Background/Purpose

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by the joint destruction. Chemokines play important roles as monocyte and neutrophil recruiters in RA. Bombina variegate peptide 8 (Bv8), one of such chemokines is highly expressed in various tissues including the brain, testis, and bone marrow. Bv8 has a diversity of functions, being involved in angiogenesis, neurogenesis, circadian rhythm, and pain threshold. We have previously reported that Bv8 expression level was elevated in the synovial tissue of collagen induced arthritis (CIA) mice.  However, it is still unknown whether Bv8 can induce arthritis. Therefore, in this study, we investigated the expressions of Bv8 and its receptors (PKR1, PKR2) in CIA mice. And, we also examined whether Bv8 recruits polymorphonuclear neutrophils (PMNs) and monocytes in vitro and induces inflammatory arthritis in vivo. 

Methods

CIA was induced in 6-week-old DBA/1j male mice. PKR1 and PKR2 mRNA expression levels of joints in CIA mice were measured by real-time PCR on days 28 and 35 and compared to those in normal mice. Immunohistochemical (IHC) staining was performed to semi-quantitate PKR1 or PKR2 expression on day 28 in the synovial tissue. We performed monocyte and neutrophil chemotaxis assays in response to recombinant Bv8 (rBv8) using Boyden chambers. Resuls were expressed as the fold increase of the number of migrated cells compared to PBS. To test Bv8 for inflammatory activity in vivo, we injected PBS or rBv8 (10^-10M, 20μl) into knee joints. The knee circumference measurements were taken in a blinded manner before and 24 hours after the intraarticular injection for all mice. IHC stainings for PMNs (Gr-1/Ly6G) and monocytes (F4/80) were performed on paraffin sections from mouse knee joints to quantify Gr-1/Ly6G and F4/80 positive cells in the Bv8 group compared to the PBS group, respectively. 

Results

In the CIA group, PKR1 mRNA expression level was significantly higher on days 35 than the control group (p<0.05), and PKR2 mRNA expression level was significantly higher on days 28 and 35 than the control group (p<0.05). IHC stainings for PKR1 and PKR2 both showed significantly higher expressions of receptors in synovial tissue in the CIA group compared to the control group. PMN chemotaxis assays showed that rBv8 had significantly increased PMN chemotactic activity at 10^-12 M compared to PBS (p<0.05). Joints injected with rBv8 had significantly increased knee circumference than those injected with PBS (p<0.05). The number of Gr-1/Ly6G positive cells was significantly higher in mouse knee joints injected with rBv8 compared to PBS (p<0.05). There was no significant difference in the number of F4/80 positive cells in both groups. 

Conclusion

As well as Bv8, PKR1 and PKR2 expression levels were elevated in the synovial tissue of the CIA group. Bv8 recruited PMNs in vitro and induced neutrophil-driven inflammatory arthritis. These results indicate that Bv8 may have a previously unrecognized pathogenesis in RA by recruiting neutrophils. Targeting Bv8 may provide a new therapeutic strategy to treat inflammatory arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bombina-variegate-peptide8prokineticin-2-a-novel-arthritis-inducible-chemokine/