A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis

Abstract

Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.

Figure 1. Expression of sPLA₂ in the SF of patients with RA and healthy controls

1. The full set of human sPLA₂ isoforms were quantified by time-resolved immunofluorescence analysis in SF from patients with RA (n = 45).

2. Groups IIA and V sPLA₂ concentrations in SF obtained from healthy volunteers (n = 12).

Figure 2. Group V sPLA₂ protects from K/BxN serum-transfer arthritis

1. Arthritis response in group V sPLA₂ null and congenic control mice in a BALB/c group IIA null background. Mice were injected with 20 µl of K/BxN serum at days 0 and 2, and disease development was monitored for 13 days.

2. Histomorphometric quantification of arthritis severity in group V sPLA₂ null and congenic control mice at experimental day 13. N = 15 mice/group. Data are mean ± SEM pooled from three independent experiments. P < 0.001 for (A).

3. Representative mid-saggital ankle sections from group V sPLA₂ null and group V sPLA₂ control mice. Upper and lower panels are 25× and 200× magnification, respectively (lower panel). White arrows demarcate the hyperplastic synovial lining surrounding a large effusion (upper) while black and white arrowheads highlight bone and cartilage erosions, respectively. Note the increased leukocytic infiltration, synovial lining hyperplasia and pannus formation in group V sPLA₂ null mice (T, tibia; S, synovial space; C, calcaneus). Figures representative of findings in 15 mice/group.

Figure 3. Systemic administration of recombinant group V sPLA₂ ameliorates K/BxN serum-transfer arthritis

1. Recombinant mouse group V sPLA₂ (50 µg in PBS) or control PBS were injected intravenously into group V sPLA₂-null mice in a BALB/c group IIA null background 2 hours prior to administration of K/BxN serum on experimental Day 0 and daily thereafter for 5 days. Arthritis was induced by injection of 35 µl and 20 µl of K/BxN serum on day 0 and 2 respectively; the development of arthritis was monitored for 7 days. Arrows indicate sPLA₂-V intravenous injections.

2. Experiment as detailed in (A) using WT BALB/c mice

3. Histomorphometric quantification of inflammation in ankle sections from mice injected with recombinant group V sPLA₂ or PBS control at experimental day 7. Note that the BALB/c mice express both group V and group IIA sPLA₂ and therefore are not the congenic controls to group V sPLA₂^−/−. N = 12 mice/group. Data are mean ± SEM pooled from three independent experiments. P < 0.001 for (A and B).

4. Representative mid-saggital ankle sections from group V sPLA₂ null mice treated with recombinant group V sPLA₂ or its diluent (PBS). Note the decreased leukocytic infiltration, synovial lining hyperplasia and pannus formation in recombinant group V sPLA₂ treated mice. Magnification = 200×. Figures are representative of findings in 12 mice/group.

Figure 4. Group IIA sPLA₂ contributes to severity of K/BxN serum-transfer arthritis

Mice were injected with 65 and 35 µl K/BxN serum on days 0 and 2, respectively, and the development of arthritis was followed for 13 days.

1. Arthritic response in group IIA sPLA₂ null and WT congenic BALB/c control mice.

2. Histomorphometric quantification of arthritis severity in group IIA sPLA₂ null and congenic BALB/c control mice at experimental day 13. N = 15 mice/group. Data are mean ± SEM pooled from three independent experiments. P < 0.001 (A).

3. Human group IIA sPLA₂ transgenic and WT C57BL/6 mice were administered a single 75 µl dose of K/BxN serum on experimental day 0 and development of arthritis was monitored for 13 days.

4. Histomorphometric quantification of arthritis severity in human group IIA sPLA₂ transgenic and WT control mice at experimental day 13. N = 15 mice/group. Data are mean ± SEM pooled from three independent experiments. P < 0.001 for (C).

Figure 5. Group V sPLA₂ promotes clearance of immune complexes in vitro

1. A. Phagocytosis of immune complexes in vitro by peritoneal macrophages from group V sPLA₂-null , group V sPLA₂-control, and FcγR-null mice was quantified cytofluorometrically using FcOxyburst. Data are mean ± SEM pooled from three independent experiments.

2. B. Phagocytosis of immune complexes in vitro by CD14+ cells in RA synovial fluid with or without addition of recombinant group V sPLA₂ or its inactive mutant H48Q was quantified cytofluorometrically using FcOxyburst. Data are mean ± SEM pooled from six independent experiments.

3. C. Phagocytosis of immune complexes by human CD14+ cells from peripheral blood incubated with recombinant group V sPLA₂ in the presence of either the cyclooxygenase (COX) inhibitor indomethacin or the FLAP inhibitor MK886. Data are mean ± SEM pooled from three experiments performed in duplicate.

4. D. CysLTs released by leukocytes from RA SF treated with sPLA₂. Group V sPLA₂, its mutant H48Q or group IIA sPLA₂ were added to leukocytes isolated from RA SF and cysLTs released into the supernatant were quantified by ELISA. Data are mean ± SEM pooled from three experiments performed in duplicate.

5. E, F. CysLTs promote phagocytosis of immune complexes by CD14+ cells. Indicated concentrations of LTC₄ (E) or LTD₄ (F) were added to peripheral blood mononuclear cells (PBMC) prior to addition of the FcOxyburst probe and phagocytosis by CD14+ cells was monitored cytofluorometrically. Data are mean ± SEM pooled from three experiments.

6. G. Phagocytosis of immune complexes by sPLA₂-stimulated CD14+ cells from peripheral blood in the presence of cysLT1 antagonist monteleukast. Data are mean ± SEM pooled from three experiments performed in duplicate.

Figure 6. Group V sPLA₂ promotes clearance of immune complexes in vivo

1. Immunofluorescent staining of IgG (Red) and complement C3 (Green) in mid-saggital cryosections of ankle tissues from group V sPLA₂-control (A) ankle joints. Nuclei (blue) are visualized by staining with DAPI. Magnification= 400X. Cartilage tissue and synovial fluid (SF) space as labeled. Mice were injected with 35 µl K/BxN serum at day 0, and ankle tissues were harvested on day 4. Data are representative of 3 independent experiments.

2. Experiment as detailed in (A) using group V sPLA₂-null ankle joints.

3. ELISA quantification of circulating immune complexes in sera from group V sPLA₂ null and group V sPLA₂ control mice 4 days after administration of 35 µl of K/BxN serum. Pooled K/BxN serum and serum from non-arthritic WT mice were included as controls. N = 10 mice/group. Data are mean ± SEM pooled from two independent experiments. P = NS.

4. Quantification of cysLTs released by human PBMC treated with recombinant sPLA₂ in the presence (white filled) or absence (black filled) of RBC. Data are mean ± SEM pooled from three experiments.

5. Phagocytosis of immune complexes in the presence of RBC. PBMC incubated in the presence or absence of RBC were treated with group V sPLA₂ and phagocytosis of immune complexes by CD14+ cells was monitored cytofluorometrically. Data are mean ± SEM pooled from three experiments.