Neutrophil-derived leukotriene B4 is required for inflammatory arthritis

Abstract

Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.

Figure 1.

Quantification of leukotrienes in arthritic joint tissues. LTB₄ (A) or LTC₄ (B) was quantified in ankle tissues from arthritic K/BxN mice (n = 5) or control B6 mice (n = 10). Values depicted are means ± SEM of three independent experiments (P < 0.001). Dotted line represents lower limit of assay sensitivity. (C) Kinetics of LTB₄ production in arthritic joint tissue. B6 mice were administered arthritogenic K/BxN serum and killed at 2-d increments thereafter for quantification of ankle tissue LTB₄ (bars). Clinical arthritis was measured throughout the experimental course (♦). Data represent means ± SEM of five mice per time point in three separate experiments.

Figure 2.

LTB₄-deficient mice are resistant to arthritis. Arthritis responses in 5-LO null (A), LTC₄S null (B), LTA₄H null (C), and control mice. n = 5 mice/group/experiment, representative of three experiments. 5-LO null and LTA₄H null versus WT, P < 0.001. (D–F) Histomorphometric arthritis quantification from 5-LO null (D), LTC₄S null (E), and LTA₄H null (F) mice. P < 0.001, 5-LO null and LTA₄H null. (G–J) Histologic sections from 5-LO null (G), LTC₄S null (H), LTA₄H null (I), and WT (J) mice at experimental day 14. Bar, 100 μm. S, synovium; C, cartilage; Bn, bone.

Figure 3.

5-LO inhibitor (L-739,010) ameliorates arthritis. (A and B) L-739,010 administered either as pretreatment (filled arrow) or as a therapeutic regimen (open arrow). n = 5 mice/group/experiment, representative of three experiments. P < 0.001 pretreatment versus mock-treated mice; P < 0.01 treatment versus vehicle. (C) Histomorphometric quantification of arthritis at experimental day 14. P < 0.01–0.001. (D) Quantification of neutrophil accumulation per 0.1-mm² synovial area. n = 5 mice/group/experiment, 20–25 fields/mouse, representative of three experiments. P < 0.01, pretreated and treated versus mock treatment.

Figure 4.

Neutrophils are a cellular source of synovial arthritogenic leukotrienes. (A) 5-LO activity derived from BM lineages. Lethally irradiated (iRAD) recipient mice were reconstituted with donor BM as labeled. n = 5 mice/group/experiment, representative of three experiments. P < 0.001. (B) Mast cell 5-LO activity is dispensable for arthritis induction. Mast cell–deficient W/W^v mice were engrafted as labeled and assessed for arthritic response. n = 10–12 mice/group, P < 0.001, WT and 5-LO null BM-derived mast cell versus W/W^v mice. (C) Neutrophils provide arthritogenic LTB₄. Purified neutrophils from WT mice were transferred daily for 5 d into 5-LO null recipient mice. n = 5 mice/group/experiment, representative of three experiments. P < 0.05. (D–H) WT donor neutrophils (CD45.1) in recipient 5-LO null joint tissues (CD45.2). Shown are: (D) hematoxylin/eosin stain, (E) isotype control, (F) CD45.1 (green), (G) Gr-1 (red), and (H) merge CD45.1 and Gr-1 with nuclear strain (blue). Bar, 25 μm.