Salbutamol inhibits trypsin-mediated production of CXCL8 by keratinocytes
Introduction
Inflammatory skin conditions such as psoriasis are characterised by enhanced proteolytic activity in the epidermis [1], infiltration of neutrophils and T-lymphocytes [2], an excessive production of pro-inflammatory chemokines [3] and hyperproliferation of keratinocytes [4]. It has become evident that, among the epidermal chemokines, CXCL8 or interleukin 8 (IL-8) contributes to the pathogenesis of a number of skin diseases [5], [6]. As a member of the CXC chemokine family, CXCL8 causes chemotaxis of neutrophils and T-lymphocytes [7], [8], degranulation of neutrophils and promotes epidermal proliferation and angiogenesis [9]. CXCL8 and its receptor CXCR2 are elevated in psoriatic lesions [10], [11] and reduction in CXCL8 levels is reported to correlate with therapeutic efficacy in patients treated with tacrolimus [12], [13].
CXCL8 is produced by diverse cell types [14], [15], [16]. Several lines of evidence have indicated that keratinocytes are a major source of CXCL8 in inflamed skin [10], [17], [18]. CXCL8 release from keratinocytes can be induced by various stimuli including UV light, IFN-γ, CD40L and phorbol esters [19], [20]. It has been reported that activation of protease-activated receptor-2 (PAR-2) can also induce CXCL8 secretion by keratinocytes [21]. This pathway of activation may be relevant to disease since high expression of PAR-2 has been detected in epidermal keratinocytes [21], [22], [23], and the severity of contact dermatitis is diminished in PAR-2−/− mice [24]. Furthermore, PAR-2 belongs to a subfamily of G-protein-coupled receptors that can be activated by trypsin-like enzymes [25]. Tryptase-containing mast cells, as a potential source for PAR-2 activation, are abundant in the dermis, the dermal–epidermal border and occasionally in the lower epidermis in psoriasis [23], and, indeed, the level of tryptase in the skin is associated with the disease severity in psoriasis [26].
Inhibition of PAR-2-mediated CXCL8 production by keratinocytes is therefore an attractive approach for the treatment of inflammatory skin diseases such as psoriasis. Keratinocytes express high levels of β2 adrenergic receptors [27], [28], [29] that are functionally active and have been shown to modulate the migratory behaviour of keratinocytes [30], [31]. While β2 agonists have been shown to influence the production of a number of cytokines by a diverse range of cell types [32], [33], [34], [35], [36], [37], the effect of β2 receptor activation on CXCL8 production by keratinocytes is unknown. Since PAR2-mediated activation of keratinocytes is thought to be relevant to the pathogenesis of inflammatory skin disease, we have therefore studied the effect of salbutamol on the production of CXCL8 by keratinocytes in response to treatment with trypsin.
Section snippets
Materials
Hank’s buffered saline solution was purchased from Invitrogen Ltd. (Paisley, UK). Trypsin, 3-isobutyl-1-methyl-xanthine (IBMX), 2′,5′-dideoxyadenosine, forskolin, dimethylsulfoxide and rolipram were obtained from Sigma–Aldrich (Dorset, UK). Salbutamol and sotalol were obtained from Tocris Bioscience (Bristol, UK). PAR-2 peptide SLIGKV-NH2 and control peptide LSIGKV-NH2 were synthesized by Sigma-Genosys Ltd. (Pampisford, UK). The HitHunter cAMP Kit II was purchased from DiscoveryX (Birmingham,
Effect of salbutamol on CXCL8 production by HEKAp keratinocytes
Treatment of primary HEKAp cells with trypsin (50 nM) led to the production of CXCL8 (⩾3.5 ng/106 cells), measured at 24 h after stimulation (Fig. 1). Heat-inactivated trypsin did not stimulate the production of CXCL8. The stimulatory effect of trypsin was mimicked by the specific PAR2-activating peptide SLIGKV-NH2 (400 μM) but not by the inactive control peptide LSIGKV-NH2 (400 μM). Salbutamol significantly inhibited trypsin-stimulated IL-8 production in a dose-dependent manner with an IC50 = 1.1 ± 0.6
Discussion
We have confirmed that treatment of keratinocytes with trypsin leads to production of CXCL8 [21]. While we cannot exclude the involvement of PAR2-independent effects of trypsin, this effect is likely to be PAR2-mediated since the effect is mimicked by the specific PAR2-activating peptide SLIGKV-NH2 but not by the inactive control peptide LSIGKV-NH2. Regardless of the precise mechanisms whereby trypsin promotes CXCL8 production, the most important observation of our study is that the effect of
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