ABSTRACT
Title
Evidence for a pro-thrombotic effect of interleukin-17A (IL-17A)
Authors
A. Parisi1, F. Maione1, F. D’Acquisto2, N. Mascolo1, C. Cicala1.
1Dept. of Experimental Pharmacology, University of Naples “Federico II”, Via D. Montesano 49, 81031, Napoli, Italy.
2William Harvey Res. Inst., Queen Mary Univ. of London, Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
1Dept. of Experimental Pharmacology, University of Naples “Federico II”, Via D. Montesano 49, 81031, Napoli, Italy.
2William Harvey Res. Inst., Queen Mary Univ. of London, Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
Abstract
IL-17A is a member of a new family of cytokines produced by Th cells recently identified as Th17, the third subpopulation of Th cells (D’Acquisto et al., 2010). IL-17A shows pro-inflammatory effect; indeed, by increasing local production cytokines and chemokines it enhances the local proinflammatory environment. Experimental a clinical evidence show that IL-17A is involved in several chronic inflammatory diseases, such as rheumatoid arthritis, BPCO, asthma, Crohn’s disease, psoriatic disease (Hoshino et al., 2000; Ivanov and Linden, 2009). There is much evidence that thrombosis and atherosclerosis might be closely associated to systemic inflammatory disorders. One common feature of patients suffering from chronic autoimmune inflammatory diseases is an increased cardiovascular risk complications. Following vascular inflammation, high levels of local and systemic of IL-17A have been found, both in humans and experimental animals and it is well known that vascular inflammation is a pathogenic mechanism for atherosclerosis; hence, IL-17A has been suggested to be a possible link between inflammation and the increased cardiovascular risk (Hansson et al., 2006; von Vietinghoff and Ley, 2010).
In the present study, we evaluated the effect of IL-17A on a model of FeCl3 – induced arterial thrombosis in rats. All experiments were performed on male Wistar rats (Harlan Nossan, 250 – 300 g). Rats were anaesthetized with urethane (10 % w/v; 10 ml/kg ip.) and placed on a surgical table; an arterial thrombus was induced by FeCl3 application onto the surface of the right carotid artery, as described by Kurz et al (1990). In brief, following surgery a piece of filter paper (Whatman n°1, 3 x 5 mm) soaked in FeCl3 (5%), or in IL-17A (100 µg/ml), was applied onto the external surface of the right carotid artery, for 30 minutes, afterward the paper was removed and the vessel left in situ for 60 minutes, to enable thrombus formation. In another set of experiments, an IL-17A (100 µg/ml), or vehicle (saline), soaked paper was applied on the vessel for 30 minutes before applying FeCl3 (5%). At the end of 60 minute period, a piece of about 1 cm in length of the right carotid artery, and of its controlateral, was removed and weighed. Thrombus size was evaluated by the difference in weight between the treated vessel and its controlateral. In another group of animals, the experiment was performed as described above and, at the end, vessels were removed, fixed in formalin (4% v/v) and morphologically analyzed by H&E staining.
In order to investigate on the proinflammatory cytokines and chemokines involved in IL17A effect, we performed the experiments with IL17A alone or with the only vehicle and at the end vessels were rapidly frozen in liquid nitrogen to be successively homogenized and analyzed for the expression of a panel of cytokines by a Proteome Profiler Cytokine Array (R&D Systems).
Application of FeCl3 (5%) did not cause an occlusive intravascular thrombus (weight 0.9 ± 0.3 mg); however, when IL-17A was applied on the vessel before FeCl3, we observed an intravascular occlusive thrombus (weight 2.13 ± 0.5 mg), also confirmed by histological analysis. Rat cytokine array panel showed the expression of MCP-1 (Monocyte Chemoattractant Protein-1) into the vessels treated with IL-17A that was absent in the control vessels.
Our results demonstrate that IL-17A facilitates arterial thrombus formation likely with the involvement of MCP-1 and further suggest the IL-17A as an important molecule at the interface between inflammation and thrombosis.
D'Acquisto et al. (2010). Biochem Pharmacol. 79, 525-534.
Hansson et al., (2006). Annu Rev Pathol Mech Dis 1, 297–329
Hoshino et al., (2000). J Allergy Clin Immunol. 105, 143-149.
Ivanov and Linden (2009). TiPS 30, 95-103.
Kurz et al., (1990). Thromb Res. 60, 269-280.
von Vietinghoff and Ley, (2010). Cytok Growth Factor Rev. 21, 463-469.
In the present study, we evaluated the effect of IL-17A on a model of FeCl3 – induced arterial thrombosis in rats. All experiments were performed on male Wistar rats (Harlan Nossan, 250 – 300 g). Rats were anaesthetized with urethane (10 % w/v; 10 ml/kg ip.) and placed on a surgical table; an arterial thrombus was induced by FeCl3 application onto the surface of the right carotid artery, as described by Kurz et al (1990). In brief, following surgery a piece of filter paper (Whatman n°1, 3 x 5 mm) soaked in FeCl3 (5%), or in IL-17A (100 µg/ml), was applied onto the external surface of the right carotid artery, for 30 minutes, afterward the paper was removed and the vessel left in situ for 60 minutes, to enable thrombus formation. In another set of experiments, an IL-17A (100 µg/ml), or vehicle (saline), soaked paper was applied on the vessel for 30 minutes before applying FeCl3 (5%). At the end of 60 minute period, a piece of about 1 cm in length of the right carotid artery, and of its controlateral, was removed and weighed. Thrombus size was evaluated by the difference in weight between the treated vessel and its controlateral. In another group of animals, the experiment was performed as described above and, at the end, vessels were removed, fixed in formalin (4% v/v) and morphologically analyzed by H&E staining.
In order to investigate on the proinflammatory cytokines and chemokines involved in IL17A effect, we performed the experiments with IL17A alone or with the only vehicle and at the end vessels were rapidly frozen in liquid nitrogen to be successively homogenized and analyzed for the expression of a panel of cytokines by a Proteome Profiler Cytokine Array (R&D Systems).
Application of FeCl3 (5%) did not cause an occlusive intravascular thrombus (weight 0.9 ± 0.3 mg); however, when IL-17A was applied on the vessel before FeCl3, we observed an intravascular occlusive thrombus (weight 2.13 ± 0.5 mg), also confirmed by histological analysis. Rat cytokine array panel showed the expression of MCP-1 (Monocyte Chemoattractant Protein-1) into the vessels treated with IL-17A that was absent in the control vessels.
Our results demonstrate that IL-17A facilitates arterial thrombus formation likely with the involvement of MCP-1 and further suggest the IL-17A as an important molecule at the interface between inflammation and thrombosis.
D'Acquisto et al. (2010). Biochem Pharmacol. 79, 525-534.
Hansson et al., (2006). Annu Rev Pathol Mech Dis 1, 297–329
Hoshino et al., (2000). J Allergy Clin Immunol. 105, 143-149.
Ivanov and Linden (2009). TiPS 30, 95-103.
Kurz et al., (1990). Thromb Res. 60, 269-280.
von Vietinghoff and Ley, (2010). Cytok Growth Factor Rev. 21, 463-469.