Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes

Role of proteinase-activated receptor-2 in anti-bacterial andimmunomodulatory effects of interferon-c on human

neutrophils and monocytes

Victoria M. Shpacovitch,1,2,* Micha

Feld,1,* Dirk Holzinger,3,* Makiko

Kido,4,1 Morley D. Hollenberg,5

Francesca Levi-Schaffer,6 Nathalie

Vergnolle,7 Stephan Ludwig,8

Johannes Roth,3 Thomas Luger1

and Martin Steinhoff1,9

1Department of Dermatology, IZKF Munster,

Boltzmann Institute for Immunobiology of the

Skin, University of Munster, 2Department of

Nephrology and Hypertension & Endocrinology

and Metabolic Disease; Otto-von-Guericke

University of Magdeburg, Magdeburg, 3Insti-

tute of Immunology, University of Munster,

Munster, Germany, 4Department of Dermatol-

ogy, Graduate School of Medical Sciences, Kyu-

shu University, Fukuoka, Japan, 5Department

of Pharmacology and Therapeutics, Canadian

Institutes of Health Research Proteinases and

Inflammation Network, University of Calgary,

Calgary, AB, Canada, 6Department of Phar-

macology and Experimental Therapeutics,

School of Pharmacy, the Hebrew University of

Jerusalem, Israel, 7INSERM, U536 Centre de

Physiopathologie de Toulouse Purpan, Tou-

louse, France, 8Department of Molecular Virol-

ogy, ZMBE, University of Munster, Germany,

and 9Department of Dermatology and Surgery,

University of California San Francisco, San

Francisco, CA, USA

doi:10.1111/j.1365-2567.2011.03443.x

Received 15 September 2010; revised 24

February 2011; accepted 15 March 2011.

*These authors contributed equally to this

work.

Correspondence: V. M. Shpacovitch,

Department of Nephrology and

Hypertension & Endocrinology and

Metabolic Disease; Otto-von-Guericke

University of Magdeburg, Leipziger str. 40,

39120 Magdeburg, Germany.

Email: [email protected]

Senior author: Martin Steinhoff,

email: [email protected]

Summary

Recent studies show that proteinase-activated receptor-2 (PAR2) contrib-

utes to the development of inflammatory responses. However, investiga-

tions into the precise role of PAR2 activation in the anti-microbial

defence of human leucocytes are just beginning. We therefore evaluated

the contribution of PAR2 to the anti-microbial response of isolated

human innate immune cells. We found that PAR2 agonist, acting alone,

enhances phagocytosis of Staphylococcus aureus and killing of Escherichia

coli by human leucocytes, and that the magnitude of the effect is similar

to that of interferon-c (IFN-c). However, co-application of PAR2-cAP and

IFN-c did not enhance the phagocytic and bacteria-killing activity of leu-

cocytes beyond that triggered by either agonist alone. On the other hand,

IFN-c enhances PAR2 agonist-induced monocyte chemoattractant protein

1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore,

phosphoinositide-3 kinase and janus kinase molecules are involved in the

synergistic effect of PAR2 agonist and IFN-c on MCP-1 secretion. Our

findings suggest a potentially protective role of PAR2 agonists in the anti-

microbial defence established by human monocytes and neutrophils.

Keywords: innate immunity; monocyte chemoattractant protein-1; phago-

cytosis; proteinase-activated receptor; Staphylococcus aureus

Abbreviations: FCS, fetal calf serum; IFN-c, interferon-c; MFI, mean fluorescence intensity; MCP-1, monocyte chemoattractantprotein-1 (other name is CCL2); PAR2, proteinase-activated receptor-2; PAR2-cAP, PAR2-tc-activating peptide; PAR2-cRP,PAR2-tc-reverse peptide.

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 133, 329–339 329

I M M U N O L O G Y O R I G I N A L A R T I C L E

Introduction

Proteinase-activated receptor-2 (PAR2) plays a role in the

development of allergic diseases of the skin1 and in cer-

tain inflammatory disorders.2 The impact of PAR2 activa-

tion on inflammation can be pro- or anti-inflammatory,

depending on the stage of disease and the primary cell

type involved in disease progression.2 During receptor

activation, serine protease cleavage of PAR2 unmasks the

N-terminal sequence of the ‘tethered ligand’. This

unmasked sequence further serves as a receptor activator.3

The PAR2 is activated by trypsin and tryptase, and also

by proteases derived from immune cells and pathogens.4

However, serine proteases cause PAR-dependent as well

as PAR-independent effects.5,6 As a result, specific syn-

thetic activating peptides are important probes for investi-

gating the role of PAR activation in different processes.

Interferon-c (IFN-c) is well-known as a mediator that

has a wide range of anti-viral, anti-bacterial and anti-

tumour or immunomodulatory activities.7 We recently

demonstrated in vitro that PAR2 activation on human

monocytes enhances the suppressive effects of IFN-c on

influenza A virus replication.8 Moreover, in vivo studies

have shown that a protective role of PAR2 against influ-

enza infection is also mediated by an IFN-c-dependent

mechanism.9 These studies revealed interplay between

PAR2 activation and IFN-c during the anti-viral response

and raise the intriguing question of whether PAR2 activa-

tion also contributes to anti-bacterial and immunomodu-

latory effects triggered by IFN-c in monocytes and

neutrophils.

Human neutrophils and monocytes are not only ‘pro-

fessional’ phagocytes, they are cells that, when activated,

secrete different chemokines and cytokines. Stimulation of

PAR2 agonist affects chemokine [IFN-inducible protein-

10, interleukin-8 (IL-8)] and cytokine (IL-1b, IL-6) secre-

tion by human neutrophils and monocytes.8,10 Among

the chemokines secreted by neutrophils there is a mole-

cule that appears to link neutrophils and monocytes dur-

ing the time-delayed immune response to local infection.

Monocyte chemoattractant protein-1 (MCP-1) is an

essential mediator for monocyte and macrophage recruit-

ment towards the site of infection.11,12 Neutrophils are a

source of MCP-1 in time-delayed responses,13 and so may

attract monocytes and macrophages. However, MCP-1 is

not only a chemotactic molecule for monocytes and mac-

rophages, it also enhances the engulfment of apoptotic

neutrophils (efferocytosis), thereby helping to resolve

acute inflammation.14 In addition, MCP-1 is involved in

fibroblast activation and influences collagen production,

which makes MCP-1 an important participant in initial

events during systemic scleroderma and skin fibrosis.15

Interferon-c is known to increase the secretion of MCP-1

by human neutrophils 48 hr after stimulation.13 However,

whether PAR2 agonists enhance MCP-1 release or influ-

ence the IFN-c-induced secretion of MCP-1 by human

neutrophils has received little study.

We therefore evaluated the contribution of PAR2 to

the anti-microbial response of isolated human innate

immune cells. We investigated whether PAR2 agonist

acting alone affects the phagocytic and bactericidal activ-

ity of human neutrophils and monocytes. We also inves-

tigated whether IFN-c enhances the effect of PAR2

agonist on the MCP-1 release by human neutrophils and

monocytes, and examined the intracellular signalling

molecules involved in the effects of PAR2 agonist on

MCP-1 secretion.

Materials and methods

Materials

Human recombinant IFN-c was purchased from TebuBio

(Offenbach, Germany). Lipopolysaccharide (LPS) from

Escherichia coli 055:B5 was purchased from Sigma

(Munich, Germany; cat.#L2880). Human PAR2 activating

peptide with the sequence trans-cinnamoyl-LIGRLO-NH2

(cAP) and reverse peptide with sequence trans-cinnamoyl-

OLRGIL-NH2 (cRP) (Peptide Synthesis Facility, Univer-

sity of Calgary, Canada, Director: Dr Denis McMaster; the

web page: http://www.ucalgary.ca/peptides).8,10 Fluorescein-

conjugated killed Staphylococcus aureus was purchased

from Molecular Probes (Karlsruhe, Germany). The E. coli

strain JM109 was obtained from Promega (Mannheim,

Germany). Cell culture reagents were purchased from Bio-

Whittaker (Aachen, Germany), PAA Laboratories (Coelbe,

Germany) and Gibco-Life Technologies (Karlsruhe,

Germany). Cell-permeable inhibitors of intracellular sig-

nalling molecules [SB203580, rottlerin, LY 294002 and

janus kinase (JAK) inhibitor I pyridone 6] were purchased

from Calbiochem (Nottingham, UK).

Cell culture and isolation of human neutrophils andmonocytes

Buffy-coats with blood cells for in vitro experiments with

human neutrophils and monocytes were obtained from

healthy adult volunteers via the German Red Cross (De-

utsches Rotes Kreuz, Munster, Germany). Neutrophils

were isolated by Biocoll (Biochrom, Berlin, Germany)

density gradient centrifugation followed by a hypotonic

shock procedure.10,16 Peripheral blood monocytes were

isolated by leukapheresis as previously described.17 Iso-

lated human monocytes were cultivated in Teflon bags in

McCoy’s medium (Biochrom) supplemented with 15%

fetal calf serum (FCS), 2 mM L-glutamine and 1% non-

essential amino acids. Monocytes were allowed to rest for

24 hr before stimulation. Isolated neutrophils were cul-

tured in RPMI-1640 medium supplemented with 0�9%

FCS, 2 mM L-glutamine and 1% non-essential amino acids

330 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 133, 329–339

V. M. Shpacovitch et al.

and allowed to recover for 2 hr before stimulation. The

following concentrations of reagents were used for stimu-

lation during experiments: LPS 100 ng/ml; IFN-c 10 or

100 ng/ml; PAR2-cAP 1 · 10)4M. The corresponding

reverse peptide with the reverse-sequence (PAR2-cRP) was

used at a concentration of 1 · 10)4M and served as a

negative control.

Bacterial killing assay

Bacterial killing assay using E. coli (strain JM109) was

performed as described previously18,19 with modifications.

In brief, E. coli bacteria were cultured into Luria broth

medium overnight at 37�. Isolated uninfected human

neutrophils were pre-stimulated with 10)4M PAR2-cAP

and/or IFN-c (100 ng/ml) for 2 hr (37�, 5% CO2).

Unstimulated neutrophils were used as control samples.

After 2 hr incubation of neutrophils with stimuli, the cell

culture medium (RPMI-1640 with 0�9% FCS, 2 mM L-glu-

tamine and 1% non-essential amino acids) with stimuli

was removed and cells were washed. Human neutrophils

(2 · 106 cells) were resuspended in 200 ll RPMI-1640

containing 2 mM L-glutamine, 1% non-essential amino

acids, 0�2% BSA, 0�01% CaCl2 and 0�01% MgCl2 (this

medium was designated the ‘assay medium’). Collected

and washed bacteria (40 · 106 cells) were opsonized for

15 min at 37�. For opsonization, bacteria were incubated

in the assay medium containing 5% human serum from

the same donor from whom the neutrophils were

obtained. After opsonization, bacteria were washed. Neu-

trophils and opsonized bacteria were co-incubated in

assay medium in the absence (for unstimulated control

samples) or presence of stimuli (10)4M PAR2-cAP and/or

100 ng/ml IFN-c) for 1 hr at 37� on a shaker. Neutrophils

were removed from samples by hypotonic lysis (for this

purpose sterile water was added to samples). A series of

dilutions were prepared from the remaining bacteria. Bac-

teria were cultured on Luria broth agar plates without

antibiotic at 37� overnight. Colonies were counted the

next day.

Phagocytosis assay with killed S. aureus

The phagocytosis assay was performed as described previ-

ously.19,20 In brief, FITC-conjugated killed S. aureus (Invi-

trogen, Darmstadt, Germany) was used for assay. The

bacteria were opsonized before the assay. For this pur-

pose, bacteria were incubated with 5% serum (from the

same donor from whom neutrophils were isolated) for

25 min at 37�. Non-infected neutrophils were pre-stimu-

lated with PAR2-cAP 10)4M, PAR2-cRP 10)4

M and/or

IFN-c 100 ng/ml for 2 hr at 37� and 5% CO2. Neutroph-

ils and opsonized bacteria were co-incubated at 1 : 20

ratio (neutrophils : S. aureus). During co-incubation of

bacteria and neutrophils, PAR2-cAP 10)4M, PAR2-cRP

10)4M and/or IFN-c 100 ng/ml were applied in the con-

centrations indicated above. Co-incubation took place in

assay medium on a shaker for 30 min at 37�. The phago-

cytosis assay was stopped by the addition of ice-cold PBS

containing 0�5 mM EDTA (500 ll PBS to 1 ml of sample

medium). Samples were then centrifuged at 169 g and

neutrophil pellets were resuspended in ice-cold PBS con-

taining 0�9% FCS and 2 mM EDTA. Trypan blue quench,

which helps to discriminate adherent and ingested bacte-

ria, was performed as described previously.21 The efficacy

of phagocytosis was estimated using flow cytometry

(FACS analysis). Measurements were performed for the

next 15 min and all samples were kept on ice during

measurements. At least 30 000 cells were analysed with

the FACSCALIBUR and CELL QUEST PRO Software (Becton

Dickinson, Heidelberg, Germany).

Phagocytosis assay with live S. aureus

Bacteria. The S. aureus (SH1000) was kindly provided

by Dr C. Eiff22 and S. aureus was grown for 18 hr in

Mueller–Hinton bouillon at 37�. Bacterial density was

measured spectrophotometrically at 540 nm, after two

PBS washings. The number of bacterial cells was calcu-

lated using a previously determined standard curve

(based on the counts of colony-forming units). Finally,

the concentration of bacteria in PBS was adjusted to

5 · 108 cells/ml. For the purpose of the quantitative

analysis of phagocytosis by flow cytometry, S. aureus was

incubated in PBS containing 0�1% FITC (Sigma Aldrich,

Munich, Germany) for 1 hr at 37�. After being labelled,

bacteria were washed three times before incubation with

pre-treated leucocytes.

Assay. During pre-treatment, human monocytes or neu-

trophils (1 · 106 cells) were cultured in medium either

without stimuli (‘control’) or containing the following

stimuli: 100 ng/ml LPS; 1 · 10)4M PAR2-cAP, 10 ng/ml

or 100 ng/ml of IFN-c. Monocytes or neutrophils were

pre-treated for 2 hr at 37� and subsequently co-incubated

with live FITC-labelled S. aureus at a ratio of 1 : 10

(cells : S. aureus) for 30 min at 37�. During co-incubation

of leucocytes and bacteria, PAR2-cAP and IFN-c were

applied in the same order and the same concentrations as

during the pre-treatment of leucocytes by these stimuli.

The efficacy of phagocytosis was determined by FACS

analysis as described previously.23

Measurement of MCP-1 release. The inhibition of cellsignalling molecules involved in the changes ofMCP-1 release

Non-infected human neutrophils (3�75 · 106 cells) and

monocytes (3 · 106 cells) were treated with PAR2-cAP


PAR2 and anti-microbial defence of human leucocytes

and/or IFN-c for 20 or 28 hr. Cell culture supernatants

were collected and used for MCP-1 ELISA. Concentration

of MCP-1 in the cell culture supernatants was measured

with a human CCL2/MCP-1 (R&D Systems, Wiesbaden-

Nordenstadt, Germany) ELISA kit according to the man-

ufacturer’s instructions.

Specific inhibitors of intracellular signalling molecules

were used to reveal which ones are involved in the effects

of PAR2-cAP and/or IFN-c at MCP-1 secretion by human

neutrophils and monocytes. The inhibitors were used in

the following concentrations: rottlerin [inhibits protein

kinase Cd (PKCd)] 5 lM; LY294002 [inhibits phosphoino-

sitide 3 (PI3) kinase] 50 lM; SB203580 (inhibits p38

kinase) 1 lM; and JAK inhibitor I pyridone 6 (pan-JAK

inhibitor) 500 nM. All inhibitors were dissolved in DMSO,

so the vehicle DMSO (1 : 1000) was used as an additional

control. Human neutrophils and monocytes were pre-

treated with the inhibitors for 30 min and then PAR2-

cAP (1 · 10)4M) alone or in combination with IFN-c

(100 ng/ml) was applied for 28 hr (the maximum effect

of the stimuli at MCP-1 release was noticed at this time-

point). After treatment, cell culture supernatants were

collected and used to measure MCP-1 concentration by

human CCL2/MCP-1 (R&D Systems) ELISA kit.

Statistical analysis

Results are expressed as mean ± SEM. At least three inde-

pendent experiments were performed. Statistical evalua-

tion was performed by paired two-tailed Student’s t-tests.

Significance was set at P < 0�05.

Results

PAR2 agonist alone enhances the phagocytic activityof human neutrophils and monocytes, but does notaffect IFN-c-stimulated phagocytosis

Neutrophils and macrophages from PAR2-deficient mice

have been shown to display a significantly reduced phago-

cytic efficiency of Pseudomonas aeruginosa compared with

cells from wild-type animals.24 However, the ability of

PAR2 agonist to enhance the phagocytic activity of

human neutrophils and monocytes and to affect IFN-c-

stimulated phagocytosis has yet to be evaluated. To inves-

tigate whether PAR2 agonist might potentially enhance

the IFN-c-induced phagocytosis we first carried out the

phagocytosis assay with FITC-conjugated killed S. aureus.

The treatment of human neutrophils with either PAR2-

cAP (1 · 10)4M) or IFN-c (100 ng/ml) alone led to a

similar enhancement of the mean fluorescence intensity

(MFI) of human neutrophils (increased by around

40 ± 7% compared with untreated cells), indicating that

the phagocytic activity of treated neutrophils increased

(see supplementary material, Fig. S1). The combined

action of PAR2-cAP and IFN-c did not enhance the phag-

ocytic activity of neutrophils above that triggered by

either agonist acting alone (combined treatment increased

phagocytic activity by around 51 ± 12% as compared

with untreated cells) (Fig. S1). Treatment with the con-

trol peptide that cannot activate PAR2 (PAR2-cRP:

1 · 10)4M) either alone or together with IFN-c did not

affect the phagocytic activity of human neutrophils (Fig.

S1).

In our next experiments, we used live FITC-conjugated

S. aureus (strain SH1000) to investigate the effect of

PAR2-cAP alone or together with IFN-c on the phagocytic

activity of human monocytes and neutrophils against via-

ble bacteria. We found that PAR2-cAP (1 · 10)4M) or

IFN-c (100 ng/ml) alone enhanced phagocytic activity

(Fig. 1a–d; a,b for neutrophils and c,d for monocytes).

Although IFN-c already appeared to stimulate phagocytic

activity of monocytes at a concentration of 10 ng/ml,

these effects were not statistically significant (Fig. 1c,d).

Interferon-c at a higher concentration (100 ng/ml) also

enhanced phagocytic activity of human monocytes and

neutrophils. The effects of IFN-c at a concentration of

100 ng/ml reached statistical significance (Fig. 1a–d).

Stimulation with IFN-c increased the number of FITC-

positive human monocytes (49 ± 13% of change com-

pared with untreated cells) and FITC-positive human

neutrophils (41 ± 7% of change compared with untreated

cells). The MFI also increased in IFN-c-treated human

monocytes (increased by 53 ± 14%) and neutrophils

(increased by 80 ± 18%) compared with untreated con-

trols. PAR2-cAP led to an increase in the amount of

FITC-positive monocytes (increased by 35 ± 7%) and

FITC-positive neutrophils (increased by 24 ± 4%) com-

pared with untreated samples. The MFI also increased in

monocytes treated with PAR2-cAP (increased by

38 ± 8%) and in neutrophils (increased by 38 ± 4%)

compared with untreated control samples. The combined

action of PAR2-cAP and IFN-c using the same concentra-

tions did not enhance the phagocytic activity of neu-

trophils or monocytes beyond that triggered by either

agonist acting alone (Fig. 1a–d).

Interferon-c is a well-known endogenous modulator of

phagocytic bacteria killing and secretory activity of

human neutrophils and human monocytes.25,26 As an

exogenous activator, LPS also affects phagocytic activity

of both cell types. We wondered whether PAR2-cAP stim-

ulation might interfere with LPS-modulated phagocytic

activity of human neutrophils and monocytes. However,

PAR2-cAP stimulation of human neutrophils as well as

monocytes did not enhance the LPS-induced phagocytic

activity against S. aureus (see supplementary material, Fig.

S2). Hence, despite the fact that PAR2-cAP alone up-reg-

ulates the phagocytic activity of human neutrophils and

monocytes against S. aureus, this agonist failed to enhance

IFN-c-induced and LPS-induced phagocytic activity.



PAR2 agonist alone increases bacterial killing byhuman neutrophils, but does not enhanceIFN-c-stimulated bacterial killing

We next investigated whether treatment of isolated

human neutrophils with PAR2-cAP alone or in combi-

nation with IFN-c affects the bactericidal activity of

these phagocytes. In accordance with biosafety limita-

tions, we used live E. coli bacteria in our experiments to

estimate neutrophil killing activity. Human neutrophils

were pre-treated for 2 hr with PAR2-cAP or IFN-ceither alone or in combination. Either PAR2-cAP

(1 · 10)4M) or IFN-c (100 ng/ml) alone had a similar

effect on bacteria killing by human neutrophils (killing

efficacy increased by 62 ± 16% after PAR2-cAP and by

72 ± 10% after IFN-c) (Fig. 2). The PAR2 agonist and

IFN-c in combination were not more effective in stimu-

lating bacteria killing activity against E. coli than either

was alone (Fig. 2).

Stimulation with IFN-c enhances PAR2-cAP-inducedrelease of MCP-1 by human neutrophils andmonocytes

It is known that MCP-1 facilitates monocyte recruitment

to the site of bacterial infection and enhances the engulf-

ment of apoptotic neutrophils (efferocytosis), thereby

helping to resolve acute inflammation.11,14 Moreover,

neutrophils may be a source of MCP-1 in time-delayed

responses.13 We therefore studied the changes of MCP-1

secretion by human neutrophils and monocytes to reveal

the effects of the PAR2 agonist acting either alone or in

combination with IFN-c. For this experiment, neutrophils

and monocytes were treated with PAR2-cAP (1 · 10)4M),

PAR2-cRP (1 · 10)4M), or IFN-c (100 ng/ml) either

alone or in combination. We found that PAR2-cAP alone

did not lead to a notable change in MCP-1 secretion by

human neutrophils after 20 hr of treatment; the level of

secreted MCP-1 was still slightly below the threshold level

250

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ntre

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Effe

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ffect

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stim

uli (

%)

(a) (b)

(c) (d)

Figure 1. Influence of proteinase-activated receptor-2 (PAR2) agonist and interferon-c (IFN-c) stimulation on phagocytic activity of human neu-

trophils (a, b) and monocytes (c, d). Isolated leucocytes were pre-stimulated with 10)4m activating protein (cAP), 10 or 100 ng/ml IFN-c, or a

combination of IFN-c and cAP for 2 hr. Subsequently, leucocytes were co-incubated for 30 min with bacteria in the presence or absence of the

stimuli mentioned. Results are presented as average values of stimuli effects on mean fluorescence intensity (MFI) of leucocytes, which engulfed

live FITC-conjugated Staphylococcus aureus, compared with unstimulated leucocytes (mean ± SEM) (a, c). Results are presented as average values

of stimuli effects on the amount of FITC-positive leucocytes (b, d) compared with unstimulated leucocytes (mean ± SEM; Student’s t-test versus

controls: *P < 0�05; **P < 0�01). Three independent experiments were performed with human neutrophils and human monocytes.



of the ELISA (Fig. 3a). However, treatment of human

neutrophils with PAR2-cAP for 28 hr resulted in a signifi-

cant increase of MCP-1 secretion by these cells (MCP-1

level in PAR2-cAP stimulated samples was 36 ± 4 pg/ml,

but was undetectable in unstimulated control samples)

(Fig. 3b). Treatment of neutrophils with IFN-c alone did

not affect MCP-1 secretion at the 20 and 28 hr time-

points. The level of secreted MCP-1 was below the thresh-

old level of the ELISA at 20 hr and at 28 hr (Fig. 3a,b).

Surprisingly, the co-application of IFN-c with PAR2-cAP

enhanced the effect of the PAR2 agonist on MCP-1 secre-

tion 20 hr after stimulation (Fig. 3a). This effect was sta-

tistically significant even at 20 hr after stimulation

(Fig. 3a). However, this effect was even more prominent

at 28 hr (MCP-1 level was 284 ± 37 pg/ml versus 36 ±

4 pg/ml in samples treated by PAR2-cAP alone) (Fig. 3b).

Treatment with the PAR2-inactive control peptide

PAR2-cRP (1 · 10)4M) alone or together with IFN-c did

not affect MCP-1 secretion by human neutrophils

(Fig. 3a,b).

We also investigated whether treatment of human

monocytes with PAR2-cAP alone or in combination with

IFN-c affects MCP-1 secretion. Here, we measured the

level of secreted MCP-1 at 28 hr after stimulation of

human monocytes with PAR2-cAP or IFN-c alone or in

combination. We found that stimulation of human neu-

trophils for 28 hr with PAR2-cAP alone, but especially in

combination with IFN-c, led to a statistically significant

increase of MCP-1 secretion. We wondered whether

monocytes would also be responsive to such stimulation

at this time-point. Indeed, PAR2-cAP enhanced MCP-1

secretion by human monocytes (Fig. 3c). The level of

MCP-1 in untreated monocytes was 71 ± 10 pg/ml, but it

was 271 ± 60 pg/ml in monocytes treated with PAR2-

cAP. When monocytes were stimulated with IFN-c alone

0

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120

Con. cAP IFN-g IFN-g+cAP

**

**

**

Live

bac

teria

com

pare

d w

ithco

ntro

l (%

)

Figure 2. Influence of proteinase-activated receptor-2 (PAR2) agonist

and interferon-c (IFN-c) stimulation on bacteria-killing activity of

human neutrophils. Neutrophils were pre-stimulated with 10)4m

activating peptide (cAP), 100 ng/ml IFN-c, or both, for 2 hr before

incubation with living Escherichia coli. The same stimuli were further

used during incubation of neutrophils and bacteria. The number of

bacterial colonies formed after co-incubation with untreated neu-

trophils was considered to be 100%. The results are presented as an

average number of bacterial colonies formed after co-incubation with

stimulated neutrophils ± SEM (results are expressed as percentages).

Student’s t-test versus controls: **P < 0�01. Eight independent exper-

iments were performed.

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(pg

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(a)

(b)

(c)

Figure 3. Analysis of changes in monocyte chemoattractant protein

1 (MCP-1) secretion by human neutrophils and monocytes after

proteinase-activated receptor-2 activating peptide (PAR2-cAP),

PAR2-reverse peptide (cRP) and interferon-c (IFN-c) stimulation.

Neutrophils were stimulated with 10)4m cAP, 10)4

m cRP, 100 ng/

ml IFN-c alone or in combination for 20 hr (a) or 28 hr (b). Mono-

cytes were stimulated with 10)4m cAP, 100 ng/ml IFN-c alone or in

combination for 28 hr (c). The sensitivity threshold of the assay was

15�6 pg/ml. This level is marked on (a, b) by dotted lines. Results

are presented as the average values of MCP-1 secretion by unstimu-

lated (control) as well as by stimulated leucocytes in pg/ml ± SEM.

Student’s t-test versus controls: **P < 0�01; ***P < 0�005. Five inde-

pendent experiments were performed for human neutrophils and

four independent experiments for human monocytes.



MCP-1 secretion was not notably affected (Fig. 3c). How-

ever, when IFN-c and PAR2-cAP were used together,

MCP-1 secretion was enhanced significantly (1686 ±

335 pg/ml versus 271 ± 60 pg/ml in samples treated by

PAR2-cAP alone) (Fig. 3c).

The inhibition of PI3 kinase and PKCd abolishes theenhancement of MCP-1 secretion after co-stimulationof human neutrophils with IFN-c and PAR2-cAP

We next investigated which intracellular signalling mole-

cules were involved in the effects of PAR2 agonist on

MCP-1 secretion by human neutrophils, when this agonist

was applied alone or in combination with IFN-c. In these

experiments, we investigated the effects of the inhibitors of

different intracellular signalling molecules: rottlerin (inhib-

its PKCd), LY294002 (inhibits PI3 kinase), SB203580

(inhibits p38 kinase), and JAK inhibitor I pyridone 6

(inhibits JAKs). Experiments were performed with neu-

trophils treated for 28 hr with PAR2 agonist alone (PAR2-

cAP 1 · 10)4M) or in combination with IFN-c (100 ng/

ml), because the maximum effect on the MCP-1 secretion

was revealed at that time-point. We found that rottlerin

and LY294002 each completely abolished the effect of

co-application of PAR2-cAP and IFN-c on MCP-1 release

by human neutrophils (Fig. 4a). These results indicate the

crucial role of PI3 kinase and PKCd in enhancing MCP-1

secretion after co-stimulation of human neutrophils with

PAR2-cAP and IFN-c. In addition, treating neutrophils

with either pyridine 6 or SB203580 only weakened the

effect of PAR2-cAP and IFN-c on MCP-1 secretion, which

shows that p38 kinase and JAKs are involved in the com-

bined action of both agonists (Fig. 4a).

We also examined which intracellular signalling mole-

cules are involved in the enhanced secretion of MCP-1 by

human neutrophils after treatment with PAR2-cAP alone

(Fig. 4b). For these experiments, rottlerin, LY294002,

SB203580 and pyridine 6 were used to check whether PI3

kinase, PKCd, p38 kinase and JAKs were involved in the

solo effect of PAR2 agonist on MCP-1 secretion by neu-

trophils. Rottlerin, LY294002 and SB203580 abolished

PAR2-cAP-induced MCP-1 secretion (Fig. 4b), indicating

a crucial role of PI3 kinase, p38 kinase and PKCd on the

effect of PAR2 stimulation. However, pyridine 6 did not

significantly affect the changes in MCP-1 release, indicat-

ing that JAKs do not participate in the effect induced by

PAR2 agonist alone (Fig. 4b).

The inhibition of PI3 kinase and JAKs in humanmonocytes reduces the enhancement of MCP-1secretion caused by co-stimulation withPAR2-cAP and IFN-c

We also investigated whether rottlerin (inhibits PKCd),

LY294002 (inhibits PI3 kinase), SB203580 (inhibits p38

kinase), and JAK inhibitor I pyridone 6 (inhibits JAKs)

affected the induction of MCP-1 secretion after stimula-

tion of human monocytes with PAR2-cAP and IFN-c(Fig. 5a). Experiments were performed with monocytes

treated with PAR2-cAP together with IFN-c for 28 hr. We

found that LY294002 and JAK inhibitor I pyridone 6 each

reduced the effect of co-stimulation with PAR2-cAP and

IFN-c on the MCP-1 secretion by human monocytes

(MCP-1 level was 1686 ± 335 pg/ml in samples treated

with both PAR2-cAP and IFN-c, 333 ± 140 pg/ml in sam-

ples treated with LY294002, and 352 ± 121 pg/ml in sam-

ples treated with JAK inhibitor) (Fig. 5a). SB203580 had

0

50

100

150

200

250

300

350

MC

P-1

pro

tein

(pg

/ml)

Unt

reat

ed

IFN

-g+

cAP

IFN

-g+

cAP

+

IFN

-g+

cAP

+

IFN

-g+

cAP

+

IFN

-g+

cAP

+

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-g+

cAP

+LY

2940

02

rottl

erin

SB

2035

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pan

JAK

inh.

DM

SO

(a)

***

***

***

0

5

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15

20

25

30

35

40

45

MC

P-1

pro

tein

(pg

/ml)

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reat

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cAP

cAP

+LY

2940

02

cAP

+ro

ttler

in

cAP

+S

B20

3580

cAP

+pa

n JA

K in

h.

cAP

+D

MS

O

(b)

***

******

Figure 4. Inhibition of the signalling molecules during co-applica-

tion of proteinase-activated receptor-2 activating peptide (PAR2-

cAP) and interferon-c (IFN-c) (a) and during application of PAR2-

cAP alone (b) affects monocyte chemoattractant 1 (MCP-1) secretion

by human neutrophils. The following inhibitors were used: rottlerin

(protein kinase-d; PKCd) 5 lm; LY294002 [phosphoinositide-3 (PI3)

kinase] 50 lm; SB203580 (p38 kinase) 1 lm; and janus kinase (JAK)

inhibitor I pyridone 6 (pan JAK) 500 nm. Cells were pre-treated with

inhibitors and vehicle (DMSO; 1 : 1000) as described in the Materi-

als and methods, and further treated with PAR2-cAP 1 · 10)4m

together with IFN-c 100 ng/ml (a) or with PAR2-cAP 1 · 10)4m

alone (b) for 28 hr. Human MCP-1 level in collected samples was

determined by ELISA. Results are presented as the average values of

MCP-1 secretion by unstimulated, as well as by stimulated neutroph-

ils in pg/ml ± SEM. Student’s pairwise t-test: *P < 0�05, **P < 0�01,

***P < 0�005. Four independent experiments were performed.



no effect on MCP-1 secretion by human monocytes

(Fig. 5a). Surprisingly, rottlerin enhanced the effect of

co-stimulation with PAR2-cAP and IFN-c on MCP-1

secretion by monocytes (Fig. 5a) and also enhanced

PAR2-cAP-induced MCP-1 release when PAR2 agonist

was used alone (Fig. 5b). However, rottlerin did not affect

MCP-1 levels in IFN-c stimulated cells (data not shown).

We were also interested in whether rottlerin alone might

affect MCP-1 secretion by human monocytes and found

that it did increase secretion (Fig. 5c). SB203580 and

JAK inhibitor each did not affect MCP-1 secretion trig-

gered by PAR2-cAP (Fig. 5b). LY294002 slightly reduced

the effect of PAR2-cAP stimulation on MCP-1 secretion

by human monocytes (the level of MCP-1 secretion

after PAR2-cAP application was 271 ± 60 pg/ml and if

LY294002 was also added, the level of MCP-1 was

154 ± 72 pg/ml) (Fig. 5b). In all cases, treatment of

monocytes with DMSO did not affect MCP-1 secretion

(Fig. 5a–c).

Discussion

The most important finding of our study is that PAR2 acti-

vation enhances phagocytic activity against Gram-positive

(S. aureus) bacteria and the killing of Gram-

negative (E. coli) bacteria by human leucocytes. The

magnitude of the bactericidal effect induced by PAR2 ago-

nist was similar to that induced by IFN-c (Figs 1 and 2; see

supplementary material, Fig. S1). Since PAR2 agonist can

synergize with IFN-c in enhancing anti-viral responses,8,9

we investigated whether co-application of PAR2-cAP and

IFN-c led to stronger anti-bacterial responses of innate

immune cells, but found that the response was no greater

than when each compound was used alone (Figs 1 and 2;

Fig. S1). In addition, PAR2 agonist stimulation also failed to

enhance LPS-stimulated phagocytic activity of neutrophils

and monocytes (see supplementary material, Fig. S2).

Hence, PAR2 stimulation might trigger additional mecha-

nisms that enhance the phagocytic activity of innate

3500

3000

2500

2000

1500

1000

800

600

400

200

0

1200

1000

500

0

MC

P-1

pro

tein

(pg

/ml)

3500

3000

2500

2000

1500

1000

500

0

MC

P-1

pro

tein

(pg

/ml)

MC

P-1

pro

tein

(pg

/ml)

Unt

reat

ed

Unt

reat

ed

IFN

-g+

cAP

IFN

-g+

cAP

+

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-g+

cAP

+

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-g+

cAP

+

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-g+

cAP

+

IFN

-g+

cAP

+

LY29

4002

LY29

4002

LY29

4002

rottl

erin

rottl

erin

rottl

erin

SB

2035

80

SB

2035

80

SB

2035

80

pan

JAK

inh.

pan

JAK

inh.

pan

JAK

inh.

DM

SO

DM

SO

DM

SO

cAP

+

cAP

+

cAP

+

cAP

+

cAP

+

cAP

Unt

reat

ed

# #

(a) (b)

(c)

Figure 5. Inhibition of the signalling molecules during co-application of proteinase-activated receptor-2 activating peptide (PAR2-cAP) and inter-

feron-c (IFN-c) (a) and during application of PAR2-cAP alone (b) affects monocyte chemoattractant protein 1 (MCP-1) secretion by human

monocytes. In addition, the effects of inhibitors of the signalling molecules alone on MCP-1 secretion were checked (c). The following inhibitors

were used: rottlerin (protein kinase d; PKCd) 5 lm; LY294002 [phosphoinositide 3 (PI3) kinase] 50 lm; SB203580 (p38 kinase) 1 lm; and janus

kinase (JAK) inhibitor I pyridone 6 (pan JAK) 500 nm. Cells were pre-treated with inhibitors and vehicle (DMSO; 1 : 1000) as described in the

Materials and methods, and further treated with PAR2-cAP 1 · 10)4m together with IFN-c 100 ng/ml (a) or with PAR2-cAP 1 · 10)4

m alone

(b) for 28 hr. Human MCP-1 level in collected samples was determined by ELISA. Results are presented as the average values of MCP-1 secretion

by unstimulated, as well as by stimulated monocytes in pg/ml ± SEM. Student’s pairwise t-test: #P < 0�05. Four independent experiments were

performed.



immune cells, and these mechanisms do not synergize with

IFN-c or LPS-triggered ones. Unfortunately, it remains

problematic to investigate whether the classic PAR2 activa-

tors trypsin and tryptase can affect phagocytic and bacteria-

killing activity of human innate immune cells. Trypsin and

tryptase are known to induce PAR-independent effects.5,6

These effects could confound the data obtained using these

enzymes as PAR2 agonists.

Cytokines and chemokines influence the recruitment of

phagocytes to the site of pathogen infection. The PAR2

agonists reportedly affect the secretion of IFN-inducible

protein-10, IL-8, IL-6 and IL-1b by human neutrophils,

monocytes and endothelial cells.8,10,27 Among chemokin-

es, MCP-1 appears to play a distinct role linking

neutrophils and monocytes during time-delayed inflam-

matory response, and helping to resolve inflammation via

activation of efferocytosis.14 In addition, IFN-c reportedly

enhances time-delayed MCP-1 secretion by human neu-

trophils.13 In our study, we investigated whether PAR2

activation interferes or synergizes with IFN-c-enhanced

MCP-1 secretion. We found that MCP-1 secretion by

human neutrophils and monocytes was enhanced 28 hr

after stimulation with PAR2-cAP (Fig. 3). Moreover, the

treatment of human neutrophils and monocytes with

IFN-c together with PAR2-cAP resulted in a synergistic

action of these agents, and so enhanced secretion of

MCP-1 by innate immune cells (Fig. 3). These findings

indicate that the combination of PAR2-cAP and IFN-c is

apparently effective at enhancing secretion of MCP-1 by

human neutrophils and monocytes.

In our study, we were interested in which intracellular

signalling molecules were involved in the synergetic action

of PAR2-cAP and IFN-c on MCP-1 secretion by human

neutrophils and monocytes. Several signalling molecules

are known to be involved in the regulation of MCP-1

secretion. For example, a serine protease plasmin induces

MCP-1 expression in human monocytes via activation of

p38 kinase and JAK/signal transducer and activator of

transcription (STAT) pathways.28 Inhibitors of PI3 kinase

attenuate IFN-c-induced expression of MCP-1 in macro-

phages.29 Moreover, IFN-c-induced activation of PI3

kinase results in down-stream activation of PKCd.30 Con-

versely, PAR2 induces some effects via signalling cascades

involving PI3 kinase and PKCd.31 Altogether, these facts

led us to hypothesize that p38 kinase, PI3 kinase, PKCdand JAKs were involved in the synergistic effect of PAR2

agonist and IFN-c on MCP-1 secretion by human mono-

cytes and neutrophils. Indeed, our experiments with

inhibitors of these signalling molecules indicate that they

all participate in synergistic effects of PAR2-cAP and IFN-

c on MCP-1 secretion by human neutrophils (Fig. 4a).

Our results show that the enhanced effect of combined

PAR2-cAP and IFN-c treatment on MCP-1 secretion by

human neutrophils appears to be associated with the sig-

nalling pathway JAK–PI3K–PKCd (Fig. 6a). Possibly,

STAT1 could be the next participant in this pathway in

neutrophils. Interferon-c is known to activate the PI3K–

PKCd axis, and activated PKCd, in turn, affects STAT1

phosphorylation.30 The PKCd is involved in a dual mech-

anism by which it participates in regulating IFN-depen-

dent responses: (i) via STAT1 phosphorylation and (ii)

via p38 mitogen-activated protein (MAP) kinase activa-

tion.32 The results of our study strongly suggest that

PKCd is the upstream activator of p38 MAP kinase dur-

ing combined action of PAR2-cAP and IFN-c on MCP-1

secretion by human neutrophils. We found that PKCdinhibition abolished the effect of co-application of PAR2-

cAP and IFN-c on MCP-1 secretion, but that p38 MAP

kinase inhibitor just weakened MCP-1 secretion by

human neutrophils (Fig. 4a). In addition, we found that

the PI3K–PKCd axis plays a crucial role for MCP-1 secre-

tion by human neutrophils stimulated with PAR2-cAP

alone (Fig. 4b).

However, our study shows that human monocytes

demonstrate not only similarities, but also differences, in

the set of signalling molecules involved in the synergistic

effect of PAR2-cAP and IFN-c on MCP-1 secretion

(Fig. 6b). The inhibition of PI3K and JAKs reduced, but

did not abolish, the enhanced MCP-1 secretion, which

was induced after monocytes were treated with PAR2-cAP

together with IFN-c (Fig. 5a). This reduced level of

secreted MCP-1 was similar to the level reached after

monocytes were stimulated with PAR2-cAP alone

(Fig. 5a,b). These data indicate that PAR2-cAP effects on

IFN-γ IFN-γ

JAK JAK

PAR2-cAP PAR2-cAP

PI3K

PKC

p38 kinase

MCP-1 release MCP-1 release

?

PI3K ?

(a) (b)

Figure 6. Suggested signalling events involved in the effects induced

by combined treatment with proteinase-activated receptor-2 activat-

ing peptide (PAR2-cAP) and interferon-c (IFN-c) on monocyte

chemoattractant protein 1 (MCP-1) secretion by human neutrophils

(a) and monocytes (b). Experiments with selective inhibitors of sig-

nalling molecules led us to suggest that the phosphoinositide 3

kinase–protein kinase d (PI3K–PKCd) axis plays a key role in

changes of MCP-1 secretion induced by co-application of PAR2-cAP

and IFN-c in human neutrophils (a). However, the inhibition of the

PI3K molecule in human monocytes just weakened the effect of

combined PAR2-cAP and IFN-c application on MCP-1 secretion.

This indicates the involvement of the PI3K-independent pathway in

the effect of PAR2-cAP and IFN-c co-application on MCP-1 secre-

tion by monocytes (b).



MCP-1 secretion by human monocytes are mediated not

only via a signalling pathway involving PI3K activation,

but also via another pathway (Fig. 6b). Surprisingly, the

PKCd inhibitor rottlerin enhanced the effect of PAR2-cAP

and IFN-c on MCP-1 release by monocytes (Fig. 5a). Rot-

tlerin also synergized with PAR2-cAP in its action on

MCP-1 secretion (Fig. 5b). Moreover, rottlerin, when

applied alone, enhanced MCP-1 secretion by human

monocytes (Fig. 5c). Treatment with the p38 inhibitor

SB203580 did not influence the increased MCP-1 secre-

tion caused by either PAR2-cAP stimulation or combined

application of PAR2-cAP and IFN-c (Fig. 5a,b). The levels

of secreted MCP-1 after IFN-c stimulation were below

the threshold in the neutrophil samples and could there-

fore not be determined (Fig. 3a,b). The treatment of

human monocytes with IFN-c yielded no significant

changes in MCP-1 levels (Fig. 3c). Hence, the effects of

the inhibitors of signalling molecules at MCP-1 release

were not studied after IFN-c stimulation of human

monocytes and neutrophils. Altogether, the results of our

experiments allowed us to suggest a possible scheme of

signalling events involved in the enhancement of MCP-1

secretion triggered after combined stimulation of human

neutrophils and monocytes with PAR2-cAP and IFN-c(Fig. 6a,b).

In summary, our study demonstrates that PAR2 agonist

acting alone can enhance a bactericidal response of human

neutrophils and monocytes in vitro. However, PAR2

agonist is unable to synergize with IFN-c in the enhance-

ment of the bactericidal response. On the other hand,

PAR2 agonist and IFN-c do synergize to increase MCP-1

secretion by human neutrophils and monocytes during

the late phase (after 24 hr) of the inflammatory response.

This synergistic action of PAR2 agonist and IFN-c on

MCP-1 release apparently involves the activation of PI3

kinase and JAKs in neutrophils and monocytes.

Acknowledgements

The work was supported by grants from the IZKF Mun-

ster (Stei3/034/09), German Research Foundation (SFB

293-A14, STE 1014/2-2), CERIES (Paris), Weston Haven

foundation San Francisco USA (to M.S.), SFB 293 (S.L.),

IMF grant SH 120709 (University of Munster, Germany)

(to V.M.S.), IMF grant FE 110905 (University of Munster,

Germany) (to M.F.) as well as Canadian Institutes of

Health Research (Operating and Proteinases and Inflam-

mation Network grants to M.D.H.), Transregional Collab-

orative Research Centre 34 (C12) (to D.H. and J.R.) and

IMF grant HO 220912 (University of Munster, Germany)

(to D.H.). The position of V.M.S at the Department of

Nephrology and Hypertension, University of Magdeburg,

Germany was supported by German Research Foundation

SFB 854 (SFB854 TP1). The authors are deeply grateful to

Pamela Derish for excellent editorial work.

Disclosures

The authors have no financial conflict of interest.

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Supporting information

Additional Supporting Information may be found in the

online version of this article:

Figure S1. Influence of proteinase-activated receptor-2

(PAR2) agonist and interferon-c (IFN-c) stimulation on

phagocytic activity of human neutrophils against killed

FITC-conjugated Staphylococcus aureus. Isolated neutroph-

ils were pre-stimulated with 10)4M activating peptide

(cAP), 10)4M reverse peptide (cRP), or 100 ng/ml IFN-c,

or a combination of IFN-c and cAP or cRP for 2 hr.

Figure S2. Influence of proteinase-activated receptor-

2(PAR2) agonist and lipopolysaccharide (LPS) stimulation

on phagocytic activity of human neutrophils (a, b) and

monocytes (c, d). Isolated leucocytes were pre-stimulated

with 10)4M activating peptide (cAP), or 100 ng/ml LPS,

or a combination of LPS and cAP for 2 hr. Subsequently,

leucocytes were co-incubated for 30 min with bacteria in

the presence or absence of the stimuli mentioned.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.



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Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes