Hemokinin-1 is a key mediator of adjuvant-induced inflammatory responses in the mouse joint and lung

Zs Helyes1,7, Zs Hajna1, E Borbely1, K Sandor1, J Kun1, I Toth1, L Kereskai2, E Pinter1,6, J Szolcsanyi1,6, CJ Paige3, JP Quinn4, A Zimmer5, A Berger3. 1University of Pecs, Pharmacology and Pharmacotherapy, H-7624, Hungary, 2University of Pecs, Pathology, H-7624, Hungary, 3Ontario Cancer Institute, University Health Network, Toronto, M5G 1X5, Canada, 4Liverpool University, School of Biomedical Sciences, L69 3BX, UK, 5University of Bonn, Laboratory of Molecular Neuroscience, Department of Psychiatry, 0228 730, Germany, 6PharmInvivo Ltd, H-7629, Hungary, 7Szentagothai Research Center, University of Pecs, H-7624, Hungary

Background: Hemokinin-1, the recently discovered neuropeptide of the tachykinin family, encoded by the preprotachykinin C (TAC4) gene, is predominantly expressed by immune cells. It has the greatest affinity to the neurokinin 1 (NK1) receptor similarly to the preprotachykinin A (TAC1) gene-derived substance P of sensory neural origin. However, based on a variety of its distinct actions, the existence of a presently not identified own hemokinin receptor has been proposed. Since there were no data on the involvement and mechanism of action of hemokinin-1 in systemic inflammatory processes, we analysed the roles of this peptide and NK1 receptors in adjuvant-induced chronic arthritis and consequent mechanical hyperalgesia, as well as lung inflammation of the mouse. Although this is a widely used mechanism model related to rheumatoid arthritis with common lung manifestations, the pulmonary inflammation has not yet been examined by others.

Methods: Complete Freund’s Adjuvant (CFA; 50 μl, 10 mg/ml) was injected s.c. into the plantar surface of the right hindpaw and into the tail root of 8-10 week-old male TAC4 and NK1 receptor gene-deleted (TAC4-/-, NK1-/-) mice and their C57Bl/6 wildtype counterparts (23-26 g; n=8-9/group) under isoflurane anaesthesia. Paw volume was measured with plethysmometry and touch sensitivity with dynamic plantar aesthesiometry every other day during 21 days. Semiquantitative histopathological scoring of the tibiotarsal joints and the lungs was performed on the basis of characteristic inflammatory changes. The inflammatory cytokine interleukin 1-beta (IL-1β) in the joint homogenate was measured with ELISA, NK1 receptor expression in the lung was determined with RT-PCR.

Results: In the wildtype group adjuvant administration induced 30-40% mechanical hyperalgesia and 80-100% swelling on the injection side throughout the experimental period. Inflammatory hyperalgesia was significantly smaller from day 11 of the study in both TAC4 -/- and NK1-/- animals (15-25%; p<0.05 repeated measures two-way ANOVA), but oedema was not altered in either group. Synovial swelling, inflammatory cell infiltration and cartilage destruction (composite histopathological score of 0.8+0.3 vs. 5.3+0.3; p<0.05 Kruskall-Wallis), as well as joint IL-1β concentration (7.4+3.8 vs. 9.3+0.6 ng/g wet tissue; p<0.05 Kruskall-Wallis) were significantly reduced in TAC4-/- mice, but not in NK1 receptor-deleted ones. Peribronchial oedema, granulocyte, macrophage and lymphocyte accumulation also decreased in the lung of TAC4-/- mice, but increased in NK1-/- animals (scores of 1.6+0.3 and 6.2+0.1, respectively vs. 4.8+0.2; p<0.05 Kruskall-Wallis). NK1 receptor expression was downregulated in the lungs of CFA-treated wildtypes.

Conclusions: Hemokinin-1 increases inflammatory mechanical hyperalgesia in the later phase of chronic arthritis through NK1 receptors. It plays a predominant role in inflammatory histopathological alterations and cytokine production in the joints via a presently not identified target. There are severe pulmonary inflammatory changes, such as oedema formation and remarkable leukocyte accumulation in wildtype mice in this model. Hemokinin-1 is also an important mediator of adjuvant-induced lung inflammation, but not by NK1 receptor activation. Interestingly, NK1 receptors play a protective role in this immune-mediated pulmonary response.