LPS challenge in healthy subjects: An investigation of neutrophil chemotaxis mechanisms involving CXCR1 and CXCR2

doi:10.1016/j.intimp.2012.04.008

International Immunopharmacology

Volume 13, Issue 3, July 2012, Pages 225-231

https://doi.org/10.1016/j.intimp.2012.04.008 Get rights and content

Abstract

LPS inhalation was used to investigate whether sputum supernatant post-LPS challenge increases neutrophil chemotactic activity and to elucidate the role of CXCR1/CXCR2 signalling in this process.

14 healthy non-smoking subjects inhaled 30 μg of LPS. Sputum was induced at baseline, 6 and 24 h post-LPS challenge. Differential cell counts were determined and supernatants CXCL8, CXCL1, IL-6 and CCL2 levels measured. Peripheral blood neutrophils obtained from healthy volunteers were used for chemotaxis experiments using sputum supernatant. To delineate signalling mechanisms, the effects of a CXCR2/CXCR1 (dual) antagonist (Sch527123) and a CXCR2 specific antagonist (SB656933) were tested.

LPS inhalation significantly increased sputum neutrophil counts from 45.3% to 76.7% and 69.3% at 6 and 24 h respectively. LPS increased CXCL8, IL-6 and CCL2 levels but not CXCL1. Neutrophil chemotaxis significantly increased (2.7 fold) at 24 h compared to baseline. Chemotaxis was inhibited by 79.0% with Sch527123 and 52.0% with SB656933.

We conclude that LPS challenge increases sputum supernatant CXCL8 levels, which is associated with increased chemotactic activity which is dependent on both CXCR1 and CXCR2.

Highlights

► LPS inhalation in healthy subjects increases the levels of CXCL8 but not CXCL1. ► LPS inhalation also increases the chemotactic capacity of sputum supernatant. ► Chemotactic capacity inhibited more using a dual CXCR1/CXCR2 antagonist. ► TLR4 signalling causes airway neutrophilia dependent on CXCL8 driven chemotaxis. ► Supports development of dual CXC1/CXCR2 antagonists for neutrophilic lung diseases

Introduction

Chronic obstructive pulmonary disease (COPD) and severe asthma are characterised by an increase in the number of neutrophils in the airways [1], [2], [3]. Neutrophils contribute to a persistent and abnormal innate immune response in these diseases, by releasing pro-inflammatory cytokines and tissue destructive proteases. Neutrophils are recruited into the lungs by chemokines such as CXCL1 (GROα) and CXCL8 (IL-8) that act through the cell surface chemokine receptors CXCR1 and CXCR2 [4]. Both CXCL1 and CXCL8 levels are increased in the airways of patients with asthma and COPD [5], [6], [7], [8], [9]. CXCL8 binds to both CXCR1 and CXCR2 [4]. In contrast, CXCL1 acts only through CXCR2 [10].

Antagonists targeted selectively against CXCR2 have been developed for the treatment of neutrophilic airway inflammation [11]. Dual antagonists targeted against both CXCR1 and CXCR2 may have a greater effect on neutrophil chemotaxis due to the additional targeting of CXCR1. Furthermore, CXCR1 is involved in neutrophil activation as well as chemotaxis [12]. It has been shown in different studies that both the selective CXCR2 antagonist SB656933 and the dual CXCR1/CXCR2 antagonist Sch527123 inhibit ozone induced airway neutrophilia in healthy subjects [13], [14].

Toll‐like receptors (TLRs) act as sensors for pathogen associated microbial peptides [15]. TLR4 is expressed on a range of structural and immune cell types, and acts as a receptor for the bacterial endotoxin lipopolysaccharide (LPS) [16]. TLR4 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF_KB) pathway and mitogen activated protein kinases (MAPK) to increase pro-inflammatory cytokine production [17]. Patients with asthma and COPD often suffer with acute and chronic bacterial infections that can amplify the innate immune response through TLR4 signalling [18]. The inhalation of LPS in healthy subjects increases the number of airway neutrophils, which is easily measured in induced sputum samples [19], [20], [21],. This model has been used to study the mechanisms of neutrophilic lung diseases [22], [23].

In vitro studies of neutrophil chemotaxis often use single chemokines as the chemoattractant [24]. Such systems do not resemble the in vivo environment containing multiple chemoattractants. Induced sputum supernatants obtained after LPS challenge in healthy subjects contain multiple chemoattractants [21], and so may be a more clinically relevant model to study chemotaxis mechanisms compared to single chemokine systems. Chemotaxis using induced sputum after LPS challenge has not been used before, and could be utilised to investigate the roles of CXCL1 and CXCL8, and their receptors CXCR1 and CXCR2 using clinically relevant chemotaxis signals.

We have used the inhaled LPS model in healthy subjects to investigate mechanisms of neutrophil chemotaxis into the airways. We studied the effect of LPS challenge on CXCL1 and CXCL8 levels in sputum supernatants, and whether LPS challenge increased the chemotactic ability of these supernatants. The roles of CXCR1 and CXCR2 in neutrophil chemotaxis caused by sputum supernatants obtained after LPS challenge were investigated by using the selective CXCR2 antagonist SB656933 and the dual CXCR1 and CXCR2 antagonist Sch527123.

Section snippets

Subjects

Fourteen healthy subjects (7 males and 7 females, mean age 33.7 years) were recruited, who had no history of smoking and had normal lung function. Exclusion criteria were: a respiratory tract infection in previous six weeks, positive skin prick test to grass, cat or house dust mite and history of chronic respiratory disease. Written informed consent was obtained and the local ethics committee approved the study.

Safety

LPS challenges were performed safely in 14 subjects, with only minor adverse events reported: headache (n = 5), myalgia (n = 1) and flu like symptoms (n = 1). All of these symptoms were resolved within 24 h. None of the subjects complained of breathlessness or wheezing during the study. Body temperature was significantly increased at 4–8 h post‐LPS inhalation with a maximum mean rise of 0.8 °C at 8 h, which had returned to normal by 24 h (Fig. 1A). The mean maximum fall in FEV1 was 5.1% (Fig. 1B). There

Discussion

We have used an in vivo model of human lung inflammation to investigate the roles of CXCL1 and CXCL8 and their receptors CXCR1 and CXCR2 in the development of pulmonary neutrophilia. We observed that LPS inhalation in healthy subjects increased the levels of CXCL8 but not CXCL1. LPS inhalation also increased the chemotactic capacity of sputum supernatant, which was more effectively inhibited using a dual CXCR1 and CXCR2 antagonist (Sch527123) compared to a selective CXCR2 antagonist (SB656933).

Conclusion

In conclusion, we have demonstrated that LPS increases neutrophil chemotaxis that appears to be driven by CXCL8 signalling rather than CXCL1. This increase in CXCL8 levels was closely associated with increased chemotaxis. Pharmacological antagonism in both CXCR1 and CXCR2 was more effective in blocking chemotaxis than antagonism of CXCR2 alone. These results support the further development of dual CXC1 and CXCR2 antagonists against neutrophilic lung diseases.

Abbreviations

LPS

Lipopolysaccharide

MAPK

Mitogen activated protein kinases

IL-6

Interleukin 6

MSD

Meso scale discovery

TLR

Toll-like receptor

NF_KB

Nuclear factor kappa-light-chain-enhancer of activated B cells

COPD

Chronic obstructive pulmonary disease

FEV1

Forced expiratory volume in 1 second

PBS

Phosphate buffered saline

Room temperature

NGS

Normal goat serum

IgG

Immunoglobulin

DAB

3,3′‐diaminobenzidine

PMN

Polymorphonuclear leukocytes

EDTA

Ethylenediaminetetraacetic acid

HBSS

Hanks balanced salt solution

BSA

Bovine serum albumin

DMSO

Competing interests

RA and JP, have no conflicts of interest to disclose. SP, SS and IK are employees of Pfizer. DS has received sponsorship to attend international meetings, honoraria for lecturing or attending advisory boards, and research grants from various pharmaceutical companies including AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Almirall, Forest, Pfizer, UCB, Novartis, and Cipla.

Funding

This work was supported by Pfizer Global R&D, Sandwich, Kent, UK.

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¹: Raminder Aul and Sheena Patel contributed equally to this work and are joint first authors.

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LPS challenge in healthy subjects: An investigation of neutrophil chemotaxis mechanisms involving CXCR1 and CXCR2

Abstract

Highlights

Introduction

Section snippets

Subjects

Safety

Discussion

Conclusion

Abbreviations

Competing interests

Funding

Chest

Respir Med

Pulm Pharmacol Ther

Cell

Pulm Pharmacol Ther

Pharmacol Ther

Airways obstruction, chronic expectoration, and rapid decline of FEV1 in smokers are associated with increased levels of sputum neutrophils

Thorax

Neutrophilic inflammation in severe persistent asthma

Am J Respir Crit Care Med

Inflammatory biomarkers in airways of patients with severe asthma compared with non-severe asthma

Clin Exp Allergy

Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines

Adv Immunol

Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma

Am J Respir Crit Care Med

Increased levels of the chemokines GROalpha and MCP-1 in sputum samples from patients with COPD

Thorax

Bronchial mucosal inflammation and upregulation of CXC chemoattractants and receptors in severe exacerbations of asthma

Thorax

Human chemokines: an update

Annu Rev Immunol

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2

Proc Natl Acad Sci U S A

SB-656933, a novel CXCR2 selective antagonist, inhibits ex-vivo neutrophil activation and ozone-induced airway inflammation in humans

Br J Clin Pharmacol