Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A₂: activation of endogenous phospholipases contributes to the pronociceptive effect

 

Renata Gonçalves Dias^1 6, Sandra Coccuzzo Sampaio^2 3, Morena Brazil Sant’Anna¹, Fernando Queiroz Cunha⁴, José María Gutiérrez⁵, Bruno Lomonte⁵, Yara Cury¹, Gisele Picolo¹

 

1 Special Laboratory of Pain and Signaling, Butantan Institute, Av. Vital Brazil, 1500, São Paulo, SP CEP 05503-900, Brazil.

2 Laboratory of Pathophysiology, Butantan Institute, São Paulo, SP, Brazil.

3 Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.

4 Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

5 Clodomiro Picado Institute, Faculty of Microbiology, University of Costa Rica, San José, Costa Rica.

6 Healthy Sciences Institute, Paulista University (UNIP), São Paulo, SP, Brazil.

 

ABSTRACT

Background

Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA₂ (Bothrops asper myotoxin II; MT-II) to induce articular inflammation.

 

Methods

The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors.

 

Results

Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF₃and PACOCF₃, inhibitors of cytosolic and Ca²⁺-independent PLA₂s, respectively, with bradykinin B₂ receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat).

 

Conclusion

These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

 

Key words: Lys49-PLA2; Myotoxin II; Arthritis; Bothrops asper; Phospholipase

 

Funding

This work was supported by the Brazilian agencies State of São Paulo Research Foundation (FAPESP – grant number 2006/03879–0) and Coordination for the Improvement of Higher Education Personnel (CAPES).

 

Received: August 19, 2016.

Revised: February 24, 2017.

Accepted: March 23, 2017.

 

Correspondence: gisele.picolo@butantan.gov.br

 

Authors’ contributions

RGD performed the behavioral experiments under the supervision of GP and YC and the in vitro assays under the supervision of SCS. They designed the assays and analyzed the results. FQC supervised the articular hypernociception evaluation. JMG and BL purified MT-II. MBS prepared the figures, revised the statistical analysis, helped with the manuscript preparation and text formatting. YC idealized the project. GP is the corresponding author and prepared the manuscript for publication. All authors read and approved the final manuscript.

 

Competing interests

The authors declare that they have no competing interests.

 

Consent for publication

Not applicable.

 

Ethics approval

All procedures were in accordance with the guidelines for the ethical use of conscious animals in pain research published by the International Association for the Study of Pain [93] and were approved by the Institutional Animal Care Committee of the Butantan Institute (Protocol number 240/05).