Elsevier

Molecular Immunology

Volume 47, Issue 10, June 2010, Pages 1905-1913
Molecular Immunology

Antimicrobial peptide rCRAMP induced glial cell activation through P2Y receptor signalling pathways

https://doi.org/10.1016/j.molimm.2010.03.012Get rights and content

Abstract

Antimicrobial peptides are part of the innate immune system of many organ systems, yet little is known about their expression and function in the brain. The antibacterial cathelicidin rCRAMP in rats (homologue of the human LL-37) not only exhibits potent bactericidal activities but also functions as a chemoattractant for immune cells. In this study, to further evaluate the role of rCRAMP in innate immunity of brain cells, we investigated the impact of rCRAMP on glial cell activation. To this end we analyzed the activation of rCRAMP-induced signalling by cytokine expression, Western blotting of certain signal transduction pathways and by cAMP level measurement in primary rat glial cells (astrocytes and microglia). We demonstrate (i) the induction of proinflammatory cytokine and neurotrophic factors and (ii) the activation of various signal transduction pathways by rCRAMP in glial cells. Moreover, (iii) we have been able to show that rCRAMP-induced IL-6 expression and ERK1/2 phosphorylation in glial cells were attenuated by the antagonists for purinergic receptor P2Y, whereas P2X and FPRL1 antagonists do not show any effects.

Our results indicate for the first time that a newly identified P2Y11 receptor participates in rCRAMP-induced signal transduction. This study provides evidence that rCRAMP participates in brain immunity by stimulating cytokine production and glial cell activation, and aid in the protection of brain cells by inducing neurotrophic factors.

Introduction

Antimicrobial peptides are effector molecules of the innate immune system that have microbicidal and proinflammatory properties (Bals, 2000, Ganz, 1999). In mammals, there are two main families of antimicrobial peptides: the defensins and the cathelicidins (Zasloff, 2002). Cathelicidins are defined by a highly conserved N-terminal cathelin pro-domain and a structurally variable antimicrobial domain at the C terminus (Lehrer and Ganz, 2002), and they have been identified in various species. In rodents and humans one gene for cathelicidin is known. This homologue gene encodes for the antimicrobial peptide LL-37 in humans and CRAMP (Gallo et al., 1997) and rCRAMP (Termen et al., 2003) in mice and rats, respectively.

However, the occurrence of antimicrobial peptides in the central nervous system (CNS) has not yet been thoroughly investigated. Recent studies have shown that rCRAMP is expressed with a distinct regional distribution in the CNS of the rat (Bergman et al., 2005). In addition, the murine CRAMP was found to be induced in endothelial cells of the blood–brain barrier (BBB) and in leptomeningeal cells after a meningococcal infection (Bergman et al., 2006). We found an increase of antimicrobial peptides (for example human cathelicidin LL-37) in the CSF of patients with bacterial meningitis but not in control CSF. Our results showed an increase of rCRAMP expression by astrocytes and by microglial as well as meningeal cells in vivo and in vitro after bacterial stimulation (Brandenburg et al., 2009b, Brandenburg et al., 2008b). Aside from their well-established antibiotic function, numerous studies have provided compelling evidence for their involvement in the complex network of immune responses and inflammatory diseases (for review, see (Zaiou, 2007)). In addition to its potent antibacterial properties (Turner et al., 1998), LL-37 can bind lipopolysaccharide (LPS) and blunt its biological activities (Larrick et al., 1995, Nagaoka et al., 2001). Despite burgeoning interest in the immunomodulatory properties of cationic peptides, little is known about their influence on brain cells or about the function of AP for the brain innate immune response. It is not clear which receptor mediates LL-37/rCRAMP activities.

For LL-37, it has been shown that the chemoattractant activity is due, at least in part, to activation of the G-protein-coupled formyl peptide receptor like 1 (FPRL1) (De et al., 2000), although this receptor is not involved in mediating other LL-37 activities (Bowdish et al., 2004). Recently, it was reported that LL-37-induced IL-1β release from monocytes via activation of the ligand-gated ion channels-coupled purinergic receptor P2X7 (Elssner et al., 2004). In addition, LL-37 can also modulate neutrophil apoptosis via activation of FPRL1 and P2X7 in bacterial infections (Nagaoka et al., 2006). Alternatively, LL-37 might work directly on the Toll-like receptors (TLR) 4 to NFκB pathways in inhibiting endotoxin-stimulated proinflammatory cytokine production (Mookherjee et al., 2006). However, the mechanism by which rCRAMP activates the brain cell and how it interacts with other functions pivotal in the innate immune response remains unclear.

In the present study, we investigate the action of rCRAMP on glial cell activation. We demonstrate the induction of proinflammatory cytokine and neurotrophic factors as well as the activation of various signal transduction pathways by rCRAMP in glial cells. Moreover, we are able to show that rCRAMP-induced IL-6 expression, ERK1/2 phosphorylation and an increase of cAMP level in glial cells were attenuated by the antagonists for P2Y receptors, whereas P2X and FPRL1 antagonists show no show. For the first time, we have been able to show the expression of P2Y11 as candidate for rCRAMP-induced signal transduction in glial cells. Our results suggest that rCRAMP is an important component of innate CNS immunity which acts in the protection of brain cells by inducing neurotrophic factors.

Section snippets

Reagents

rCRAMP (GLLRKGGEKFGEKLRKIGQKIKDFFQKLAPEIEQ) and FPRL1 antagonist WRW4 were purchased from Dr. P. Henklein (Charité, Berlin, Germany). Peptides were dissolved at 10 mM concentration in DMSO. Mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK1/2)-specific inhibitor PD98059 (10 μM), p38 MAPK-specific inhibitor SB203580 (10 μM), the specific inhibitor of NFκB (which is also an inhibitor of other pathways, for example the lipoxygenase pathway; (Sud’ina et al., 1993)), CAPE

rCRAMP-induced various cytokine expressions in glial cells

In a first set of experiments, we investigated the induction of various cytokines by primary astrocytes and microglia after stimulation with various rCRAMP concentrations. After 0, 6, 12 and 24 h incubation with 2, 5 and 10 μM rCRAMP, the gene expression of IL-1β, IL-6, IL-10, TNF-α, and TGF-β was analyzed by the RT-PCR technique. As shown in Fig. 1, the results of these experiments showed that maximal IL-1β mRNA expression was seen in astrocytes after 12 h of incubation with 5 μM (8.1 ± 3-fold

Discussion

The present investigation shows, for the first time, activation of glial cells by the antimicrobial peptide rCRAMP (rat homologue of the human LL-37) (Termen et al., 2003). These peptides are mainly expressed on epithelial surfaces and in neutrophils, and seem to provide a first line of defence against infection by the innate immune system (Zasloff, 2002). Our previous results shown an increase in LL-37/rCRAMP expression in bacterial meningitis and has localized this to astrocytes, microglia

Acknowledgments

We thank Rosemarie Sprang, Regine Worm, Marie Pradella, Susanne Echterhagen and Michaela Nicolau for excellent technical assistance.

Grant support: This study was supported by the Hensel Foundation (University of Kiel, Germany, to L.O.B.) and in part by the SFB 617 of the Deutsche Forschungsgemeinschaft (DFG; to D.V.) and the Swiss National Science Foundation (632-66057.01 to S.L.L).

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    These authors contributed equally to this study.

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