of 5-LO^–/– neutrophils and WT FLSs in the presence of 1 μM A23187 for 30 min. C, LTB₄ formation after coculture of LTA₄H^–/– neutrophils and WT FLSs in the presence of 1 μM A23187 for 30 min. LTB₄ was analyzed as described in Materials and Methods. Values represent the mean ± SEM of data pooled from three independent experiments. The p values are indicated on the top of the each bar. The assay limit of detection is denoted by the horizontal dotted line.

FIGURE 4

Metabolism of exogenous LTA₄ in FLSs pretreated with TNF. WT FLSs (8 × 10⁴ cells per milliliter) were pretreated with or without TNF (10 ng/ml) for 2 h followed by incubation with exogenous LTA₄ (1 μM). LTB₄ elaborated into the culture supernatants was quantified by ELISA as described in Materials and Methods. Values are the mean ± SEM of pooled data from three independent experiments. The p values are indicated on the top of each bar. The assay limit of detection is denoted by the horizontal dotted line.

FIGURE 5

BLT1 expression on radioresistant cells contributes to arthritis severity. A, Clinical arthritis severity in radiation chimeric mice: WT → iRAD WT, WT → iRAD BLT1^–/–, and BLT1^–/– → iRAD BLT1^–/–. Shown are the mean ± SEM from one of four independent experiments (n = 5 mice per group per experiment). The p value was determined by two-way ANOVA. B, Histomorphometric quantification of inflammation, bone erosion, or cartilage erosion on day 14 after K/BxN serum transfer in recipient mice. Values are the mean ± SEM of pooled data from three experiments. The p value was determined by the Student unpaired two-tailed t test.

FIGURE 6

LTB₄ promotes synovial fibroblast migratory and invasive behavior. A, Lamellipodium formation in WT FLSs stimulated by LTB₄. WT FLSs were plated on coverslips and stimulated and stained as labeled. Nuclei are visualized with a Hoechst dye (blue). Arrows indicate lamellopodia in FLSs stimulated by LTB₄. Data are representative of three independent experiments. B, Percentage of FLSs demonstrating lamellipodium formation. The results represent the mean ± SEM of data pooled from three experiments. The p value was determined by the Student unpaired two-tailed t test. C, LTB₄-induced invasion of FLSs. Primary FLSs placed in Matrigel-coated transwell dishes were stimulated as labeled and cells migrating to the lower membrane surface were enumerated. Results shown are the mean ± SEM of data pooled from four independent experiments. D, LTB₄-induced invasion in FLSs requires BLT1 expression. WT or BLT1^–/– FLSs were placed in Matrigel-coated transwells and stimulated and enumerated as in C. E, BLT1 antagonist CP-105,696 inhibits LTB₄-driven FLS invasion. WT FLSs were pretreated with CP-105,696 for 30 min prior to measuring LTB₄- or PDGF-driven invasion as in C. Data shown in D and E are the mean ± SEM of data pooled from three experiments. The p value was determined by the Student unpaired two-tailed t test.