A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis

Abstract

Neutrophil recruitment into tissue plays an important role in host defense and disease pathogenesis, including the inflammatory arthritides. A multitude of diverse chemoattractants have been implicated in neutrophil recruitment, suggesting that they have overlapping functions in mediating this critical biological response. However, here we demonstrate a unique, non-redundant role for the leukotriene B4 receptor BLT1 in mediating neutrophil recruitment into the joint in the K/BxN mouse model of inflammatory arthritis. We demonstrate that neutrophil expression of BLT1 was absolutely required for arthritis generation and chemokine production in this model, and that specific BLT1 inhibition reversed established disease. Adoptive transfer of wild-type (WT) neutrophils restored arthritis and chemokine production in BLT1(-/-) mice. Surprisingly, the primary effect of the transferred WT neutrophils into BLT1(-/-) mice was to promote the entry of endogenous BLT1(-/-) neutrophils into the joints of these mice. However, continued joint inflammation was dependent on the presence of WT neutrophils, indicating an ongoing specific requirement for BLT1-activated neutrophils in mediating BLT1(-/-) neutrophil recruitment by other chemoattractants. These experiments demonstrate that neutrophil BLT1 functions in a novel and essential non-cell-autonomous manner to enable the recruitment of additional neutrophils not expressing this receptor, thereby amplifying the inflammatory response in autoantibody-induced arthritis.

Figure 1.

BLT1-deficient mice are resistant to K/BxN serum transfer arthritis. (a) Ankle thickness and (b) clinical score were determined in WT and BLT1^−/− mice after injection of K/BxN serum (n = 5 each group). Data are representative of three independent experiments. (c) Representative histopathology of ankle joints from WT and BLT1^−/− mice during early onset, early peak, and resolving disease activity. WT joints display synovial inflammation, cartilage, and bone erosions (filled arrow), and synovial hypertrophy (asterisk), whereas BLT1^−/− joints are free of inflammation and the synovium retains its relatively acellular composition (open arrows). Bar, 400 μm. (d) Histopathological scoring of ankles from WT and BLT1^−/− mice during early onset, early peak, and resolving disease activity (n = 6–12 in each group). All error bars represent SEM. *, P < 0.001 (WT vs. BLT1^−/−).

Figure 2.

The BLT1 inhibitor CP-105,696 prevents and treats arthritis. The specific BLT1 inhibitor CP-105,696 was administered daily to C57BL/6 mice before the induction of disease on day −1 (preventative, filled arrows) or as disease was approaching maximal activity on day 5 (delayed, open arrows), and ankle thickness (a) and clinical score (b) were assessed daily (n = 4 in each group). Data are representative of three independent experiments. In a separate experiment, mice received sham gavage of vehicle lacking CP-105,696 without any effect on arthritis production (not depicted). (c) Histopathological scoring of ankles from control untreated mice compared with mice treated with preventative or delayed CP-105,696 dosing strategies (n = 10–15 in each group). Error bars represent SEM. (d) Total number of synovial cells and neutrophils within the joints of control untreated mice and mice treated with preventative or delayed CP-105,696 dosing strategies. *, P ≤ 0.005; **, P < 0.0001 (control vs. CP-105,696 treated).

Figure 3.

BLT1 is required for synovial inflammatory chemokine and cytokine production in inflammatory arthritis. (a) K/BxN serum was administered to WT and BLT1^−/− mice, and ankles were harvested at 7 d. Frozen ankle sections were stained with FITC-labeled F(ab′)₂ fragment directed against the Fc portion of mouse IgG. A WT mouse that did not receive any serum was used as a control. Bar, 230 μm. (b–e) Chemokine expression was measured by qPCR of total RNA isolated from synovial tissue on postserum transfer days 3 (early onset disease) (b) or 7 (early active disease) (c). Chemokine receptor expression was quantified by qPCR of total RNA isolated from synovial fluid at days 3 (d) and 7 (e). Total RNA was extracted from eight individual ankles in each experimental group, and qPCR reactions were run separately on each sample. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0005 (WT vs. BLT1^−/−).

Figure 4.

WT BLT1-expressing neutrophils are required for the generation of arthritis by promoting entry of BLT1^−/− neutrophils into the joint. Bone marrow neutrophils from WT and BLT1^−/− mice were adoptively transferred to BLT1^−/− mice on days 0 and 2, and (a) ankle thickness and (b) clinical score were assessed daily. n = 3–4 in each group, and data are representative of three independent experiments. (c) Total numbers of Gr-1⁺ neutrophils within the ankle synovial fluid of WT mice, BLT1^−/− mice that received WT neutrophils (WT PMN→BLT1^−/−), BLT1^−/− mice, and BLT1^−/− mice that received BLT1^−/− neutrophils (BLT1^−/− PMN→BLT1^−/−) at 20 h and 4 d. (d) Representative flow cytometric analysis of Gr-1⁺ neutrophils in the blood and ankle synovial fluid from WT PMN→BLT1^−/− mice at 20 h and 4 d after initial serum and neutrophil transfer. For these experiments, transferred CD45.1⁺ WT neutrophils were used to distinguish from endogenous CD45.2⁺ BLT1^−/− neutrophils. For comparison, synovial fluid analysis from WT mice is depicted in Fig. S2. (e) Percentage of transferred Gr-1⁺ WT neutrophils out of total neutrophils in the blood and synovial fluid of WT PMN→BLT1^−/− mice at 20 h and 4 d. (f) Total numbers of transferred WT and endogenous BLT1^−/− neutrophils within the synovial fluid of WT PMN→BLT1^−/− mice at 20 h and 4 d. (g and h) Ankle synovial tissue was harvested, and total RNA was isolated from WT PMN→BLT1^−/− mice at 3 (g) and 12 (h) d after initial serum and adoptive neutrophil transfer. Chemokine expression was measured by qPCR. RNA from six to eight individual ankles was isolated. Error bars represent SEM. *, P < 0.05; **, P < 0.005 (WT vs. WT PMN→BLT1^−/− mice).