Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1beta-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice.
BACKGROUND
Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.


METHODS
Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1beta stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)alpha, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.


RESULTS
PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1beta-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1beta-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.


CONCLUSIONS
Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.
Thwin
Maung-Maung
MM
Department of Anatomy, Yong Loo Lin School of Medicine, 4 Medical Drive, National University of Singapore, 117597 Singapore. antmmt@nus.edu.sg
Douni
Eleni
E
Arjunan
Pachiappan
P
Kollias
George
G
Kumar
Prem V
PV
Gopalakrishnakone
Ponnampalam
P
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000188:drug therapy* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000096:biosynthesis* / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic; D010455:Peptides / Q000494:pharmacology; D054497:Phospholipases A2, Secretory / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D054497:Phospholipases A2, Secretory; D020782:Matrix Metalloproteinases