Priming of Polymorphonuclear Neutrophils' by Atrial Natriuretic Peptide In VitroChristian J. Wiedermann, Monika Niedermihlbichler, and Herbert BraunsteinerDepartment of Internal Medicine, Faculty of Medicine, University of Innsbruck, A-6020 Innsbruck, Austria

Abstract

In ischemia-reflow states of coronary artery disease, the activa-tion of PMNprecedes the initiation of tissue damage. Releaseof atrial natriuretic peptide (ANP) from myocytes occurswithin minutes after the onset of myocardial ischemia, whichsuggests a possible role of ANPin PMNactivation. To investi-gate this possibility, we tested the effects of ANPon functionsof PMNin vitro. ANPis a potent signal for priming the PMNrespiration burst to secrete superoxide anion. Phorbol 12-myr-istate 13-acetate, opsonized zymosan, or FMLPcould all beused as triggering stimuli to demonstrate the priming of PMNactivation by ANP. Only ANPfragments 1-28 and 7-28 en-hanced respiration burst activity but identical preparations ofANPfragments 13-18 or 1-11 failed to do so. This structure-activity relationship is typical of receptors for ANPfound inother tissues. In addition, ANPstimulated the release of t-gluc-uronidase from PMNtriggered by FMLP. The observed inhibi-tion by ANPof FMLP-stimulated chemotaxis of PMNmay bedue to their enhanced adhesiveness. These data show that aclassic cardiac hormone is involved in regulating importantfunctional activities of PMN. These data support the possibil-ity that ANPcould act as a preinflammatory substance in isch-emia-reperfusion states and myocardial necrosis. (J. Clin. In-vest. 1992. 89:1580-1586.) Key words: chemotaxis * coronaryartery disease - degranulation - ischemia-reperfusion injury.respiratory burst

Introduction

The observation that leukocyte depletion was associated with asignificant reduction in infarct size in dogs subjected to coro-nary occlusion (1) suggests a role for polymorphonuclear neu-trophils (PMN) in causing myocardial necrosis. Within min-utes of occlusion of a major coronary artery, the PMNareactivated (2), whereat they adhere to the vascular endotheliumand migrate through the endothelial layer. Interactions withthe endothelium can promote increased vascular resistance (3),diminished collateral flow, capillary blockade (4), predisposi-tion to vasospasm (5), as well as enhanced vascular permeabil-ity (6). Onsubsequent reperfusion, entrapped PMNgain accessto the previously ischemic region. Reperfusion of the ischemicmyocardium increases the number of PMN distributedthroughout the damaged region (7, 8).

Address reprint requests to Dr. Wiedermann, Department of InternalMedicine, Anichstrasse 35, A-6020 Innsbruck, Austria.

Receivedfor publication 13 May 1991 and in revisedform 5 De-cember 1991.

J. Clin. Invest.©The American Society for Clinical Investigation, Inc.0021-9738/92/05/1580/07 $2.00Volume 89, May 1992, 1580-1586

In ischemia-reflow states of coronary artery disease with thedevelopment of arrythmias and myocardial infarction, reintro-duction of oxygen to the ischemic or hypoxic tissue can causean additional insult that is mediated in part by oxygen radicals(9). Generation of oxygen-derived radicals by PMNenteringreoxygenated tissue is one of the potential sources of damage(10). Activated PMNdepress both the uptake of Ca2' by myo-cytes and the activity of Ca2'-stimulated, Mg2e-dependentATPase. Because both of these activities can be prevented byfree radical scavengers, it can be presumed that they are causedby oxygen-derived free radicals (11).

Activation and adhesion of PMNprecede the initiation oftissue damage as determined histologically (2, 4, 12, 13). Thisobservation suggests that PMNactivation is not a response totissue injury, but rather reflects other, more subtle changes re-sulting from the ischemic episode. Thus preinflammatory cyto-kines, such as tumor necrosis factor-a (TNF),' are releasedearly in myocardial infarction (14, 15) and may contribute toPMNactivation (16). PMN-activating substances may be pro-duced by endothelial cells upon hypoxia which dramaticallymodulates endothelial cell function (17, 18). In addition, su-peroxide anions that are produced by affected cells during isch-emia (9) may directly promote PMNadhesion (19) and act onplasma to produce PMNchemotactic lipids (20). Other factorsthat could act as modulators of injury and inflammation in theischemic heart are polypeptide hormones derived from perivas-cular nerves and/or other sources (21).

Atrial natriuretic peptide (ANP) may significantly contrib-ute to the pathophysiological changes occurring in ischemia-re-flow states of the heart in vivo, in that measurable release ofANP into coronary sinus blood and systemic circulation butnot of other substances such as endothelin, vasoactive intes-tinal peptide, tachykinins, P-endorphin, and TNF-a, has beenreported to occur within minutes of balloon occlusion of amajor coronary artery for angioplasty (22, 23). ANP is a 28-amino acid polypeptide, secreted primarily by atrial myocytesin response to local wall stretch (i.e., increased intravascularvolume or central venous pressure). The combined actions ofANPon vasculature, kidneys, and adrenals serve to feducesystemic blood pressure as well as intravascular volume (24).The heart atria represent the major site of synthesis of ANP.However, more recently ANPhas been found in organs notimmediately related to cardiovascular physiology: e.g., the ner-vous system, adrenals, lungs, and gut, as well as tissues belong-ing to the lymphatic, reproductive, or endocrine systems (25).Thus, ANPmight have more physiological roles than originallythought of, one of them being modulation of inflammatorycells. However, the functional significance of ANP in tissuesother than the cardiovascular system is, at present, poorly un-derstood.

1. Abbreviations used in this paper: ANP, atrial natriuretic peptide;GM-CSF, granulocyte/macrophage colony-stimulating factor, NBT,nitroblue tetrazolium; TNF, tumor necrosis factor.

1580 C. J. Wiedermann, M. Niedermithlbichler, and H. Braunsteiner

Because of the important role of the PMNin responsesoccurring in ischemic events, we studied the in vitro effects ofANPon PMN. In this report we show that ANPprimes PMNfor an enhanced respiration burst activity in response to solublestimuli. Weshow that: (a) over the time course studied ANPwithout the additipnal presence of known PMN-activating fac-tors such as FMLP, opsonized zymosan, or phorbol 12-myris-tate 13-acetate (PMA) does not activate PMNrespiration burstactivity, migration, or granule release; (b) ANPactivates PMNto increased respiration burst activity and granule release in-duced by coincubation with FMLPand zymosan, respectively,and decreases migration of PMNtoward maximally stimulat-ing concentrations of FMLPwhen simultaneously exposed tothese agents; (c) preincubation of PMNwith ANPsignificantlyenhances FMLP- or PMA-induced respiration burst activity;(d) the structure-activity relationship of ANPsuggests a spe-cific receptor-mediated effect of ANPon PMN, as ANP 1-28and ANP7-28 are active, whereas ANP 13-18 and ANP 1-11are inactive as stimulants; (e) because ANPdoes not stimulatecyclic GMPaccumulation in the PMNafter 10 min of expo-sure, the signaling mechanism for ANPmay involve the ANPreceptor subtype, which lacks guanylate cyclase activity on thecytoplasmic domain.

Methods

Materials used in these studies include ANP and ANP fragments(Sigma Chemical Co., St. Louis, MO; or Peninsula Laboratories, Inc.,Belmont, CA), the recombinant human cytokines TNF-a (sp act 4X 107 U/mg protein; Genentech Inc., South San Francisco, CA), gran-ulocyte/macrophage colony-stimulating factor (GM-CSF; sp act 5X 107 U/mg protein; Behring AG, Marburg, FRG), PMAand FMLP(Sigma Chemical Co.). ANPand its fragments were reconstituted indistilled water or 0.02 Nacetic acid to stock solutions of 10 mmol/liter,stored at -200C, and freshly diluted to final concentrations in me-dium. FMLPwas stored at -20'C in DMSOat a concentration of 10mmol/liter and was diluted into assay medium before use. PMAwasreconstituted to 1 mg/ml in ethanol and also stored at -20'C. Zymo-san was opsonized with fresh serum as follows: 100 mgwas suspendedin 10 ml of saline and heated at 100IC in a boiling water bath for 1 hwith occasional shaking. The particles were washed three times in sa-line and incubated with 6 ml of fresh human serum for 20 min at 37°Cin a shaking water bath. The material was washed twice with saline andresuspended in saline to 50 mg/ml. Opsonized zymosan was storedfrozen at -70°C.

RPMI-1640, BSA, zymosan, nitroblue tetrazolium (NBT), Percoll,EDTA, dextran 485,000, iodoacetamide, ferricytochrome c, SOD, 4-methylumbelliferone, 3-isobutyl-L-methylxanthine, and 4-methylum-belliferyl-fl-D-glucuronide were from Sigma Chemical Co. Addition-ally, the following were purchased: Triton X-100 (New England Nu-clear, Dreieich, FRG); Hanks' balanced salt solution (HBSS; Gibco,Vienna, Austria); sodium acetate (E. Merck & Co., Darmstadt, FRG);and phosphoramidone (Peninsula Laboratories, Inc.).

Purification of human neutrophils. From the peripheral blood ofhealthy volunteers (anticoagulated with EDTA), PMNwere obtainedafter discontinuous density gradient centrifugation of whole blood onPercoll or from buffy coat residues (mixed with normal saline in a ratioof 3:1) by dextran sedimentation and centrifugation through a layer ofFicoll-Hypaque as described (26), followed by hypotonic lysis of con-taminating erythrocytes using sodium chloride solution. The cell prepa-rations (> 95% neutrophils by morphology in Giemsa stains, > 99%viable by trypan dye exclusion) were resuspended in RPMI-1640/0.5%BSAfor chemotaxis experiments, or in HBSSfor respiration burst activ-ity and exocytosis assays. The assays were performed in these mediawhich also contained the test substances or their appropriate vehicles.

Respiration burst activity. NBTreduction by PMNwas measuredas described (26). To the cells (100 ,d of 2 X 106 PMN/ml in HBSSperwell in 96-well microplates), NBTwas added (final concentration (f/c)of 0.5 mg/ml) with medium or test substances to a final volume of 200!tl per well. One vertical row of eight wells, in which the cells werepreincubated for 10 min at 370C with a 10-mmol/liter solution of io-doacetamide in HBSSserved as reference. After gentle agitation on amicroplate shaker, the amount of formazan accumulating in PMNduring incubation in a humidified incubator at 370C in 95% air 5%CO2 for the indicated time intervals was repeatedly quantitated in anELISA reader at 550-nm wave length (model MR700; Dynatech Labs,Guernsey, UK).

Measurement of the production of °2 was based on the reductionof ferricytochrome c by O°, the specificity of reduction being con-trolled by its inhibition by SOD. Immediately after preparation ofPMNwith or without exposure to test substances, 100 ML per well of 2X 106 PMN/ml were immersed in a 160 MMsolution of ferricy-tochrome c in phenol red-free HBSScontaining 10 MFMLPor vehi-cle. To one vertical row, cytochrome c containing 300 U/ml SODwasadded. This row served as a blank. The plates were covered with lidsand placed in a 370C humidified incubator with a 95% air-5% CO2atmosphere. At indicated time intervals, the plates were transferred tothe ELISA reader and absorbances were read at 550 nm. The absor-bance values at 550 nmwere converted to nanomoles of °- based onthe extinction coefficient of (reduced minus oxidized) cytochrome c:OD5jonm = 21 X 103 M-'cm-'. In that the vertical light path passingthrough 100 Ml of cytochrome c was 3 mm,calculated concentration of°2 was (nmol °2 per well) = (absorbance at 550 nmX 15.87) accord-ing to reference 27.

Chemotaxis. The chemotactic response of PMNto different gra-dients of FMLPand/or ANPwas tested in a modified multiwell Boy-den chamber as previously described (26). To the upper wells of thechemotaxis chamber 50,000 PMNwere then added and the bottomwells were filled with 25 Ml of FMLPor ANPalone and in combination,or with 25 Ml of medium. The two wells were separated by a 3-,umpore-sized nitrocellulose filter (Sartorius, Gottingen, FRG) presoakedwith medium. Chambers were incubated for 35 min at 37°C (5% CO2,fully humidified). Migration depth of the cells into the filter was micro-scopically measured (leading-front assay) in triplicates, after the cellsand filters were fixed, dehydrated, and stained with hematoxylin (26).

Release of PMNgranules. PMNgranule release was determined bymeasuring exocytosis of fl-glucuronidase (EC 3.2.1.31) as described(28). PMNwere suspended in 0.9 ml of HBSSat 2.5 X 107/ml andpreincubated in a shaking water bath for 10 min at 37°C. The cells werethen stimulated by addition of 0.1 ml of FMLP(1.0 Mimol/liter), ANP1-28 (1.0 Mmol/liter), or a combination of FMLP(1.0 Mmol/liter) andANP1-28 (1.0 Mmol/liter) in HBSS, and the incubation was continuedfor 15 min under mild agitation in a water bath at 37°C. The reactionwas stopped by rapid cooling in ice followed by centrifugation at 800 gfor 10 min at 4°C. The supernatant was aspirated and diluted with aequal volume of HBSScontaining 0.1% Triton X-100. A suspension ofthe pellet was prepared with 1 ml of HBSScontaining 0.2% TritonX- 100, kept on ice during 20 min with occasional vortexing for extrac-tion, diluted with 3 ml of HBSS, and centrifuged at 800 g for 10 min at4°C. For assaying granule release, (i-glucuronidase was measured insupernatants and pellet extracts by mixing 0.1 ml of sample with 0.1 mlof 10 mmol/liter 4-methylumbelliferyl-8-D-glucuronide in 0.1 mol/liter sodium acetate, pH 4.0, containing 0.1% Triton X-100, and incu-bated for 15 min at 37°C. The reaction was stopped by addition of 3.0ml of 0.05 mol/liter glycine and 5 mmol EDTA, pH 10.4. Liberated4-methylumbelliferone was measured fluorimetrically (excitation 365nm, emission: 460 nm) (Eppendorf, Hamburg, FRG). For standardcurves, fluorimetric data obtained from dilutions of stock solutionscontaining 5 mmol/liter 4-methylumbelliferone in ethanol (stored inthe dark at 4°C) were used.

Cyclic GMPassay. The cyclic GMPlevels in the PMNwere deter-mined by means of a commercial radioimmunoassay kit (New EnglandNuclear). Samples containing 5 x 106 PMNwere resuspended in HBSS

Neutrophil Priming by Atrial Natriuretic Peptide 1581

0

FMLP[106 M]

ANP 1-28[10-6M]ANP 1-28 [10-8 M]ANP1-28 [101M]ANP 1-28 [1012M]

2

0

15 min 30 min 60 min 90 min

Figure 1. Stimulation of NBT reduc-tion in PMNat 15, 30, 60, and 90min of incubation (37°C) by 10-6 Mof FMLPvs. 10-6, 10-8, 10-'°, and10-12 Mof ANP 1-28. Bars representthe mean and SEMof determinationsin replicates of eight from three inde-pendent experiments.

containing 0.5 mM3-isobutyl-L-methylxanthine and incubated in a

shaking water bath at 37°C. After 10 min, 100 nmol/liter of ANP, 100ng/ml of PMA, or vehicle controls were added for additional 7 min.Incubations were then terminated with centrifugation of the samples at4°C for 3 min at 400 g. Pellets were resuspended in 750 Ml of ice-cold6% trichloroacetic acid (TCA) and centrifuged at 2,500 g for 20 min,allowing the supernatants to be collected. TCA was eliminated bywashing the extract five times with 5 ml of water-saturated ethyl ether.Then, 500 Ml of the samples was lyophilized and acetylated and cyclicGMPlevels (fmol/106 PMN)were determined by radioimmunoassay,as given by the manufacturer.

Calculations. The data is expressed as the mean and standard error

of the mean (SEM). The nonparametrical analysis of variance was per-formed for independent samples after Kruskal-Wallis. Differenceswere compared using the Wilcoxon-Mann-Whitney test and a signifi-cant level designated 2 a = 0.05. Statistics were performed usingthe StatView512+ software package (Abacus Concepts, Inc., Berke-ley, CA).

Results

Effects ofANP alone on PMNfunctions. WhenPMNare incu-bated at 37°C for 0-90 min in the presence of 1 pmol/liter to 1

Mmol/liter of ANP1-28, no significant iodoacetamide-inhibit-able reduction of NBT(Fig. 1) or SOD-inhibitable reduction of

w/o 3 wI Phosphoramidone

20

Z

C) X

CX c

z 0

15

10

5

0

ANPplus

FMLP

Figure 2. Stimulation of NBTreduction in PMNat 30 min of incu-bation (37°C) by 10-8 Mof ANP1-28 in the presence and absenceof 10-6 Mof FMLP, with and without lO-' Mof phosphoramidone.Bars represent the mean and SEMof determinations in replicates ofeight from three independent experiments.

ferricytochrome c (not shown) occurs. Similarly, during thesame time period 0.1 tmol/liter of ANP 1-28 does not causechemotaxis of PMNinto nitrocellulose micropores (see Fig. 6),nor does it induce release from PMNof f3-glucuronidase(2.49% of total cellular (3-glucuronidase at 100 nmol/liter ofANP1-28 compared to 2.45% for control untreated PMNafter15 min of incubation; mean, n = 5; P > 0. 1; see Fig. 7).

Effects ofANP on agonist-induced PMNfunctions. BecausePMNpriming agents can enhance PMNresponses to chemo-tactic factors, we examined the effect of ANP 1-28 on respira-tion burst activity of PMNin response to the chemotactic fac-tors FMLP, zymosan, and PMA. Fig. 2 shows that, at a concen-tration 10 nmol/liter ANP1-28 enhances the reduction of NBTin PMNafter 30 min of stimulation by 1 gmol/liter of FMLP(enhancement after 15 and 60 min not shown). Addition of 10umol/liter of phosphoramidone to the incubation media doesnot affect NBTreduction in PMNexposed to ANP1-28 alone,FMLP alone, or the combination of FMLPand ANP 1-28.NBT reduction was also enhanced by ANP 1-28 when PMNwere exposed for a similar time period to 0.5 mg/ml of acti-vated zymosan (Table I).

The dose dependency of the effect of ANPon NBTreduc-tion is shown in Fig. 3. Both ANP 1-28 and a fragment withsimilar cardiovascular bioactivity, ANP7-28 (29), increase in adose-dependent fashion NBTreduction by PMNat 0.1 tmol/liter of FMLP. Concentrations of ANP 1-28 or 7-28 that en-hance FMLP-induced respiration burst activity were in therange of 0.1 nmol/liter to 1.0 jumol/liter. In comparison toANP1-28 and ANP7-28, which at a concentration of 10 nmol/liter stimulate NBT reduction at 0.1 umol/liter of FMLPby128% and 147%, respectively, ANP1-11 and ANP13-18 at the

Table L Stimulation of FMLP- or Opsonized Zymosan-inducedReduction of NBTby PMNin the Absence or Presence of ANP

NBT reduction at 30 min (OD550 .,, x 0.1)

FMLP Opsonized zymosan(10-7 M) (0.5 mg/ml)

Control 0.249±0.011 0.175±0.010ANP 1-28 (10-7 M) 0.330±0.011 0.247±0.012P value* 0.0209 0.0209

Values are given as mean±SEMof four experiments each performedin replicates of eight. * Wilcoxon-Mann-Whitney test, n = 4.

1582 C. J. Wiedermann, M. Niedermiuhlbichler, and H. Braunsteiner

12

I--

O o-_ C)

x= 'LU

m 0Z 0_-S

10

8

6

4

Z 140-0'-a ° 120

CDo FMLPZ 100 .. .. .. . .. .. .

0 -17 -15 -13 -11 -9 -7

Concentration [log M]

Figure 3. Stimulation by 10-' Mof FMLPof the reduction of NBTat30 min of incubation (370C) by PMNin response to the presence ofvarious concentrations of ANP1-28 (o; n = 4) or ANP7-27 (-; n = 3).Each point represents the mean percent of control values obtainedin independent experiments each performed in replicates of eight.

same concentration do not enhance respiration burst activity(Fig. 4). Under these test conditions, the known PMNprimingcytokines, TNF-a and GM-CSF(16), significantly increasedFMLP-induced reduction of NBT.

To confirm priming activity of ANP for respiration burstactivity, we examined the effect of ANP preincubation onPMNreduction of NBTor ferricytochrome c. Table II showsthat preincubation of PMNwith 10 nmol/liter of ANP1-28 at370C for 40 min significantly enhances the reduction of NBTupon addition of 0.1 smol/liter of FMLP. This enhancementof FMLP-induced NBT reduction was not reversible becausePMNpreincubated with ANP1-28, 10 nmol/liter, at 370C andthen washed before stimulation with FMLPstill demonstratedenhancement of NBTreduction (not shown). Fig. 5 shows thatin PMNpreincubated for 40 min with 0.1 fmol/liter to 0.1,umol/liter of ANP7-28 there is a bell-shape-curved, dose-de-pendent enhancement in the O2 production in response to 0.1,gmol/liter of FMLP. Table III shows that ANP7-28 signifi-cantly augments the secretion of O- in PMNtriggered withPMA. The enhanced rates of O- secretion or NBT reductionclearly demonstrate the priming ability of ANPto augment the

Table II. Effect of ANPor GM-CSFPretreatment on Reductionof NBTby PMNStimulated with FMLP

NBT reduction at 30 min (OD550 X 0.1) withFMLP(10-7 M)

Preincubation Percent(45 min, 37'C) Mean±SEM control P value*

Control 0.132±0.053 100ANP1-28 (10-" M) 0.153±0.054 127.9 0.0463ANP7-28 (10-8 M) 0.167±0.054 138.6 0.0414ANP 1-11 (10-8M) 0.132±0.047 106.4 0.9784ANP13-18 (10-8 M) 0.155±0.059 113.2 0.2050GM-CSF(10 ng/ml) 0.161±0.055 135.5 0.0414

Spontaneous NBT reduction at 30 min of incubation at 370C in theabsence of FMLPof PMNthat were preincubated for 40 min at370C in medium was 0.0014±0.0012 (mean±SEM), n = 6. * Stu-dent's t test (two-tailed, paired), n = 6.

respiration burst activity after addition of FMLP, zymosan,or PMA.

Wenext determined the directed migration of PMNintonitrocellulose micropore filters stimulated by FMLPin the pres-ence or absence of ANP 1-28. Maximal locomotion of PMNthat were stimulated by FMLPoccurs (after 35 min of incuba-tion) at concentrations of 10-100 nmol/liter. Higher FMLPconcentrations are less effective. In the presence of 0.1 emol/liter of ANP1-28, significant reduction of maximal FMLP-in-duced chemotaxis can be observed (Fig. 6).

To further assess the effect of ANPon PMNstimulus-in-duced function, we evaluated PMNenzyme release during 15min at 370C, measuring both total cellular and releasedamounts of fl-glucuronidase in the absence of cytochalasin B.Fig. 7 shows that PMNtreated with a combination of 0.1 tmol/liter of FMLPand 0.1 mol/liter of ANP 1-28 release signifi-cantly more lysosomal enzyme than PMNtreated with 0.1,smol/liter of FMLPalone.

Effect ofANP on cyclic GMPaccumulation in PMN. Thereare two known receptor classes for ANP, one of which containsguanylate cyclase on the cytoplasmic domain and stimulates

Figure 4. Stimulationby lIO- Mof FMLPofthe reduction of NBT

* * at 30 min of incubation(370C) by PMNin re-sponse to the presenceof 10-8 Mof ANP, ANPfragments, or the aceticacid vehicle control, andto 100 U/ml of TNF-aor 10 ng/ml GM-CSF.Bars represent the mean

and SEMof determina-tions in replicates ofat75 Lo eight from five indepen-

CL - a) Li tt dent experiments; *PZ CLX Z C < 0.05 (Student's t test;

<< two-tailed, paired).

E0V-

0Ec

Z0Z

w0

w.,

en)

o5 - 2

° Go-cml

N

ZI-Ea

O- -.c -

c

-

V- W 0 0 - d.CU C CX

X Z a Z L X,Z < Z < Z Z<c < < <

Figure 5. Stimulation of10-7 MFMLP-triggeredproduction of superox-ide anion at 10 min ofincubation (370C) byPMNby preincubationfor 40 min with variousconcentrations of ANP7-28 or 100 U/ml ofTNF-a. Each point rep-resents the mean andSEMobtained in fourseparate experimentseach performed in repli-cates of eight. *P < 0.05(Wilcoxon-Mann-Whit-ney test).

Neutrophil Priming by Atrial Natriuretic Peptide 1583

60 -

Q oo

- X

0CD C

U,0a

m c]Z o

40 -

20 -

CJ ('C Zr

Z Z

r.

Table III. Stimulation by Pretreatment with ANPor TNFof PMA-triggered Superoxide Anion Production in PMN

Superoxide anion secretion PMA(50 ng/ml)Preincubation

(45 min, 370C) Mean SEM P value*

nmo/1 06 PMNat S min

Control 7.22 0.64ANP7-28 (10-8 M) 10.62 0.55 0.0012TNF (100 U/ml) 10.97 0.68 0.0002

Spontaneous reductions of cytochrome c at 5 min of incubation at370C in the absence of PMA(50 ng/ml) by PMNthat were preincu-bated for 45 min at 370C in medium was 0.03±0.02 mmol/ 106 PMN(mean±SEM, n = 5). * Student's t test (two-tailed, paired), n = 6.

cellular cyclic GMPaccumulation (24). The level of cyclicGMPin PMNbefore addition of test substances was 29.3±2.61fmol/106 PMN(mean±SEM, n = 3). At 10 min of incubationwith the vehicle control, levels were 31.7±2.19 (n = 3). In thepresence of 100 ng/ml of PMA, cyclic GMPaccumulated tolevels of 60.3±5.21 fmol/106 PMN(n = 3), whereas 10 nmol/liter of ANP 1-28 did not affect cyclic GMPcontent of PMN(32.3±2.96 fmol/106 PMN, n = 3).

Discussion

In this study we have demonstrated that although ANPin theabsence of additional stimuli does not induce PMN°2 produc-tion, brief incubations of PMNwith ANPmarkedly enhancethe O2 production in response to PMA, FMLP, or zymosan. Aserum-free system was particularly critical for these experi-

220 -

200 -

E 180-

.2 160-

0)2 140-

120 -

- Control- -- ANP1-28 [10 7 MJ

Control -12-10 I -I

-10 -8 -6

FMLP [log M]Figure 6. Migration of PMNinto nitrocellulose micropore filters in-duced by different concentrations of FMLPin the presence (m) orabsence (o) of 10-7 Mof ANP 1-28. Mean±SEMdistance on the yaxis of four separate experiments each performed in triplicate. Mi-gration is significantly stimulated by FMLP(Kruskal-Wallis analysisof variance, P = 0.0005) but not by I08 Mof ANP1-28 (control vs.ANP1-28, n = 4; P> 0.05, Wilcoxon-Mann-Whitney test). Migrationof PMNat 10-1 Mof FMLPis significantly inhibited by ANP(*P< 0.05; Wilcoxon-Mann-Whitney test). Control is equal to migrationin the absence of FMLP.

P=0.0459 Figure 7. Effect of ANPon(D

4 p=O.0O13 FMLP-induced release ofX ~ n.s. fl-glucuronidase from PMN.a) i 3 Cells (2.5 X 10'/ml) were incu-

2,1 1 Xbated for 10 min at 370C with

Uc X medium, I0-' Mof ANP 1-28,2 lO` Mof FMLP, or a combi-2 - nation of ANP1-28 and FMLP

ez6 1 - S W at concentrations of 10-' MX each. In six separate experi-

0 ments, the total amount of5no a- 0- f3-glucuronidase present in un-

z -I zO< :E < stimulatedcells(0.121±0.017° c nmol/min X 106 PMN;

E mean±SEM), the amount re-LL leased and that remaining in

the cells were determined (re-coveries were 96.6±1.68% for ANP 1-28; 97.9±1.74% for FMLP;and 98.4±2.60% for ANP-plus-FMLP). The released activity is repre-sented as the percentage of the total cell enzyme content(mean±SEM). P, probability by Student's t test (two-tailed, paired).

ments because serum contains ANP(24). Using this approach,we have clearly demonstrated that ANPis similar to TNF-a interms of its ability to prime PMNin vitro for enhanced respira-tion burst activity. After priming by ANP 1-28 or ANP7-28,enhanced levels of NBT reduction or °2 secretion could betriggered by either the soluble stimulant PMAor the more phys-iologic stimulants FMLPor opsonized zymosan.

The enhancement is dependent on the concentration ofANPand the conservation of the carboxy terminus of ANP.The concentrations of ANP, which were found to be active invitro (0.1-1 nmol/liter) are well within the range of concentra-tions of ANP found in coronary sinus blood of humans inmyocardial ischemia (0.1-0.5 nmol/liter) (22, 24). The intact-ness of its disulfide bond may be important, inasmuch as onlyANP 1-28 and ANP7-28, but not ANP 1-11 or ANP 13-18were seen to be active. It is known from previous studies thatthe natural hormone ANP 1-28 binds indiscriminately to thecyclase-coupled and noncoupled receptors of ANP(24). Theamino-terminal domain of ANPcontributes to the biologicalactivity to a limited extent, and the removal of six amino-ter-minal residues up to Cys7 is tolerated without a dramaticchange in the relaxation of smooth muscles or natriuresis (re-viewed in reference 29). Our studies on activation of PMNrespiration burst activity demonstrated no significant differ-ence in the potencies of ANP1-28 and ANP7-28. Truncationof the peptide at the carboxy terminus of natural ANPwithdestruction of the ring structure abolished activity on PMN, asis known from studies on ANPstructure-activity requirementsemploying other tissues for bio- or ligand-binding assays (29).Data obtained from the present study on levels of cyclic GMPin PMNafter stimulation by ANP1-28 suggest that the effect ismediated by the non-guanylate cyclyase-coupled receptor ofANP, inasmuch as ANPfailed to stimulate cyclic GMPaccu-mulation in PMN.

The significance of the finding of PMNactivation by ANPis underlined by demonstration of other functions of PMNthatare also affected by ANP. Although ANPalone does not inducerelease of significantly greater amounts of PMNlysosomal en-zyme than the control untreated cells, it enhances the release oflysosomal enzyme in response to FMLP. Finally, presence of

1584 C. J. Wiedermann, M. Niedermuhlbichler, and H. Braunsteiner

ANP in chemoattractant incubation media inhibited theFMLP-induced chemotaxis of PMN. Ward and Becker (30)described deactivation of chemotaxis toward a chemoattrac-tant by incubation of PMNwith a second stimulus, a phenome-non that, with FMLP, was shown to be due to cellular hyperad-hesiveness (31). Similarly, PMNpreincubated or incubatedwith potent PMNpriming cytokines, such as TNFor GM-CSF(16), demonstrated a decrease in chemotaxis to FMLPthroughcellulose nitrate (32). Wealso observed that, in addition toTNF, other PMNpriming factors such as human growth hor-mone, tuftsin, and tuftsin-like tachykinins, but not IL-2 or IL-3inhibit chemotaxis through cellulose nitrate induced by FMLPor recombinant human IL-8 (26, 33). It was concluded that thedecrease in chemotaxis by neutrophil priming cytokines de-scribed above is due to in vitro hyperadhesiveness of PMNtoartificial surfaces causing reduced migration toward standardstimuli (32). The observed migration inhibition by ANPmaythus reflect enhancement of PMNadhesiveness by ANP.

Although the molecular mechanisms involved in ANPacti-vation of PMNfor enhanced function are not yet defined, thefact that priming could be elicited with a variety of triggeringagents that utilize distinct signal transduction pathways suggestsome possible mechanisms. Previous studies have demon-strated effects of PMN-priming factors alone on PMNfunc-tion. For example, TNF alone is a weak stimulant of H202production, iodination, and lysozyme release; incubationtimes up to 90 min with the stimulant were required for theeffects of the stimulant on PMNfunctional events to becomeevident (reviewed in reference 34). More potent effects of prim-ing factors on PMNrespiration burst activity were seen in re-cent studies when PMNwere coincubated with priming factorsand FMLPor PMA(35), or pretreated with factors and subse-quently stimulated with soluble stimuli (36). In the presentstudy, we observed that ANPenhanced oxidative burst andgranule exocytosis in PMNwhen coincubated or when stimu-lated after pretreatment of PMNwith FMLP, opsonized zymo-san, or PMA.

GM-CSFup-regulates FMLPreceptors on PMN, and GM-CSF priming can be elicited with FMLPbut not with PMA(37). Because enhanced O2 secretion could be triggered byFMLP, zymosan, and PMA, it is unlikely that ANPenhancesexpression of receptors for all of these stimulating molecules.Instead, the priming induced by ANPmay be related to en-hanced signal transduction: e.g., enhanced expression of pro-tein kinase C or augmented activity of the respiration burst orexocytotic systems.

The physiological role of preinflammatory factors in re-sponse to brief recurrent ischemia is the subject of intensivestudies (for review, see reference 38). Endotoxin treatment inrats seems to be associated with protective effects on reperfu-sion injury (39). It will be important to see whether the activecomponents of endotoxin, IL-1 and TNF-a, can protect theischemic or reperfused heart and/or cause the expression ofprotective stress proteins (40). A rise in oxygen-derived freeradicals after successful thrombolysis in myocardial infarctionwas recently reported (41) and such free radicals can inducesynthesis of heat-shock proteins (42). However, it is not knownwhether ANPcan induce stress proteins in vitro or in vivo. Ourresults add to the evidence for an important role of PMNinmyocardial ischemia by demonstrating for the first time thatANPwhich is released from cardial myocytes may efficientlyactivate the functions of PMN.

Acknowledgment

The work was supported by the Austrian Science Funds, grantP-7476-Med.

References

1. Romson, J. L., B. G. Hook, S. L. Kunkel, G. D. Abraham, M. A. Schork,and B. R. Lucchesi. 1983. Reduction of the extent of ischemic myocardial injuryby neutrophil depletion in the dog. Circulation. 67:1016-1023.

2. De Servi, S., A. Mazzone, G. Ricevuti, A. Fioravanti, E. Bramucci, L.Angoli, G. Stefano, and G. Specchia. 1990. Granulocyte activation after coronaryangioplasty in humans. Circulation. 82:140-146.

3. Lipowsky, H. H., S. Usami, and S. Chien. 1980. In vivo measurement of"apparent viscosity" and microvessel hematocrit in the mesentery of the cat.Microvasc. Res. 19:297-319.

4. Engler, R. L., G. W. Schmid-Schoenbein, and R. S. Pavelec. 1983. Leuko-cyte capillary plugging myocardial ischemia and reperfusion in the dog. Am. J.Pathol. 111:98-111.

5. Kohchi, K., S. Takebayashi, T. Hiroki, and M. Nobuyoshi. 1985. Signifi-cance of adventitial inflammation of the coronary artery in patients with unstableangina: results at autopsy. Circulation. 71:709-716.

6. Engler, R. L., G. W. Schmid-Schoenbein, M. Dahigren, and D. Morris.1985. Leukocyte depletion prevents edema formation during one hour of myo-cardial ischemia. Fed. Proc. 44:821. (Abstr.)

7. Sommers, H. M., and R. B. Jennings. 1964. Experimental acute myocardialinfarction: histologic and histochemical studies of early myocardial infarcts in-duced by temporary or permanent occlusion of a coronary artery. Lab. Invest.13:1491-1503.

8. Mullane, K. M., R. Kraemer, and B. Smith. 1985. Myeloperoxidase activityas a quantitative assessment of neutrophil infiltration into ischemic myocardium.J. Pharmacol. Methods. 7:733-738.

9. Braunwald, E., and R. A. Kloner. 1985. Myocardial reperfusion: a double-edged sword? J. Clin. Invest. 76:1713-1719.

10. Grisham, M. B., L. A. Hernandez, and D. N. Granger. 1986. Xanthineoxidase and neutrophil infiltration in intestinal ischemia. Am. J. Physiol.25 1:G567-G574.

11. Rowe, G. T., N. H. Manson, M. Caplan, and M. L. Hess. 1983. Hydrogenperoxide and hydroxyl radical mediation for activated leukocyte depression ofcardiac sarcoplasmic reticulum: participation of the cyclooxygenase pathway.Circ. Res. 53:584-591.

12. Mullane, K. M., N. Read, J. A. Salmon, and S. Moncada. 1984. Role ofleukocytes in acute myocardial infarction in anesthetized dogs: relationship tomyocardial salvage by anti-inflammatory drugs. J. Pharmacol. Exp. Ther.228:510-522.

13. Tillmanns, H., H. Leinberger, F.-J. Neumann, M. Steinhausen, N. Pa-rekh, and R. Zimmerman. 1984. Early microcirculatory changes during briefischemia-primary events in ischemic myocardial injury? Circulation. 70(Suppl.II):88. (Abstr.)

14. Maury, C. P. J., and A. M. Teppo. 1989. Circulating tumor necrosisfactor-alpha (cachectin) in myocardial infarction. J. Intern. Med. 225:333-336.

15. Tilg, H., J. Mair, M. Herold, W. E. Aulitzky, P. Lechleitner, F. Dienstl,and C. Huber. 1990. Acute phase response after myocardial infarction: correla-tion between serum levels of cytokines and C-reactive protein. Klin. Wochenschr.68:1083.

16. Arai, K., F. Lee, A. Miyajima, S. Miyatake, N. Arai, and T. Yokota. 1990.Cytokines: coordinators of immune and inflammatory responses. Annu. Rev.Biochem. 59:783-836.

17. Vane, J. R., E. E. Anggard, and R. M. Botting. 1990. Regulatory functionof the vascular endothelium. N. Engl. J. Med. 323:27-36.

18. Pohl, U. 1990. Endothelial cells as part of a vascular oxygen-sensing sys-

tem: hypoxia-induced release of autacoids. Experientia. 46:1175-1179.19. Skoglund, G., I. Cotgraeve, J. Rincon, M. Patarroyo, and M. Ingelman-

Sundberg. 1988. H202 activates CDl lb/CD18-dependent cell adhesion. Bio-chem. Biophys. Res. Commun. 157:443-449.

20. Tainer, J. A., S. R. Turner, and W. S. Lynn. 1975. Newaspects of chemo-taxis: specific target cell attraction by lipid and lipoprotein fractions of Esche-richia coli chemotactic factor. Am. J. Pathol. 81:401-410.

21. Said, S. I. 1990. Neuropeptides as modulators ofinjury and inflammation.Life Sci. 47:PLl9-PL-21.

22. Wiedermann, C. J., Wiedermann, F. J., Herold, M., Tiug, H., Moes, N.,Muhlberger, V., Beimpold, H., and Knapp, E. 1991. Plasma levels of atrial natri-uretic peptide and tumor necrosis factor-a during transient myocardial ischemiain patients with stable angina. Klin. Wochenschr. 69:944.

23. Falcone, C., M. Totorici, C. Tosti Croce, C. Greco, R. Rinaldi, A. Salviati,A. Berni, A. Nigri, and A. Reale. 1990. Changes of beta-endorphin plasma levels

during PTCAin patients with and without anginal pain. J. Am. Coll. Cardiol.15:226A. (Abstr.)

Neutrophil Priming by Atrial Natriuretic Peptide 1585

24. Brenner, B. M., B. J. Ballermann, M. E. Gunning, and M. L. Zeidel. 1990.Diverse biological actions of atrial natriuretic peptide. Physiol. Rev. 70:665-699.

25. Vollmar, A. M. 1990. Atrial natriuretic peptide in peripheral organs otherthan the heart. Klin. Wochenschr. 68:699-708.

26. Wiedermann, C. J., M. Niedermiihlbichler, H. Beimpold, and H. Braun-steiner. 1991. Priming of normal human neutrophils by recombinant growthhormone. Br. J. Haematol. 78:19-22.

27. Pick, E. 1986. Microassays for superoxide and hydrogen peroxide produc-tion and nitroblue tetrazolium reduction using an enzyme immunoassay micro-plate reader. Methods Enzymol. 132:407-421.

28. Dewald, B., and M. Baggiolini. 1986. Methods for assessing exocytosis byneutrophils leukocytes. Methods Enzymol. 132:206-277.

29. Bovy, P. R. 1990. Structure activity in the atrial natriuretic peptide (ANP)family. Med. Res. Rev. 10:115-142.

30. Ward, P. A., and E. L. Becker. 1968. The deactivation of rabbit neutro-phils by chemotactic factor and the nature of activatable esterases. J. Exp. Med.127:693-709.

31. Fehr, J., and C. Dahinden. 1979. Modulating influence of chemotacticfactor-induced cell adhesiveness on granulocyte function. J. Clin. Invest.64:8-16.

32. Schleifenbaum, B., R. Moser, and J. Fehr. 1990. A defect in chemotaxisfrom interleukin-2. N. Engl. J. Med. 323:682-683. (Lett.)

33 Wiedermann, C. J., M. Niedermtihlbichler, U. Zilian, D. Geissler, I. Lind-ley, and H. Braunsteiner. 1991. Priming of normal human neutropils by tachy-kinins: tuftsin-like inhibition of in vitro chemotaxis stimulated by formylpeptideor interleukin-8. Regul. Pept. 36: 359-368.

34. Steinbeck, M. J., and J. A. Roth. 1989. Neutrophil activation by recombi-nant cytokines. Rev. Infect. Dis. 1 1:549-568.

35. Shalaby, M. R., M. A. Palladino, Jr., S. E. Hirabayashi, T. E. Eessalu,G. D. Lewis, H. M. Shepard, and B. B. Aggarwal. 1987. Receptor binding andactivation of polymorphonuclear neutrophils by tumor necrosis factor-alpha. J.Leukocyte Biol. 41:196-201.

36. Berkow, R. L., D. Wang, J. W. Larrick, R. W. Dodson, and T. H. Howard.1987. Enhancement of neutrophil superoxide production by preincubation withrecombinant human tumor necrosis factor. J. Immunol. 139:3783-3791.

37. English, D. H., H. E. Broxmeyer, T. G. Gabig, L. P. Akard, D. E. Williams,and R. Hoffman. 1988. Temporal adaptation of neutrophil oxidative responsive-ness to n-formyl-methionyl-leucyl-phenylalanine: acceleration by granulocyte-macrophage colony stimulating factor. J. Immunol. 141:2400-2405.

38. Editorial. 1991. Stress proteins and myocardial protection. Lancet.377:271-272.

39. Brown, J. M., M. A. Grosso, L. S. Terada, G. J. R. Whitman, A. Banedjee,C. W. White, Alden H. Harken, and J. E. Repine. 1989. Endotoxin pretreatmentincreases endogenous myocardial catalase activity and decreases ischemia-reper-fusion injury of isolated rat hearts. Proc. Nati. Acad. Sci. USA. 86:2516-2520.

40. Tilg, H., J. Mair, M. Herold, and F. Dienstl. 1991. Stress proteins andmyocardial protection: is there a role for tumor necrosis factor? Lancet. 337:677(letter).

41. Davies, S. W., K. Ranjadayalan, and D. G. Wickens. 1990. Lipid peroxi-dation associated with successful thrombolysis. Lancet. 335:741-743.

42. Polla, B. S. 1988. A role for heat shock proteins in inflammation? Im-munol. Today. 9:134-137.

1586 C. J. Wiedermann, M. Niedermiihlbichler, and H. Braunsteiner