Sphingosine-1-phosphate increases human alveolar epithelial IL-8 secretion, proliferation and neutrophil chemotaxis

doi:10.1016/j.ejphar.2009.03.012

European Journal of Pharmacology

Volume 609, Issues 1–3, 1 May 2009, Pages 132-139

https://doi.org/10.1016/j.ejphar.2009.03.012 Get rights and content

Abstract

Sphingosine-1-phosphate (S1P) has been presented recently as a pro-inflammatory agent in the airway epithelium since S1P levels are increased in bronchoalveolar lavage fluid of human asthmatics. However, the effects of S1P over the alveolar epithelium and neutrophil interactions are poorly understood. Here, we show that S1P increased interleukin 8 (IL-8) gene expression and protein secretion and proliferation in alveolar epithelial cells A549 at physiological concentrations (1 μM). At the same time, S1P increased intracellular Ca²⁺ concentration (potency 17.91 μM, measured by epifluorescence microscopy), phospholipase D (PLD) activity (measured by chemiluminiscence method) and extracellular matrix-regulated kinase1/2 (ERK1/2) phosphorylation (measured by western blot) via G_i-coupled receptor (inhibited by pertussis toxin 100 ng/ml) in A549 cells. Both, IL-8 secretion and A549 proliferation were dependent of PLD activity (inhibited by 1-butanol 0.5%), intracellular Ca²⁺ (inhibited by acetoxymethyl 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) 100 μM), ERK1/2 phosphorylation (inhibited by 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD98059) 10 μM) and G_i-coupled receptors (blocked by pertussis toxin 100 ng/ml). Moreover, S1P increased intercellular adhesion molecule I (ICAM-1) expression and failed in vascular cell adhesion molecule I (VCAM-1) modification (measured by flow cytometer) in A549. Indirectly, A549 supernatant fluids arising from A549-S1P 1 μM stimulation decreased L-selectin expression without CD11b/CD18 integrin modification in human neutrophils. In the same way, A549-S1P supernatant fluids increased neutrophil chemotaxis (Boyden chamber), which was inhibited by antibody against IL-8. This study demonstrates for the first time that S1P participates in the alveolar epithelial interactions in vitro.

Introduction

The airway epithelium plays a crucial role in initiating and augmenting pulmonary host defense mechanisms by synthesizing and releasing a variety of inflammatory mediators. Interleukin-8 (IL-8), a member of the CXC family of chemokines, is a potent chemoattractant and activator of neutrophils at sites of acute inflammation (Fuke et al., 2004). Studies supporting the involvement of IL-8 in pulmonary inflammatory diseases reported elevated levels of IL-8 in the bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease and asthma among other pulmonary disorders (Car et al., 1994). Alveolar epithelium–neutrophil interactions are partly mediated by adhesion molecules such as selectins, and CD11b/CD18 integrin in neutrophils (Burns et al., 2003), but adhesion of neutrophil granulocytes to epithelial intercellular adhesion molecule I (ICAM-1) has also been reported (Colgan et al., 1995). ICAM-1 is expressed on type I and type II pneumocytes (Cunningham et al., 1994) and is up-regulated in the presence of pro-inflammatory cytokines (Barton et al., 1995).

In addition to inflammatory changes, pulmonary disorders present a balance between cellular proliferation and cell death of airway epithelial cells that may explain the structural remodelling changes noted in severe asthma or chronic obstructive pulmonary disease (Cohen et al., 2007, Pilette et al., 2007). The proliferative response in the airway epithelium may be triggered by viral infection or injury in the genetically susceptible individual, but this mechanism remains unknown.

Recently, the bioactive phospholipid Sphingosine-1-phosphate (S1P) has been presented as a pro-inflammatory agent since S1P levels are increased in the bronchoalveolar lavage fluid of asthmatics after antigen challenge (Ammit et al., 2001). S1P is found in nanomolar to micromolar concentrations in human plasma and serum (Yatomi et al., 1997), and activates intracellular signalling pathways by ligating 5 members of a G protein-coupled lysophospholipid receptor family (SIP₁, SIP₂, SIP₃, SIP₄, SIP₅), regulating calcium levels and cell growth and survival among other functions. Actually there is growing evidence that S1P is involved in the complex tissue-specific process of inflammation, but the role of S1P on the alveolar epithelium and in the neutrophil–epithelial interaction remains unknown. In the present work we have investigated the effect of physiologic concentrations of S1P over IL-8 secretion and proliferation in human alveolar epithelial cells A549, as well as the role in the alveolar epithelial–neutrophil interactions.

Section snippets

Materials

Sphingosine-1-phosphate (S1P), 1-butanol, fura 2-Acetoxy-Methyl esther (AM), pertussis toxin, N-formylmethionyl-leucyl-phenylalanine (fMLP), α-actinin and acetoxymethyl 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) were obtaioned from Sigma Chemical (Madrid, Spain). 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD 98059) was obtained from Calbiochem Corp. (BioNova, Madrid, Spain). Polyclonal antibodies against phospho-extracellular-signal-regulated kinase 1/2 (ERK 1/

Expression of S1P receptors in alveolar epithelial cells A549

We examined the mRNA expression of different S1P receptors in A549. The semiquantitative amount of each one referred to housekeeping, GAPDH, was quantified as different band intensities as shown Fig. 1A. S1P receptor SIP₃ was the most highly expressed with lesser amount of SIP₁, SIP₂, SIP₄ and SIP₅.

S1P induces IL-8 expression and alveolar epithelial cells A549 proliferation

A549 treatment with S1P at physiologic concentrations (1 μM) resulted in a time-dependent IL-8 mRNA increase (Fig. 1B, n = 4), with meaningful expression at 4 h, maximal expression at 12 h and

Discussion

Our findings demonstrate that in human alveolar type II A549 cells, extracellular S1P stimulates proliferation, IL-8 production and adhesion molecule modification in A549, and indirectly, modulates chemotaxis and adhesion molecule expression in human neutrophils. Although human A549 cells have been widely used in studies of the interaction leukocyte–epithelium (Rosseau et al., 2000) these tumour epithelial cells are not phenotypically normal which represents a limitation of this study.

S1P₁, S1P₃

Acknowledgements

This work was supported by grants SAF2006-01002 (EJM), SAF2005-00669 (JC) and SAF20083113 (JC) from CICYT (Ministry of Education and Science, Spanish Government), and GV2007-287 from Regional Government (‘Generalitat Valenciana’). JM has a research contract from FIS of Health Institute ‘Carlos III’ of Ministry of Health (Spain). This work has not received financial support from drug companies.

References (35)

ColganS.P. et al.
Receptors involved in carbohydrate binding modulate intestinal epithelial–neutrophil interactions
J. Biol. Chem.
(1995)
CummingsR.J. et al.
Phospholipase D activation by sphingosine 1-phosphate regulates interleukin-8 secretion in human bronchial epithelial cells
J. Biol. Chem.
(2002)
JollyP.S. et al.
The roles of sphingosine-1-phosphate in asthma
Mol. Immunol.
(2002)
KawaS. et al.
Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate
FEBS Lett.
(1997)
LeeS. et al.
Actin directly interacts with phospholipase D, inhibiting its activity
J. Biol. Chem.
(2001)
LeyK. et al.
Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo
Blood
(1991)
MeacciE. et al.
Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells
J. Biol. Chem.
(1999)
PedruzziE. et al.
Analysis of choline and phosphorylcholine content in human neutrophils stimulated by f-Met-Leu-Phe and phorbol myristate acetate: contribution of phospholipase D and C
Cell. Signal.
(1998)
ShimamuraK. et al.
Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells
Eur. J. Pharmacol.
(2004)
SpiegelS. et al.
Functions of a new family of sphingosine-1-phosphate receptors
Biochim. Biophys. Acta
(2000)

WilkinsonP.C.

Assays of leukocyte locomotion and chemotaxis

J. Immunol. Methods

(1998)

AmmitA.J. et al.

Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma

FASEB J.

(2001)

BartonW.W. et al.

Disparate cytokine regulation of ICAM-1 in rat alveolar epithelial cells and pulmonary endothelial cells in vitro

Am. J. Physiol.

(1995)

BerridgeM.J. et al.

The versatility and universality of calcium signalling

Nat. Rev. Mol. Cell Biol.

(2000)

BoyumA.

Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g

Scand. J. Clin. Lab. Invest. Suppl.

(1968)

BuenestadoA. et al.

Olive oil-based lipid emulsion's neutral effects on neutrophil functions and leukocyte–endothelial cell interactions

JPEN J. Parenter Enteral Nutr.

(2006)

BurnsA.R. et al.

Unique structural features that influence neutrophil emigration into the lung

Physiol. Rev.

(2003)

Cited by (40)

An in vitro alveolar model allows for the rapid assessment of chemical respiratory sensitization with modifiable biomarker endpoints
2022, Chemico-Biological Interactions
Citation Excerpt :
In a recent study, it was shown that DCs and AMs are specific sources of IL-6 relating to allergic airway inflammation and sensitization [54]. Interleukin 8 (IL-8), a cytokine secreted during early inflammatory responses by both T1EC and AMs, is a potent neutrophil attractant and activator within the inflamed space [55–57]. IL-8 is elevated in many respiratory conditions [34,58–60].
Diisocyanates are commonly used in polyurethanes where use includes industrial, commercial, and residential applications and can exist as respirable contaminants. These respirable contaminants exist in the air we breathe. Yet, there is no rapid assay available to test for potential respiratory sensitizers. To assess these hazards, as well as to decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. We describe an easy, reliable, verified cell culture model that can be adopted by any lab capable of performing molecular toxicology. The architecturally relevant alveolar model consists of epithelial cells, macrophage cells, and dendritic cells in a simply maintained submerged system ideal for high-throughput testing. Exposures to contaminants that verify biomarker identification include a known pulmonary sensitizer (isophorone diisocyanate) and a positive control for cellular activation (phorbol 12-myristate 13-acetate/ionomycin). The mitochondrial reactive oxygen species generation and cytostructural changes were assessed with confocal laser scanning microscopy; cell morphology was assessed with scanning electron microscopy; biochemical reactions were assessed via protein arrays; genetic alterations were assessed via gene arrays; and cell surface activation markers were assessed via flow cytometry. Results showed that compared to untreated cultures, isophorone diisocyanate increased markers for dendritic cell activation, trafficking, and antigen presentation; number and length of dendritic protrusions; oxidative stress; and genetic and cytokine expression of neutrophil chemoattractants. The chemokines and cytokines CCL7, CXCL5, IL-6, and IL-8 were identified as biomarkers indicative of respiratory sensitization. By including multiple methods to assess endpoints, the in vitro model described can serve as a high-throughput assay to identify substances which may lead to respiratory sensitization.
Sphingosine 1-phosphate in sepsis and beyond: Its role in disease tolerance and host defense and the impact of carrier molecules
2021, Cellular Signalling
Citation Excerpt :
Sphingolipids play multifaceted roles in neutrophil development and function, which was excellently reviewed in [151]. S1P is involved in the chemotaxis of neutrophils by regulating IL-8 expression [152,153], and a few data suggest that S1P and its receptors play an important role in survival, resolution, and efferocytosis of neutrophils [154]. There is also evidence that S1P/S1PR4 signaling suppresses leukotriene synthesis by inactivating the 5-lipoxygenase (Fig. 3).
Sphingosine 1-phosphate (S1P) is an important immune modulator responsible for physiological cellular responses like lymphocyte development and function, positioning and emigration of T and B cells and cytokine secretion. Recent reports indicate that S1P does not only regulate immunity, but can also protect the function of organs by inducing disease tolerance. S1P also influences the replication of certain pathogens, and sphingolipids are also involved in pathogen recognition and killing. Certain carrier molecules for S1P like serum albumin and high density lipoproteins contribute to the regulation of S1P effects. They are able to associate with S1P and modulate its signaling properties. Similar to S1P, both carrier molecules are also decreased in sepsis patients and likely contribute to sepsis pathology and severity.
In this review, we will introduce the concept of disease tolerance and the involvement of S1P. We will also discuss the contribution of S1P and its precursor sphingosine to host defense mechanisms against pathogens. Finally, we will summarize current data demonstrating the influence of carrier molecules for differential S1P signaling. The presented data may lead to new strategies for the prevention and containment of sepsis.
Phosphorylation and inhibition of ceramide kinase by protein kinase C-β: Their changes by serine residue mutations
2019, Cellular Signalling
Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), and various roles for the CerK/C1P pathway in the regulation of cellular/biological functions have been demonstrated. CerK is constitutively phosphorylated at several serine (Ser, S) residues, however, the roles of Ser residues, including their phosphorylation, in CerK activity, have not yet been elucidated in detail. Therefore, we conducted the present study to investigate this issue. In A549 cells expressing wild-type CerK, a treatment with phorbol 12-myristate 13-acetate (PMA) decreased the formation of C1P in a protein kinase C (PKC)-βI/II-mediated manner. In the Phos-tag SDS-PAGE analysis, CerK existed in its phosphorylated form and was further phosphorylated by the PMA treatment in a PKC-βI/II-mediated manner. We examined the effects of the displacement of Ser residues (72/300/340/403/408/427) in CerK by alanine (Ala, A) on its activity and phosphorylation. Triple mutations (S340/408/427A), but not a single or double mutations (S340/408A), in CerK significantly decreased the formation of C1P. PMA-induced phosphorylation levels in S340/408A- and S340/408/427A-CerK were significantly and maximally reduced, respectively, but were similar in CerK with a single mutation and wild-type CerK. Ser residue mutations tested, including six mutations, did not affect PMA-induced decreases in C1P formation more than expected. Treatments with the protein phosphatase inhibitors, okadaic acid and cyclosporine A, decreased the formation of C1P. These results demonstrated that the activity of CerK was regulated in a phosphorylation-dependent manner in cells.
Sphingolipids as targets for inhalation treatment of cystic fibrosis
2018, Advanced Drug Delivery Reviews
Citation Excerpt :
Extensive histology did not show any dose responsive toxicity up to an inhalation of a 1 mM sphingosine solution [67]. Further, sphingosine inhalation did not induce any signs of inflammation, a potential side effect if inhaled sphingosine were converted to sphingosine 1-phosphate [97]. None of these effects was observed after prolonged inhalation of even very high doses of sphingosine by wild-type or CF mice [56,67], suggesting the potential safety of this approach.
Studies over the past several years have demonstrated the important role of sphingolipids in cystic fibrosis (CF), chronic obstructive pulmonary disease and acute lung injury. Ceramide is increased in airway epithelial cells and alveolar macrophages of CF mice and humans, while sphingosine is dramatically decreased. This increase in ceramide results in chronic inflammation, increased death of epithelial cells, release of DNA into the bronchial lumen and thereby an impairment of mucociliary clearance; while the lack of sphingosine in airway epithelial cells causes high infection susceptibility in CF mice and possibly patients. The increase in ceramide mediates an ectopic expression of β1-integrins in the luminal membrane of CF epithelial cells, which results, via an unknown mechanism, in a down-regulation of acid ceramidase. It is predominantly this down-regulation of acid ceramidase that results in the imbalance of ceramide and sphingosine in CF cells. Correction of ceramide and sphingosine levels can be achieved by inhalation of functional acid sphingomyelinase inhibitors, recombinant acid ceramidase or by normalization of β1-integrin expression and subsequent re-expression of endogenous acid ceramidase. These treatments correct pulmonary inflammation and prevent or treat, respectively, acute and chronic pulmonary infections in CF mice with Staphylococcus aureus and mucoid or non-mucoid Pseudomonas aeruginosa. Inhalation of sphingosine corrects sphingosine levels only and seems to mainly act against the infection. Many antidepressants are functional inhibitors of the acid sphingomyelinase and were designed for systemic treatment of major depression. These drugs could be repurposed to treat CF by inhalation.
Sphingolipids in neutrophil function and inflammatory responses: Mechanisms and implications for intestinal immunity and inflammation in ulcerative colitis
2017, Advances in Biological Regulation
Citation Excerpt :
S1P secreted from airway epithelial cells indirectly promoted neutrophil recruitment by increasing IL-8 production and ICAM-1 expression in A549 alveolar epithelial cells (Fig. 2). Paracrine signaling by S1P was mediated by signaling via the extracellular matrix-regulated kinase 1/2 (ERK1/2) pathway and subsequent activation of PLD and [Ca2+] flux (Milara et al., 2009). The compartmentalization of S1P in blood and tissues is a critical regulatory mechanism of lymphocyte trafficking and is dependent on S1PR signaling (Degagne and Saba, 2014).
Bioactive sphingolipids are regulators of immune cell function and play critical roles in inflammatory conditions including ulcerative colitis. As one of the major forms of inflammatory bowel disease, ulcerative colitis pathophysiology is characterized by an aberrant intestinal inflammatory response that persists causing chronic inflammation and tissue injury. Innate immune cells play an integral role in normal intestinal homeostasis but their dysregulation is thought to contribute to the pathogenesis of ulcerative colitis. In particular, neutrophils are key effector cells and are first line defenders against invading pathogens. While the activity of neutrophils in the intestinal mucosa is required for homeostasis, regulatory mechanisms are equally important to prevent unnecessary activation. In ulcerative colitis, unregulated neutrophil inflammatory mechanisms promote tissue injury and loss of homeostasis. Aberrant neutrophil function represents an early checkpoint in the detrimental cycle of chronic intestinal inflammation; thus, dissecting the mechanisms by which these cells are regulated both before and during disease is essential for understanding the pathogenesis of ulcerative colitis. We present an analysis of the role of sphingolipids in the regulation of neutrophil function and the implication of this relationship in ulcerative colitis.
Sphingosine 1-phosphate regulates IL-8 expression and secretion via S1PR<inf>1</inf> and S1PR<inf>2</inf> receptors-mediated signaling in extravillous trophoblast derived HTR-8/SVneo cells
2015, Placenta
Citation Excerpt :
After a screening of selected cytokines, we found that unlike BeWo cells, which apparently secreted IL-8 at very low level, HTR-8/SVneo cells secreted IL-8 after S1P treatment. Previously, S1P has been shown to promote IL-8 secretion from lung epithelial cells and ovarian cancer cells [35–37]. In these cells, multiple S1P receptor subtypes (S1PR1–3) were expressed similar to HTR-8/SVneo cells.
Both villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown.
IL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used.
We found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR₁, induced IL-8 gene expression but not protein secretion. The specific S1PR₂ inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR₁/S1PR₃ antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells.
In this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR₁ and its secretion is regulated through S1PR₂ receptor subtype_. Rho GTPases signaling is essential for S1P-induced IL-8 secretion.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Immunopharmacology and InflammationSphingosine-1-phosphate increases human alveolar epithelial IL-8 secretion, proliferation and neutrophil chemotaxis

Abstract

Introduction

Section snippets

Materials

Expression of S1P receptors in alveolar epithelial cells A549

S1P induces IL-8 expression and alveolar epithelial cells A549 proliferation

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Mol. Immunol.

FEBS Lett.

J. Biol. Chem.

Blood

J. Biol. Chem.

Cell. Signal.

Eur. J. Pharmacol.

Biochim. Biophys. Acta

J. Immunol. Methods

Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma

FASEB J.

Disparate cytokine regulation of ICAM-1 in rat alveolar epithelial cells and pulmonary endothelial cells in vitro

Am. J. Physiol.

The versatility and universality of calcium signalling

Nat. Rev. Mol. Cell Biol.

Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g

Scand. J. Clin. Lab. Invest. Suppl.

Olive oil-based lipid emulsion's neutral effects on neutrophil functions and leukocyte–endothelial cell interactions

JPEN J. Parenter Enteral Nutr.

Unique structural features that influence neutrophil emigration into the lung

Physiol. Rev.

Immunopharmacology and Inflammation
Sphingosine-1-phosphate increases human alveolar epithelial IL-8 secretion, proliferation and neutrophil chemotaxis