Received: 6 October 2017 Revised: 22March 2018 Accepted: 22March 2018

DOI: 10.1002/JLB.3A1017-398RR

ART I C L E

𝜷2-Adrenoceptors inhibit neutrophil extracellular traps inhuman polymorphonuclear leukocytes

FrancaMarino1 Angela Scanzano1 Laura Pulze2 Monica Pinoli1

Emanuela Rasini1 Alessandra Luini1 Raffaella Bombelli1 Massimiliano Legnaro1

Magda de Eguileor2 Marco Cosentino1

1Center of Research inMedical Pharmacology,

Varese, Italy

2Department of Biotechnology and Life

Sciences, University of Insubria, Varese, Italy

Correspondence

FrancaMarino, PhD,Center forResearch in

Medical Pharmacology,University of Insubria,

Via,OttorinoRossi n. 9, 21100VareseVA, Italy.

Email: franca.marino@uninsubria.it

AbstractThis study tests the hypothesis that in isolated human polymorphonuclear leukocytes (PMN)

adrenergic ligands can affect neutrophil extracellular trap (NET) formation. We have previously

shown that, in PMN, adrenaline (A), through the activation of adrenergic receptors (AR), reduces

stimulus-dependent cell activation; we have, therefore, planned to investigate if AR are involved

inNETproduction. PMNwereobtained fromvenousbloodof healthy subject. The ability of adren-

ergic ligands to affect reactive oxygen species (ROS) production, NET production, and cell migra-

tion was investigated in cells cultured under resting conditions or after activation with N-formyl-

methionyl-leucyl-phenylalanine (fMLP), LPS, or IL-8. Stimuli-induced NET production measured

as ROS, microscopic evaluation, and elastase production was reverted by A and this effect was

blocked by the selective 𝛽2–AR antagonist ICI-118,551. The stimulus-induced ROS generation

andmigrationwas prevented by A and by isoprenaline (ISO), and these effects were counteracted

only by ICI-118,551 and not by the other two selective ligands for the 𝛽1 and 𝛽3–AR. Finally, the

presence of the 𝛽–ARs on PMNwas confirmed, by means of microscopy and flow cytometry. The

data of the present study suggest that adrenergic compounds, through the interaction of mainly

𝛽2–AR, are able to affect neutrophil functions. These data are suggestive of a possible therapeutic

role of 𝛽2–AR ligands (in addition to their classical use), promoting the possible therapeutic rele-

vance of adrenergic system in the modulation of innate immunity proposing their possible use as

anti-inflammatory drugs.

K EYWORDS

adrenergic receptor, adrenaline, neutrophils, neutrophil extracellular traps, reactive oxygen

species

1 INTRODUCTION

Polymorphonuclear neutrophils (PMN) are usually considered as the

first line of defense against invading microorganisms and as contribu-

tors to the orchestrated response after tissue invasion by pathogens.

PMN are able to migrate into inflamed tissues, and in loco provide

to the immune response through a number of different mechanisms,

including adhesion to endothelial layers, migration into tissues, and

subsequently phagocytosis, production of reactive oxygen species

(ROS) aswell as other pro-inflammatorymediators suchas chemokines

and cytokines, degranulation leading to release of neutrophil elastase

(NE) ormetalloproteases (MP), and formation of neutrophil extracellu-

lar traps (NET).1,2

During the last 3 decades, however, the involvement of these cells

in non-infectious diseases has been progressively unveiled, and now

PMN are considered pivotal cells in a wide range of inflammatory

diseases as, for example, cardiovascular and autoimmune diseases.3,4

In particular, although a key product of PMN, the NET, play a key

role in the killing of pathogens,1 compelling evidence indicates that

they are also involved in other clinical conditions such as auto-

immune diseases.2

Ultrastructural analysis shows that NET composition mainly

consists of nuclear chromatin, together with granular and cyto-

plasmic proteins.3 In particular, the presence inside the NET of

myeloperoxidase (MPO), NE, and cathepsin G suggests their involve-

ment in different inflammatory diseases, including cardiovascular

J Leukoc Biol. 2018;1–12. c©2018 Society for Leukocyte Biology 1www.jleukbio.org

2 MARINO ET AL.

diseases (CVD)5; in fact, MPO and NE are present in human

atherosclerotic plaques and contribute to their destabilization

and rupture.6,7 NET are also involved in auto-inflammatory diseases

such as systemic lupus erythematosus, Wegener’s granulomatosis,

and psoriasis.4,8,9

The catecholamines (CA) noradrenaline (NA), adrenaline (A), and

dopamine (DA) in addition to being key neurotransmitters and neu-

rohormones in the brain and in peripheral tissues are also trans-

mitters connecting the nervous and the immune system.10–14 Nerve

fibers positive for tyrosine hydroxylase (TH; enzyme limiting the syn-

thesis of CA) extensively innervate lymphoid organs.15,16 TH is also

expressed in immune cells,17,18 where it subserves endogenous pro-

duction of CA, which in turn affect immune cell functions.17,19 CA

can modulate hematopoiesis20–22 and their content in immune cells is

affected by several diseases19,23; cells of acquired and innate immu-

nity express both adrenergic receptors (AR) and dopaminergic recep-

tors (DR) and it has been shown that the activation of these recep-

tors results in changes of different cell functions.19,24–27 Several

studies have shown that these receptors are involved in immune-

mediated diseases.23,28,29

We have previously shown that, during activation with pro-

inflammatory stimuli, human PMN undergo morphological and func-

tional changes,30–35 including changes in AR expression.27 We have

also shown that these changes are followed by increased produc-

tion of chemokines, such as IL-8,30,31 and increased generation of

ROS.27 Finally, we have shown that these functional and morphologi-

cal changes can be prevented by pharmacological treatments31,33 and

involve the activation of specific AR-operated pathways.27

The aim of the present study was to investigate the ability of

adrenergic agents to affect NET production in human PMN and to

characterize the AR pathways eventually involved. Since in previ-

ous studies,27 we have identified a key role by 𝛽-AR-operated path-

ways, in the present study, we have particularly explored the ability

of A and of selective 𝛽-AR ligands to affect the NET production as

well as migration and ROS generation, two key functions involved in

the PMN-dependent tissue protection. Finally, by means of fluores-

cence microscopy and flow cytometry, we have investigated the 𝛽-

AR expression and the changes occurring following the exposition to

activating stimuli.

2 MATERIALS AND METHODS

2.1 PMN isolation

Experiments were performed on buffy coats of healthy subjects

obtained by the local blood bank (Circolo Hospital, Fondazione Mac-

chi, Varese, Italy). PMN were isolated by standard density-gradient

centrifugation as previously described.27 Cells were examined using

light microscopy in order to exclude the presence of platelets or

erythrocytes. Cell purity and viability were always assessed either

by light microscopic examination or by flow cytometric analysis and

experimentswereperformedonly in cellswhich presented apurity and

viability higher than 95%.

2.2 Cell culture

Cellswere cultured, according to our previous studies,27,34 under stan-

dard conditions, in RPMI medium, and subsequently, allowing to the

experimental protocols, harvested for the following analysis.

After harvesting, cells were stained with trypan blue and viability

was assessed by an automatic cell counter “Cellometer Auto T4” (Nex-

celomBsioscience LLC, Lawrence, MA, USA).

Stimuli employed to induce NET formation included (i) the chemo-

tactic peptide N-formyl-Met-Leu-Phe (fMLP, 0.1 𝜇M), which acts on

membrane receptors resulting in PMN activation36; (ii) LPS (1 𝜇g/ml;

extracted from E. coli serotype O127:B8), a strong inducer of NET and

able to induces an inflammatory response through the activationof dif-

ferent receptors, in particular the TLR437; and (iii) IL-8 (100 ng/mL),

a key physiological activator of PMN which induces extensive

NET-osis.3

2.3 Cell apoptosis

To evaluate the effect of A (1 nM–1 𝜇M) on PMN viability, cells

were cultured for 30 min or 3 h, then samples were recovered and

centrifuged (600 × g, 5 min, room temperature) to remove super-

natant, and washed with 1 mL of PBS. Apoptosis was evaluated by

means of flow cytofluorimetric assay using an FITC Annexin V detec-

tion Kit I (Becton Dickinson, Milan, Italy) according to the manufac-

turer’s instructions. Briefly, the cells were resuspended in 100 𝜇L of

Annexin V Binding Buffer (present in the kit) and stained with 5 𝜇L

of FITC-conjugated Annexin V (ANX-FITC) and 5 𝜇L of propidium

iodide staining solution (PI) in the dark for 15 min. After the incu-

bation, 250 𝜇L of binding buffer was added and samples were ana-

lyzed by a BD FACSCanto II Flow Cytometer (Becton Dickinson Italy,

Milano, Italy) and data were analyzed using BD FACSDiva software

(version 6.1.3). PMNwere identified based on FSC and SSC properties,

and at least 15.000 events were collected from each sample. Viable

(ANX−/PI−), early apoptotic (ANX+/PI−), late apoptotic (ANX+/PI+),necrotic (ANX−/PI+) PMNwere identifiedonabiparametric plotANX-

FITC versus PI with a log scale.

2.4 Morphological and quantitative evaluation of

NET production

2.4.1 ROS generation

Intracellular ROS levels were assessed in PMN under resting or acti-

vated conditions by using the redox sensitive dye C-DCDHF-DA

(Molecular Probes, Eugene, OR, USA) as previously described.27 Flu-

orescence was detected by means of a spectrofluorimeter (Perkin-

Elmer LS-50B, Perkin-Elmer Instruments, Bridgeport, CT, USA), with

excitationwavelength of 488 nm. Fluorescence emissionwas collected

at 525 nm and intracellular ROS levels were finally expressed as differ-

ence (Δ) between resting values measured at 60 s and after 30 min or

3 hmonitoring as area under the curve (AUC) of the complete intervals

of measurement. In particular, AUC represents the final AUC obtained

after the measurement of the complete 30 min (or 3 h) analysis, while

the delta represents the peak difference between values measured

MARINO ET AL. 3

after 60 s analysis and the final value reached after 30 min (or 3 h)

of evaluation.

The different time of data recording (30 or 180 min) were selected

according to the experimental procedures. In particular, NET-related

ROS evaluation were recorded for 180 min according to the other

procedures used to evaluate NET that were optical microscopy and

immunocytochemical characterization of myeloperoxidase expression

and elastase production (all of these data were recorded for 180 min).

On the contrary, the ability of adrenergic ligands to affect ROS gen-

eration through the activation of 𝛽-AR was evaluated during 30 min

according to our previous study.27

2.4.2 Optical microscopy

Isolated PMNwere seeded at the concentration of 8× 104 cells/mL on

rounded glass coverslips (12 mm diameter, treated with 0.1% gelatin)

in RPMI-1640 medium and incubated for 3 h at 37◦C under resting

or activated conditions. After the incubation, cells were fixed with

4% paraformaldehyde (PFA) in PBS. In order to evaluate the pres-

ence of NET, PMN were stained with crystal violet and basic fuchsin.

Coverslips were mounted in Eukitt (medium for microscopy) and cells

were observed under a light microscope (Eclipse Nikon, Amsterdam,

the Netherlands).

2.4.3 Immunocytochemical characterization of

myeloperoxidase expression

PMN, cultured as above described for optical microscopy, were fixed

with 4%PFA on rounded glass coverslips and pre-incubated for 30min

with a blocking/permeabilizing solution made of PBS containing 2%

BSA (Sigma–Aldrich, Milan) and 0.1% Tween20 (Sigma–Aldrich). The

presence of MPO was assessed using anti-human MPO monoclonal

primary Ab (1:100 dilution; Cell Signaling Technology, the Nether-

lands). Incubation with appropriate secondary Ab conjugated with

cyanin3 (anti-rabbitCy31:300dilution, ThermoFisher Scientific,USA)

was performed for 1 h in a dark humid chamber at room temperature.

Nuclei were counterstained with DAPI (Sigma–Aldrich). All the anti-

bodies were diluted in blocking solution. In control samples, primary

Ab was omitted and cells were treated with BSA/Tween20 containing

PBS. Coverslips were mounted in CitiFluor (CitiFluor Ltd, London, UK)

and slides were observed under an Eclipse Nikonmicroscope.

2.4.4 Evaluation of NET elastase activity

PMNwere cultured for 3 h (5%CO2 and 37◦C) in RPMI 1640 enriched

with 1% BSA and 0.1% calcium chloride (NET buffer) and stimulated

as appropriate. At the end of culture, cells were washed with NET

buffer; NET DNA was digested by S7 nuclease (Roche 1010721001

from Sigma–Aldrich, Merck Group; 15 U/mL) for 15 min at 37◦C. The

nuclease was inactivated using EDTA (0.5 M). The suspension was

centrifuged and the supernatant was added to the elastase substrate

N-methoxysuccinyl-Ala-Ala-Pro-Val- p-nitroanilide (Cayman 17601–

50 from Vinci-Biochem srl, Florence, I; 0.52 mM), which is selectively

cleaved by elastase to yield a 4-nitroaniline product. The absorbance

was measured at 405 nm (spectrophotometric assay), and NET

elastase production was finally quantified by plotting the absorbance

versus a standard curve of elastase at different concentrations (Vinci-

Biochem srl, Florence, I; 0–36 mU/mL) and expressed as mU/mL con-

tent of the product.

2.5 Cell migration

Cell migration wasmeasured by a Boyden Chamber assay with modifi-

cations, as previously described.27 Migration was quantified by means

of optical microscopy, by measuring the distance (𝜇m) from the sur-

face of the filter to the leading front of cells. IL-8 (100 ng/mL) was used

as stimulus for cell migration (and added to the lower chamber) and

migration wasmeasured after 90min of incubation of PMN alone or in

the presence of IL-8 (and adrenergic ligands as appropriate, according

to the experimental design).

2.6 Characterization of 𝜷-AR expression on human

PMN

2.6.1 Real time PCR evaluation ofmRNA expression

𝛽-AR mRNA expression was evaluated in isolated PMN cultured for

3 h in the alone or in the presence of IL-8 or LPS. Total mRNA was

extracted from1× 106 cells by PerfectPure RNACell KitTM (Hamburg,

Germany) and quantity and quality of RNA extractedwas estimated by

spectrophotometrywith the 260/280 nm ratio. Total RNAwas reverse

transcribed using the high-capacity cDNAArchive Kit (Applied Biosys-

tems, Foster City, CA, USA) according to the manufacturer’s instruc-

tions and Real-time PCR was performed (StepOne R© System; Applied

Biosystems) using assay-on-demand kits. Gene expression level in a

given sample was represented as 2−ΔCt where ΔCt = [Ct (sample)

- Ct (housekeeping gene)]. Relative expression was determined by

normalization to 18S cDNA (analyzed by StepOne softwareTM 2.2.2,

Applied Biosystems).

2.6.2 Immunocytochemical characterization of 𝜷-AR

expression

PMN, fixed with 4% PFA on rounded glass coverslips, were pre-

incubated for 30 min with a blocking/permeabilizing solution made of

PBS containing 2% BSA (Sigma–Aldrich) and 0.1% Tween20 (Sigma–

Aldrich). The presence of the different 𝛽-ARwas assessed using the fol-

lowing primary Abs: anti-human 𝛽1-AR polyclonal Ab (1:100 dilution;

LifeSpan Biosciences, USA), anti-human 𝛽2−AR mAb (1:100 dilution;

Abcam, UK), anti-human 𝛽3-AR polyclonal Ab (1:100 dilution; Novus

Biologicals, USA). Incubations with appropriate secondary antibod-

ies conjugated with cyanin 3 (anti-rabbit Cy3 [1:300 dilution, Thermo

Fisher Scientific, USA]; anti-mouse Cy3 (1:200 dilution [KPL, USA])

were performed for 1 h in a dark humid chamber at room temperature.

Nuclei were counterstained with DAPI (Sigma–Aldrich, Milan). All the

antibodies were diluted in blocking solution. In control samples, pri-

maryAbswereomitted and cellswere treatedwithBSA/Tween20 con-

taining PBS. Coverslips were mounted in CitiFluor (CitiFluor Ltd, Lon-

don, UK) and slideswere observed under an EclipseNikonmicroscope.

4 MARINO ET AL.

2.6.3 Flow cytometric evaluation of 𝜷-AR

Expression of 𝛽1− and 𝛽2−AR on PMN was assessed in whole blood

samples, either under resting conditions or after stimulation with

fMLP, LPS, or IL-8, by using an indirect immunofluorescence stain-

ing and flow cytometry. Cells were first labeled with the following

primary Abs (first Ab): anti-human rabbit polyclonal Ab recognizing

an extracellular epitope of 𝛽1-AR (anti-𝛽1-AR, LifeSpan Biosciences,

USA) or anti-human mouse mAb directed against an extracellular epi-

tope of 𝛽2-AR (anti-𝛽2-AR, Abcam, UK), respectively. This step was fol-

lowed by a second labeling with fluorochrome-conjugated secondary

antibodies (second Ab): AlexaFluor647-conjugate F(ab’)2 donkey anti-

rabbit IgG specific for anti-𝛽1-AR (DAR-AF647, Biolegend, USA) and

eFluor660-conjugatedF(ab’)2 goat anti-mouse IgGspecific for anti-𝛽2-

AR (GAM-eFluor660, eBioscience-Invitrogen, USA). For PMN stimula-

tion the following procedure was performed: from each whole blood

sample, aliquots of 50 𝜇L were diluted with 950 𝜇L RPMI medium and

incubated for 30min and 3 h at 37◦C in amoist atmosphere of 5%CO2,

aloneorwith oneof the following stimuli: fMLP, LPS, or IL-8 (at the con-

centrations above detailed). At the end of the incubation time, samples

were centrifuged at 600 × g for 5 min at room temperature and pro-

cessed for the 𝛽-AR staining. To this end, blood samples were treated

with 3mLof a lysis buffer containing (g/L)NH4Cl (8.248), KHCO3 (1.0),

and EDTA (0.0368) in order to remove the erythrocytes and incubated

for 5 min at room temperature, during which the samples were gently

vortexed. After one centrifugation at 600 × g for 5 min, supernatants

were removed and the leukocytes were thenwashed once in 1mL PBS

(pH 7.4) and finally resuspended in 100 𝜇L PBS supplemented with 1%

BSA (PBS/BSA). Before first Ab labeling, the cells were incubated with

5 𝜇L of Human TruStain FcX-Fc Blocking Solution (Biolegend USA) for

10 min at room temperature to prevent unspecific binding to human

FcRs expressed on PMN. After this incubation and without washing

the cells, the labeling of the first Ab was performed by incubating the

samples with 10 𝜇L of anti-𝛽1-AR (final dilution 1:100) or anti-𝛽2-AR

(final dilution 1:50) for 30min at room temperature. The samples were

then washed with 1 mL PBS/BSA, centrifuged, resuspended in 100 𝜇L

PBS/BSA, and second Ab DAR-AF647 or GAM-eFluor660 (final dilu-

tion 1:400) were added and incubated for 30min at room temperature

in the dark. After the end of staining, the samples were washed again,

resuspended in 400 𝜇L PBS and kept on ice until acquisition. In each

evaluation, appropriate negative control samples in which primary Ab

were omitted were included. Acquisition and analysis were performed

on a BD FACSCanto II flow cytometer (Becton Dickinson Italy, Milan,

Italy) with BD FACSDiva software (version 6.1.3). PMN were identi-

fied by their classical morphological parameters by using a forward-

scatter (FSC) versus a side-scatter (SSC) biparametric dot-plot and at

least 20,000 cells from each sample were collected in the gate. Data

were analyzedwithBDFACSDiva software and resultswereexpressed

as both % of granulocytes positive for 𝛽1- and 𝛽2-AR, and ratio of the

median fluorescence intensity (MFI), corresponding to specific staining

for each 𝛽-AR, overMFI of a negative control (second Ab alone).

2.7 Statistical analysis

Data are presented asmeans± SEM or SD,with n indicating the number

of observations, as appropriate. Gaussian distributionwas analyzed by

means of D’Agostino & Pearson omnibus normality test. Parametric

continuous variables were compared by means of Mann–Whitney or

Wilcoxon test and statistical significancewas set at P< 0.05. Statistical

significance of concentration-response curve was evaluated by means

of one-way-ANOVA followed by t-test. Calculations were performed

using a commercial software (GraphPad Prism version 5.00 for Win-

dows, GraphPad Software, San Diego, CA, USA, www.graphpad.com).

3 RESULTS

3.1 Apoptosis

As shown in Table 1, culture of isolated PMN in the presence of differ-

ent concentrations ofA (1 nM–1𝜇M)did not induce appreciable differ-

ences in term of viability (ANX-/PI−), early apoptosis (ANX+/PI−), lateapoptosis (ANX+/PI+), and necrosis (ANX−/PI+; Table 1).

3.2 Morphological and quantitative evaluation of

NET production

ROS generation is functionally related with NET production.38 We

have investigated the ability of A to modulate NET production. To

this end, we have measured NET production through the evalua-

tion of ROS generation, optical microscopic evaluation of cell mor-

phology, MPO expression, elastase activity, and all these parameters

were analyzed after incubation of cells for 3 h with the different

activating stimuli.

TABLE 1 Effect of A on PMNviability (ANX-/PI−), early apoptosis (ANX+/PI−), late apoptosis (ANX+/PI+), and necrosis (ANX−/PI+) measuredby flow cytometry. PMNwere cultured for 30 min or 3 h alone or in the presence of A (10 nM–1 𝜇M). Data are expressed as percent of total cellsand are represented asmean± SD of 6 separate experiments

30min (% of positive cells) 3 h (% of positive cells)

ANX−/PI− ANX+ /PI- ANX+/PI+ ANX-/PI+ ANX−/PI− ANX+/PI− ANX+/PI+ ANX-/PI+

Control 84.6± 7.7 12.1± 7.3 1.7± 0.7 1.7± 1.0 81.9± 9.3 15.8± 9.3 1.6± 0.7 0.7± 0.6

A 10 nM 82.7± 9.3 13.3± 8.5 2.1± 0.8 1.9± 1.0 81.5± 7.1 15.6± 6.3 2.0± 1.1 0.9± 1.1

A 100 nM 83.2± 8.6 12.5± 7.4 2.1± 1.1 2.2± 2.1 81.7± 7.6 15.4± 6.6 2.0± 1.1 0.9± 1.0

A 1 𝜇M 81.3± 11.3 15.0± 9.9 2.0± 1.0 1.7± 0.9 80.3± 7.8 16.6± 6.4 2.2± 1.6 0.9± 1.1

MARINO ET AL. 5

F IGURE 1 Effect of A on NET-related ROSgeneration in isolated PMN. 3 h ROS generationexpressed as AUC (left panel) or Δ (right panel)in human PMN cultured alone (empty columns)or in the presence of A (hatched columns) underresting conditions (R) or during activation withfMLP, IL-8, and LPS. Data are presented asmean ± SEM of at least 3–6 separate experi-ments. Statistical analysis was performed usingMann–Whitney test and “D’Agostino & Pearsonomnibus normality test. *P < 0.05 and **P < 0.01versus absence of A; §versus R conditions

3.2.1 NET-related ROS generation

As shown in Fig. 1, the presence of A in the culture medium does not

significantly influence the 3 h resting ROS production (measured both

as AUC and delta [Δ]).Activation for 3 h with fMLP, increased the ROS generation, while

the effect of LPS and IL-8 although evident, did not reached the sta-

tistical significance. A (1 𝜇M) was able to prevents the fMLP and

IL-8 induced ROS generation (measured both as AUC and Δ) and only

slightly, but not significantly by LPS (Fig. 1).

In order to better evaluate the role of 𝛽-AR activation in the abil-

ity of A to prevent the NET-related ROS increase induced by acti-

vating stimuli, we have preincubated cells with the different 𝛽-AR

antagonist to counteract the effects of A. As shown in Fig. 2, the abil-

ity of A to decrease fMLP-induced ROS generation was contrasted

(both as AUC and Δ) by the nonselective 𝛽-AR antagonist propra-

nolol and by the selective 𝛽2-AR antagonist ICI-118,551, but not

by the 𝛽1-AR antagonist CGP-12177 and by the 𝛽3-AR antagonist

L-74,8337 (fig. 2).

3.2.2 Microscopic evaluation of cell morphology

Morphological analysis (Fig. 3) showed that, as expected, 3 h incuba-

tion with fMLP (panel C), IL-8 (panel E), and LPS (panel G) induced a

change in NETs production and a change in cellular phenotypes, com-

pared to resting (panel A). The pre-incubation with A (1 𝜇M, panel B)

although did not affect the resting conditions, partially prevents both

the NET formation and the morphological changes induced by the dif-

ferent stimuli (panel D, F, and H).

3.2.3 Immunocytochemical analysis of NET-related

myeloperoxidase expression

As shown in Fig. 4, MPO was expressed at low levels in neutrophils

under resting conditions (panel A); after 3 h activation with fMLP

(panel C), IL-8 (panel E), and LPS (G), a rise in MPO production was

observed both in the cytoplasm and also on NETs. The pre-incubation

with A (1 𝜇M) although did not affect the resting conditions, prevented

NETs formation andMPO expression, compared to resting conditions,

was slightly increased (panels D, F, and H).

3.2.4 NET elastase activity

The quantification of elastase, one of the NET component (Fig. 5),

corroborated the morphological data showing, that activation with

fMLP (exerting, in our model, the strongest PMN activation) induced

an increase in elastase production and that A was able to prevents

this effect; similarly, the pre-incubation with ICI-118,551 but not with

CGP-12177 reverted the A-induced inhibition of fMLP-induced elas-

tase production.

3.3 ROS generation

We have previously shown that, PMN when activated for 30 min

with fMLP, produce abundant ROS, and that the preincubation with

F IGURE 2 Effect of treatment with adren-ergic ligands on NET-related ROS generationin isolated PMN. Effect of A alone or in thepresence of selective 𝛽-AR antagonists on NET-related fMLP-inducedROSgenerationmeasuredas AUC (left panel) or Δ (right panel). Dataare represented as mean ± SEM of at least3–5 separate experiments. Statistical analysiswas performed using Mann–Whitney test and“D’Agostino & Pearson omnibus normality test.”*P < 0.05 and **P < 0.01 versus fMLP alone;#P< 0.05 versus A+fMLP

6 MARINO ET AL.

F IGURE 3 NET evaluation trough optical microscopy in PMNsunder resting and stimulated conditions. PMNs under resting con-ditions (A) or in the presence of A (B; 1 𝜇M) show similar pheno-type. Most of the cells are characterized by roundish profile (whitearrowheads) and noNET can be visualized. PMNs activated with fMLP(0.1 𝜇M; panel C), IL-8 (100 ng/mL; panel E), and LPS (1 𝜇g/mL; G) showchanges in morphology and the production of NETs (black arrows)can be observed. The treatment with A retains for the most part theunstimulated conditions and partially prevents the formation of NETs(D, F, and H)

A, was able to prevent this increase. In addition, we have shown

that the A-induced effects were counteracted by the nonselective

𝛽-AR antagonist propranolol.27 In the present study, we aimed to

deepen which kind of 𝛽-AR was specifically involved in the above

described effects. As shown in Fig. 6, the ability of A to prevent

the 30 min fMLP-induced ROS generation was contrasted only by

ICI-118,551 (selective 𝛽2-AR antagonist) while CGP-12177 (selective

𝛽1-AR antagonist) and L-74,8337 (selective 𝛽3-AR antagonist) were

ineffective.

3.4 Cell migration

In our previous paper, we have shown that A was able to prevent

the fMLP-induced cell migration and that this effect was reverted

by the 𝛽-AR antagonist propranolol and not by the 𝛼1 and -𝛼2-AR

F IGURE 4 Immunocytochemical characterization of myeloperox-idase expression. PMNs under resting conditions (A) or in the pres-ence of A (B) show low levels ofMPOexpression. PMNs activatedwithfMLP (0.1 𝜇M; panel C), IL-8 (100 ng/mL; panel E), and LPS (1 𝜇g/mL; G)show that NETs production (white arrowheads) and MPO expressionis higher both in the cytoplasm and also on NETs (white arrows). Thepre-incubation with A before fMLP (D), IL-8 (F), and LPS (H) preventsNETs formation and MPO levels are only slightly increased, comparedto control (panel B). MPO positivity is visible in red; nuclei, counter-stainedwith DAPI, are in blue

antagonists.27 Considering the key role of IL-8 as chemoattractant

stimulus for PMN and on the basis of the previous mentioned effects

exerted by propanolol, in the present study, we have explored the abil-

ity of A and of the 𝛽-AR agonist isoprenaline (ISO) to modulate IL-8-

induced PMNmigration.

Neither A nor ISO (1 𝜇M) were able to affected PMN spontaneous

migration (15 ± 7 𝜇m and 12 ± 2 𝜇m, respectively versus R; n = 4,

P > 0.05). As expected, IL-8 increased PMN migration and both A

and ISO prevented, in a concentration-dependent manner, the IL-8

induced migration (Fig. 7, panel A). According to our previous results,

we have investigated which kind of 𝛽-AR was involved in the ISO-

induced effects.

As shown in Fig. 7 (panel B), the ability of ISO (1 𝜇M) to

prevents the IL-8-induced cell migration was counteracted only

by propranolol and ICI-118,551, while CGP-12177 and L-74,8337

were ineffective.

MARINO ET AL. 7

F IGURE 5 Elastase production in isolated PMN. Elastase produc-tion in isolated neutrophils cultured alone or in the presence of fMLP,A, the 𝛽2 selective antagonist ICI-118,551, and the selective 𝛽1 antag-onist CGP-12177. Data are presented as mean ± SEM of 5 separateexperiments. Statistical analysis was performed using Mann–Whitneytest and “D’Agostino&Pearsonomnibus normality test” **P<0.01 ver-sus resting; #P< 0.005 and ##P< 0.01 versus fMLP alone

F IGURE 6 Effect of treatmentwith adrenergic ligands onROS gen-eration in isolated PMN. Treatment with A and selective 𝛽-AR antago-nists on 30 min fMLP-induced ROS generation. Data are presented asmean ± SEM of at least 3–6 separate experiments. Statistical analysiswas performed using Mann–Whitney test and “D′Agostino & Pearsonomnibus normality test.” **P < = 0.01 versus A + fMLP; #P < 0.05 and##P< 0.01 versus fMLP alone

3.5 Characterization of 𝜷-AR expression on PMN

3.5.1 Real time PCR evaluation ofmRNA expression

In our previous study, we have shown that all the AR are expressed at

the mRNA level and in particular that the highest expression was for

the 𝛽-AR subtypes. We have also shown that the 3 h activation with

fMLP induced an increase in themRNAexpression of all the AR.27 Sim-

ilarly, our previous study showed that the effects exerted by A on PMN

functions were for the most part related to 𝛽-AR activation.27 In the

present study, starting from this observation, we have explored the

ability of other PMN activators namely IL-8 and LPS to influence the

𝛽-AR expression. As shown in Fig. 7, neither IL-8 (100 ng/mL) or LPS

(1𝜇g/mL)were able to significantly affect themRNA levels of the three

𝛽-ARs; in fact, both induce a slight increase of the expression for all the

3 receptors, but this increase did not reach the statistical significance.

3.5.2 Immunocytochemical analysis of 𝜷-AR expression

As shown in Fig. 9, 𝛽1-AR (A), 𝛽2-AR (E), and 𝛽3-AR (I) are expressed on

isolated PMN, but at low intensity; after activation with fMLP (panels

B,F, and J), IL-8 (panels C, G, and K), and LPS (panels D, H, and L), the

expression of all the three 𝛽-AR subtypes was increased.

3.5.3 Flow cytometric evaluation of 𝜷-AR expression

According to our previous data and on the basis of the present results,

we have evaluated the membrane receptor expression of the 𝛽1 and

𝛽2-ARbothafter30minand3h incubation.As shown inFig. 10, both 𝛽1and 𝛽2-ARare expressed andno substantial differenceswere observed

for the expression at the 2 times of incubation. The expression how-

ever was quite different for the two subtypes, showing higherMFI and

percent of positive cells for the 𝛽1-AR (P< 0.05) both after 30min and

180min of incubation.

Activation with fMLP, IL-8, or LPS did not significantly altered the

membrane expression, measured as MFI of both receptors with the

only exception of a slight decrease after activation with LPS for the

𝛽1-AR (P < 0.05 vs. resting) after 180 min of incubation both as MFI

and percent of positive cells (lower panels).

4 DISCUSSION

NET primarily identified, produced by neutrophils to kill microorgan-

isms and todefend against pathogens,1 are demonstrated tobe formed

also during inflammatory events,2 as for example, in cystic fibrosis,39

thrombosis,40 and chronic obstructive pulmonary disease.41 In the

last years, the list of diseases in which these neutrophil products are

demonstrated to be involved was certainly increased42 and a key role

ofNET in non-infective diseaseswas recently outlined.43 NETare com-

monly classified in 2 main types: lytic and vital NET44 and usually it is

documented that vital NET are produced rapidly by cells in which via-

bility and functional activities aremaintained.44

In the present study, we have shown that the stimulus-inducedNET

production can be modulated by A and other adrenergic ligands. In

particular, we have shown, for the first time, that A is able to pre-

vent the stimulus-induced NET production and this effect seems to

involve receptor activation, in particular the 𝛽-AR subtypes, as sug-

gested by the ability of 𝛽-AR antagonists to counteract the A and ISO

induced effects.

8 MARINO ET AL.

F IGURE 7 Effect of treatment with adren-ergic ligands on PMN migration. (A) Effect oftreatment with A and ISO on the IL-8 inducedcell migration. (B) Effect of pretreatment withthe selective 𝛽-AR antagonists on the abilityof ISO to prevent the IL-8-induced cell migra-tion. In both panels, data are presented asmean ± SEM of at least 3–7 separate experi-ments. Statistical analysis was performed usingMann–Whitney test and “D’Agostino & Pearsonomnibus normality test”. *P < 0.05, **P < 0.01,and ***P < 0.001 versus R; #P < 0.05, and##P < 0.01, and ### = P < 0.0001 versus IL-8;$P < 0.05 versus IL-8 alone; #P < 0.05 versusISO+ IL-8

F IGURE 8 mRNA levels of 𝜷-AR expressionin isolatedPMN. mRNA levels of 𝛽1-AR (left), 𝛽2-AR (middle), and 𝛽3-AR (right) in isolated PMNcultured for 3 h under resting conditions orafter activation with IL-8 (100 ng/mL) or LPS(1 𝜇g/mL). Data are presented as mean ± SD of4 separate experiments

F IGURE 9 Immunocytochemical characteri-zation of 𝜷-AR expression. 𝛽-AR expression inPMN under resting conditions (A, E, and I) andafter activation with fMLP (B, F, and J), IL-8 (C,G, and K), and LPS (D, H, and L). (A, E, and I): neu-trophils under resting conditions showing basallevel of 𝛽-AR expression. PMNs after activationwith different type of stimuli show increasedexpression of 𝛽1-AR (BC andD), 𝛽2-AR (F, G, andH), and 𝛽3-AR (J, K, and L). 𝛽-AR positivity is visi-ble in red; nuclei, counterstained with DAPI, arein blue

MARINO ET AL. 9

F IGURE 10 Flow cytometric evaluation of 𝜷1- and 𝜷2-AR expression. (A) Representative flow cytometric analysis of 𝛽1-AR or 𝛽2-AR fromwhole blood sample performed for each donor showing the morphological dot-plot (FSC vs. SSC signals) used to identify granulocyte population(red region) and the histogram overlays for 𝛽1-AR (left) or 𝛽2-AR (right). The ratio (MFI), calculated by dividing the MFI corresponding to specificstaining for 𝛽-AR over MFI corresponding to a negative control, was used to quantify the expression level of each 𝛽-AR and is shown above eachhistogram line.(B) Analysis of 𝛽1-AR (left) and 𝛽2-AR (right) expression after 30 or 180 min culture in the presence of fMLP (0.1 𝜇M), IL-8 (100 ng/mL), and LPS(1 𝜇g/mL). Data are expressed as ratio (MFI; in each time-set, upper panels) or percent of positive cells (in each time-set, lower panels) and arerepresented asmean± SEM of at least 4–5 separate experiments. Statistical analysis was performed usingMann–Whitney test and “D ′Agostino &Pearson omnibus normality test”. *P< 0.05 versus respective resting conditions

In our previous paper, we have extensively characterized, in human

neutrophils, the ROS-induced generation and the ability of A, and in

general of adrenergic compounds, to modulate this production.27

In the present study, we have investigated the ability of 𝛽-AR lig-

ands to affect NET production. To this end, we have measured the

3 h-induced ROS generation (ROS are necessary for NETosis3) related

to NET production and other different methods, such as microscopic

analysis and colorimetric assays. The main relevant finding is that the

3 h-NET-induced production was reduced by activation of 𝛽2-AR; in

particular, we have shown that A prevented the stimulus-induced NET

production; in addition we have shown, and to our knowledge this is

the first study showing a contribution of 𝛽2-AR in the modulation of

NET production, that this effect was receptor mediated; in fact, the

A-induced effect was reverted by the addition of the 𝛽2-AR antag-

onist ICI-118,551. We have considered the possibility that the abil-

ity of A to prevent the fMLP-induced effects can be due to an apop-

totic effect exerted by the CA in culture; we have shown no significant

apoptosis suggesting that the observed effects on NET, ROS genera-

tion, andmigration are receptor-mediated. Considering the recent evi-

dence showing thatNET can be produced by living neutrophils and this

function is not only an end stage of these cells43,44 or thatNET are pro-

duced only by a small part of these cells,43 this evidence supports the

idea that it is possible to interferes pharmacologicallywith thesemech-

anisms without affecting cell viability.

This result is of greater interest considering the pivotal role

of NET in several immune-mediated diseases and the possibility

offered by different safe drugs widely used in therapy known to

act through the interaction with these receptors (e.g., beta agonists

used in asthma and cardiovascular diseases) and this observation is

in line with literature data that propose 𝛽2-AR agonists as safe anti-

inflammatory drugs.27,45,47

ROS production can induce a negative process that can result

in chronic inflammation or cancer.48 According to our previous

data showing the ability of A to counteract the stimuli-induced

ROS generation and by the ability of propranolol (a nonselective

𝛽-AR antagonist) to counteract this effect, we have deepened this

aspect investigating specifically which kind of 𝛽-AR was involved in

this effect. The role of 𝛽2-AR in this effect is outlined by the fact that

only ICI-118,551 andnotCGP-12177 (𝛽1-AR antagonist) or L-74,8337

(𝛽3-AR antagonist) was able to revert the A-induced effects.

10 MARINO ET AL.

Wehavealso characterizedmigration, another key functionof these

cells, that allows them to reach different tissues and to starts inside

the production of the inflammatory mediators devoted to contrast tis-

sue invasion by pathogen and finally, resolves inflammation. All these

events are key points aimed to the prevention of the chronicity of the

inflammatory processes.48

According to what observed for ROS production, also migration

seems to be affected by adrenergic ligands, confirming our previous

data showing that A was able to prevent the fMLP-induced migration

and that PRO counteracted this effect.27 In the present study, we have

shown that not only the fMLP but also the IL-8 induced migration is

affected by A and by the selective 𝛽-AR agonist ISO. Likewise to what

observed for ROS generation, also in this case, the 𝛽-AR involved is the

𝛽2-AR, as suggested by the ability of ICI-118,551 and by the lack of the

other 2 receptor antagonists to revert the A and ISO-induced effects.

These data together with the data on ROS generation suggest a strong

role of the 𝛽2-AR activation in the modulation of pivotal functions in

human neutrophils.

In line with these evidences, we have previously shown that

neutrophils after the migration into inflamed tissues produce pro-

inflammatory mediators contributing to the orchestration and main-

tenance of the inflammatory status typically found in all the immune-

mediated diseases33 and this observation is confirmed by the data on

long-term culture (24 h) showing a response to inflammatory stim-

uli and increased production of key mediators, such as IL-8, vascular

endothelial growth factor (VEGF), and elastase.34 The production of

NET can be modulated by different substances. In addition, according

to these observations, the possibility to produce NET while remain-

ing alive and functional43,44 can be considered as possible approach to

revert this phenomenon.

These data are in line with recent evidences showing that 𝛽2-AR

activation is linked with inhibition, in innate immune cells, of NF-𝜅B,

an intracellular transcription factor involved in the production of the

main proinflammatory products.45 This finding, together with other

publisheddata,44;27;29 canopennewquestions about the possible new

alternative use of drugs acting on these receptors aimed at contrast-

ing inflammation in diseases usually not treated with these medica-

tions; these data are also in linewith previous evidences suggesting the

ability of adrenergic compounds to affect immune cell functions44–47

but, more interestingly, that these drugs can be safely used in other

context, promoting the possible therapeutic relevance for their anti-

inflammatory properties.

The possible clinical relevance of an adrenergic tone is also sus-

tained by the presence, on PMN of the 𝛽-ARs. By means of immuno-

cytochemical analysis, real time PCR evaluation, and flow cytometry,

we have evaluated the expression of the 3 𝛽-AR. According to the func-

tional data we have evaluated, the protein expression after 30 min of

cell activation and both immunocytochemical and cytometric evalua-

tion (after 30min and after 3 h) show that all the 3 𝛽-AR are expressed,

although the microscopic evaluation shows an increased expression

after cell activation while flow cytometry not. In order to better define

this aspect, we have also explored the mRNA levels and the mem-

brane expression after 3 h activation. Interestingly, the mRNA expres-

sion was not changed and similarly the protein expression after 3 h

was superimposable to the values measured after 30 min activation.

The possible explanation of this discrepancy can be due to the differ-

ent technical approach; the flow cytometric evaluation of AR expres-

sions is performed in whole blood without cell isolation procedures

that can influence the final results. In addition, flow cytometry anal-

ysis is performed without membrane permeabilization and therefore

we measured only ARs expressed on membrane; on the contrary, the

immunocytochemical method presents a step that includesmembrane

permeabilization and the possible “recognition” of internalized recep-

tors. It is well known in fact that 𝛽-ARs are receptors which can rapidly

internalize as consequence of different kind of stimuli or during par-

ticular conditions.49 Interestingly, the present immunocytochemical

data are in line with our previous data performed with real time PCR

analysis showing an increased expression of all the 𝛽-AR after cell

activation with fMLP.27 On the contrary, the present data measured

by real time PCR receptor expression after activation with LPS or

IL-8 shows unchanged expression. But in any case, although it is not

clear why microscopic data are different from both flow cytometry

and current real time PCR data, our functional data are suggestive

of an involvement of the 𝛽2-AR in the observed effects. In line with

the functional evidences of this study recently, other researchers have

described a safe antinflammatory profile for 𝛽2-AR ligands on differ-

ent cell types44–47; interestingly, it is remarkable that the range of con-

centrations used for our in vitro studies are physiologically relevant

considering that A used, for example, in emergency is applied in the

range of 0.3–1 mg and salmeterol or other 𝛽2-AR agonists are used in

the range of 200 𝜇g–1.5 mg. Not all data of literature reports antin-

flammatory effects. For example, in animal models, chronic treatment

with 𝛽2-AR agonists increases 𝛽-amyloid generation50 and the use of

𝛽2-AR blockers can revert acute stress-induced beta amyloid produc-

tion suggesting a possible protective role of drugs acting on these path-

ways in Alzheimer’s disease.51 Similarly, the relevance of 𝛽2-AR acti-

vation in wound healing was recently demonstrated in a stressed ani-

mal model,52 suggesting that drugs acting on these receptors can be

proposed as alternative choice in several other diseases beside their

classical use.

ACKNOWLEDGMENTS

This study was technically supported by Centro Grandi Attrezzature

(CGA) core facilities of University of Insubria.

The authors’ research was funded by CARIPLO (Fondazione Cassa

di Risparmio delle Province Lombarde)—2016-0835 Ricerca malattie

invecchiamento 2016: FRAMYEVO and by the University of Insubria

(Fondo Ateneo Ricerca [FAR] 2015–2016 -Marino).

M.P. was supported by fellowship grants from the Ph.D. pro-

gram in Clinical and Experimental Medicine and Medical Humanities,

University of Insubria; A.S. and L.P. were supported by post-doc fellow-

ships of University of Insubria (CARIPLO grants).

AUTHORSHIP

Study conception and design: F.M., A.S., andM.C.

Acquisition of data: A.S., E.R., L.P., M.P., A.L., R.B., andM.L.

MARINO ET AL. 11

Analysis and interpretation of data: E.R., R.B., A.S., M.P., M.L., and

M.D.E.

All authors were involved in drafting the article or revising it criti-

cally for important intellectual content, and all authors approved the

final version to be published.

All authors agree to be accountable for all aspects of the work in

ensuring that questions related to the accuracy or integrity of any part

of the work are appropriately investigated and resolved, and declare

to have confidence in the integrity of the contributions of their co-

authors.

DISCLOSURE

The authors declare no conflict of interests.

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How to cite this article: Marino F, Scanzano A, Pulze L,

et al. 𝛽2-Adrenoceptors inhibit neutrophil extracellular traps

in human polymorphonuclear leukocytes. J Leukoc Biol. 2018;1-

12. https://doi.org/10.1002/JLB.3A1017-398RR