1985 66: 1379-1383   

 B Meyrick, RJ Workman, MG Frazer, M Okamoto, JE Hazlewood and KL Brigham into bovine pulmonary artery intimal explantsEndothelial prostacyclin production is a late event in granulocyte migration

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Blood, Vol 66, No 6 (December), 1985: pp 1379-1383 1379

Endothelial Prostacyclin Production Is a Late Event in Granulocyte MigrationInto Bovine Pulmonary Artery Intimal Explants

By Barbara Meyrick, Robert J. Workman, Marshall G. Frazer, Masanori Okamoto,

Jeffrey E. Hazlewood, and Kenneth L. Brigham

Whether migration of granulocytes across pulmonary vas-

cular endothelium in the absence of structural evidence of

endothelial injury causes increased production of throm-

boxane or prostacyclin is not known. Using bovine pulmo-

nary artery intimal explants mounted in Boyden chambers

and homologous separated granulocytes. concentrations of

thromboxane B2 and 6-keto-PGF1,, in the upper-well fluid

were measured by radioimmunoassay over a three-hour

period under the following conditions: (1 ) granulocyte

chemotaxis (zymosan-activated plasma in the lower well,

granulocytes in the upper well); (2) unstimulated granulo-

cyte migration (serum or plasma in the lower well. granulo-

cytes in the upper well); (3) granulocyte activation without

migration (zymosan-activated plasma and granulocytes in

the upper well); (4) granulocyte chemotaxis in the absence

of endothelium (identical to condition 1 above except that

endothelium was scraped from the explant surface); and

(5) explants incubated in the absence of granulocytes.

Minimal increases in thromboxane B2 concentrations in

upper-well fluid occurred under all conditions. In contrast,

granulocyte chemotaxis was accompanied by large

A SINGLE IN FUSION of Escherichia co/i endotoxin

into sheep is followed by a two-phase response in the

pulmonary circulation. There is an initial phase of pulmo-

nary hypertension and a later increase in protein-rich lung

lymph flow. The latter change is interpreted as an increase in

pulmonary vascular permeability.’ Measurements of the

stable metabolites of thromboxane A2 and prostacyclin in

lung lymph show an early peak in the level of thromboxane

B, (TxB,) that is coincident with the marked increase in

pulmonary artery pressure.2 The increase in prostacyclin

production reaches its peak at approximately one hour, prior

to the increase in pulmonary vascular permeability.34 Struc-

tural studies have shown early and sustained sequestration of

granulocytes and lymphocytes in the pulmonary microcircu-

lation and migration of leukocytes into the alveolar intersti-

tium, followed, at one hour, by evidence of endothelial cell

damage.5 The increased levels of prostacyclin seen during

endotoxemia have been suggested to be the result of endothe-

hal damage5 possibly caused by activated granulocytes.67

Our recent studies using bovine pulmonary artery intimal

explants have shown, however, that granulocyte chemotaxis

and migration across an intact endothelial layer can occur

without structural or functional evidence of endothelial

damage.8

In light ofsuch findings, the present study was undertaken

to determine whether granulocyte migration across pulmo-

nary vascular endothelium in the absence of structural

evidence of endothelial injury is associated with increased

production of prostacyclin and/or thromboxane by vascular

tissue. Thus, the purpose of the present study was to deter-

mine the time course of prostacyclin and thromboxane

production by pulmonary artery intimal explants during

stimulated and unstimulated granulocyte migration and to

increases in concentrations of 6-keto-PGF1a evident by two

hours of incubation and increasing markedly by three

hours, to 524.3 ± 69.0 ng/mL (m ± SEM). Unstimulated

migration of granulocytes toward serum or plasma and

granulocyte activation without migration were accompa-

nied, at three hours, by more modest increases in 6-

keto-PGF1a (296.5 ± 46.4; 128.0 ± 38.6, and 236.7 ± 47.0

ng/mL. respectively) and, in the absence of granulocytes or

in the absence of endothelium. only minimal increases in

this prostacyclin metabolite occurred (137.2 ± 16.9 and

53.9 ± 12.6 ng/mL, respectively). The large rises in pros-

tacyclin metabolite occurred at a time when the majority of

granulocytes had migrated through the endothelial layer

rather than during their adherence or transendothelial

passage. We conclude that chemotaxis of granulocytes

through pulmonary vascular endothelium causes endothe-

hal production of large amounts of prostacyclin. but thisoccurs late in the chemotactic process. after granulocytes

have transversed the endothelium.

S 1985 by Grune & Stratton, Inc.

relate these findings to the rate of granulocyte migration into

the explants.8’9

Our studies show that increased prostacyclin but not

thromboxane production is a late event in granulocyte che-

motaxis across an endothelial cell layer, and that the prosta-

cyclin originates from endothelium and requires migration of

granulocytes through endothelium rather than interaction of

activated granulocytes with the endothelial surface.

MATERIALS AND METHODS

Bovine main pulmonary artery and blood were collected from alocal slaughterhouse on each day of experimentation.

Preparation of intimal explants. The main pulmonary arterywas slit with care to avoid any undue damage to the endothelial cell

layer and placed on a cork board. Discs ( I 3 mm in diameter) were

cut through the arterial wall with a brass cork borer and placed in

medium 199 (GIBCO, Grand Island, NY) in a sterile Petri dish. By

careful manipulation with dissecting forceps, the intimal layer was

stripped from these discs.

The discs of intima were floated onto nitrocellulose filters, connec-

From the Departments of Pathology and Medicine. Pulmonary

Circulation Center, Vanderbilt University School of Medicine,

Nashville, Tenn.

Submitted Dec 20, /984; accepted June 21. 1985.

Supported by grant No. HL 19153 (Specialized Center of

Research in Pulmonary Vascular Diseases) from the National

Heart, Lung and Blood Institute and by a grant from the Kroc

Foundation.

Address reprint requests to Dr Barbara Meyrick. Pulmonary

Circulation Center, B-1308, Medical Center North, Vanderbilt

University School ofMedicine, Nashville, TN 37232.

(r) / 985 by Grune & Stratton, Inc.

0006-4971/85/6606-0031$03.OO/O

For personal use only. by guest on July 11, 2011. bloodjournal.hematologylibrary.orgFrom

4*

1380 MEYRICK ET AL

tive tissue down. The filters were 1 3 mm in diameter and had a pore

diameter of 12 �zm (Sartorius Filters, Hayward, Calif). The filters

and explants were placed in chemotaxis chambers, endothelium

uppermost. The lower well of the chamber contained either 20%

homologous zymosan-activated plasma, 20% homologous heat-mac-

tivated plasma, 20% homologous bovine plasma, or 10% fetal calfserum in medium 199. Since the results with heat-inactivated

plasma were similar to those for plasma, only the latter are shown.

Medium 199 with the addition of 10% fetal calfserum was added tothe upper wells and the chambers were incubated at 37 #{176}Cfor 60

minutes in 5% CO2 in air to allow the endothelial cell layer to

equilibrate prior to the start of experimentation.Preparation of zymosan-activated plasma. Heparinized (20

U/mL) bovine blood was centrifuged and the plasma was removed

and incubated with zymosan (10 mg/mL; Sigma Chemical Co. St

Louis) at 37 #{176}Cfor 45 minutes. After incubation, the zymosan was

removed by centrifugation (2,000 rpm for 15 minutes) followed byMillipore filtration (0.45-�sm pore size; Millipore Corp, Bedford,

Mass). Some plasma was heat-inactivated by incubation at 60 #{176}Cfor

45 minutes. This plasma was then centrifuged and filtered as

outlined above.

Separation and labeling of bovine granulocytes. Granulocytes

were isolated by a modification of the method of Weening et al.’#{176}Heparinized blood was centrifgued and the buffy coat was removed

and added to an equal volume of 6% dextran in Hanks’ balanced salt

solution (HBSS). This suspension was floated onto Histopaque-1077

(Sigma Chemical Co) and centrifuged. RBCs that passed through

the gradient were lysed with ammonium chloride. The remaining

granulocytes were washed twice in H BSS. Direct counts of granulo-

cyte numbers were made using a hemocytometer. Wright’s stained

smears of the preparations showed that the pellets contained >95%

granulocytes, of which >95% were viable as judged by trypan blue

dye exclusion.

Radioimmunoassay for 6-keto-PGF,,, and TxB2. After appro-

priate dilutions, 6-keto-PGF,,, and TxB2 levels in the aliquots taken

from the upper well of the chemotaxis chambers were determined by

radioimmunoassay using 6-keto-PGF,a-(’251) histamine and TxB2-

(1251) histamine as tracers. The specific activity of these tracers is

�2,500 �sCi/izg. Each assay was carried out in triplicate. The

antibodies to 6-keto-PGF,a bovine serum albumin (BSA) and TxB2BSA were produced as recently described.”2 The detection limits

are 0.4 and 4.0 pg and the B/Bo at 50% displacement is 8 and 22 pg

for 6-keto-PGF,,, and TxB2, respectively. The cross-reactivity of the

antiserum to 6-keto-PGF,,, is 6.8% dinor-6-keto-PGF,a; 5.5% PGF�,;0.2% 6,15-diketo-13,14-dihydro-PGF,a; 0.12% 4,13-diketo-1 1,12-

dihydroprostanoic acid; 0.01% PGE2; 0.009% 6-keto-PGE,; and

0.006% TxB2; that of the antiserum to TxB2 is 0.39% POD2; 0.05%PGE2; 0.067% PGF�,,; 56% dinor-TxB2; 0.1% 6-keto-PGF,a; 0.23%6-keto- 13,1 4-dihydro-PGF,�; 0.03% 6, 15-diketo-PGF,�; 0.009%6,1 5-diketo-PGF,,; and 0.009% 6,1 5-diketo-l 3,14-dihydro-PGF,,,.

Experimental protocols. After equilibration, the medium in the

upper well of the chemotaxis chambers was replaced with 0.5 mL of

either 10% fetal calf serum or 20% zymosan-activated plasma in

medium 199 with the addition of granulocytes to give a final

concentration of 5,000 cells per cubic millimeter. In addition, some

chambers were prepared without the addition of granulocytes and

some with explants from which the endothelial layer had been gently

scraped away with a scalpel blade. The chambers were incubated for

30, 60, 1 20, or I 80 minutes; at the end of each time, a chamber from

each group was removed from the incubator. The fluid in the upper

wells was mixed, removed, and centrifuged at 1 ,000 rpm for ten

minutes. The supernatant was frozen for later measurement of the

stable metabolites of prostacyclin and thromboxane by radioimmu-

noassay.

Statistics. The means and SEMs were calculated for eicosa-noids and variables for each group at each time studied. Since the

experiments carried out with homologous heat-inactivated serum in

the lower well gave similar results to those with 10% fetal calf serum,

these data were pooled. The data were subjected to a split plot in

time design BMDP2V of the Biomedical Computing Programs(BMDP), computer series (analysis of variance and covariance with

repeated measures).’3 A P value of <0.5 was taken as significant.

RESULTS

Accumulation of6-keto-PGF,a. Chemotaxis of granulo-

cytes across an intact endothelial cell layer was accompanied

by marked accumulation of the prostacyclin metabolite,

6-keto-PGF,5, in the upper well ofchemotaxis chambers (Fig

I ). When zymosan-activated plasma was placed in the lower

well and granulocytes in the upper well of the chamber,

accumulation of 6-keto-PGF,,, was modest over the first 30

minutes but its accumulation became striking with longer

incubation periods, reaching a mean value of 524.3 ± 69.0

ng/mL (m ± SEM) at three hours (Fig 1 ). Unstimulated or

random migration toward either serum or plasma in the

lower well was associated with more modest increases in

6-keto-PGF,5, the levels being strikingly and significantly

less after three hours’ incubation than seen with granulocyte

chemotaxis (serum, 296.5 ± 46.4 ng/mL; plasma,

128.0 ± 38.6 ng/mL). When granulocytes were stimulated

but migration was inhibited (zymosan-activated plasma and

granulocytes in the upper well), the rate of 6-keto-PGF,a

accumulation was similar to that seen with random migra-

tion (three hours, 236.7 ± 47.0 ng/mL) (Fig 2). Prostacyclin

production by the intimal explant in the absence of granulo-

cytes was minimal over the three-hour period of incubation

6- keto- PGF,0(ng/mI)

600-

400-

200-

- ._Q,*

I 2 3

HOURS OF INCUBATION

Fig i . Accumulation of 6-keto-PGF1,. in the upper well of

chemotaxis chambers over three hours of incubation during che-motaxis (zymosan-activated plasma [ZAPI in the lower well (LWIand granulocytes in the upper well luwl of chemotaxis chamber;

ZAP . LW/PMN . UW [#{149};n = 6�), during random migration (SE-RUM . LW/PMN . UWEO; n = 5]) and from the intact intimalexplant alone (SERUM OR ZAP . LW/NO PMN [�J; n = ii]).Accumulation of 6-keto-PGF1,, is striking under conditions ofgranulocyte chemotaxis (ANOVA P < .OOi for ZAP . LW!

PMN . UW V both SERUM AND ZAP . LW/NO PMN). , P < .05

when compared to#{149}.

For personal use only. by guest on July 11, 2011. bloodjournal.hematologylibrary.orgFrom

600

6 -keto -PGF1�Ir�g/mII

I 2 3

HOURS OF INCUBATION

20

0�

A

1123 m ±S . E.

TxB2

(ng/mI)

1*82(ng /mI)

3

PROSTACYCLIN AND GRANULOCYTE MIGRATION 1381

Fig 2. Accumulation of 6-keto-PGF1,, in the upper well ofchemotaxis chambers over three hours of incubation during che-motaxis (zymosan-activated plasma [ZAP] in the lower well [Lw]and granulocytes [PMN] in the upper well [uw] of chemotaxis

chamber; ZAP . LW/PMN . UW [�; n 6]). when granulocytemigration is inhibited (ZAP + PMN . UW [L\; n = 5]) and duringchemotaxis when the endothelial layer of the explant has beenscraped away (ZAP . LW/PMN . UW-scraped Ix; n = 9]). In theabsence of endothelium, granulocyte chemotaxis into the intimalexplant gives rise to minimal 6-keto-PGF1,, production. Additional-ly. when chemotaxis is inhibited. little 6-keto-PGF1,, is released

(ANOVA P < .001 for ZAP . LW/PMN UW v both ZAP +PMN . UW and ZAP . LW/PMN . UW-scraped). , P < .05 whencompared to#{149}.

HOURS OF INCUBATION

Fig 3. Accumulation of TxB2 in the upper well [uw] ofchemotaxis chambers over three hours of incubation (A) when

granulocyte migration is inhibited (ZAP + PMN - UW [A; n = 5])and from the intact intimal explant (SERUM . LW/NO PMN [0;

n = i i ]), and (B) during granulocyte chemotaxis (zymosan-activated plasma (ZAP] in the lower well [Lw] and granulocytes in

the upper well of chemotaxis chamber; ZAP . LW/PMN . UW [#{149};n - 6]) and random granulocyte migration (SERUMLW/PMN . uw [0; n = 5]). Accumulaton of TxB2 is minimal and isnot significantly different under all conditions of incubation.

whether fetal calfserum (three hours, 1 18.2 ± I 1.7 ng/mL)

or zymosan-activated plasma (three hours, 169.7 ± 18.9

ng/mL) was in the lower well. Since 6-keto-PGF,,, concen-

trations were not significantly different between the groups,

the combined data from both of these groups is shown in Fig

When granulocytes were incubated either alone or in the

presence of 20% zymosan-activated plasma, even after three

hours of incubation, accumulation of either 6-keto-PGF,a

and TxB2 in the supernatant was <0.05 ng/mL.

To ascertain whether the endothelial cells per se were the

source of the increased levels of prostacyclin, intimal

explants whose endothelial layer had been removed with a

scalpel blade prior to experimentation were studied under

conditions of granulocyte chemotaxis (zymosan-activated

plasma in the lower well and granulocytes in the upper well).

Accumulation of 6-keto-PGF1� was minimal over the three

hours of incubation (53.9 ± 1 2.6 ng/mL) (Fig 2).

Accumulation of TxB2. Chemotaxis and/or random

migration of granulocytes across an intact endothelial layer

did not cause a significant increase in TxB2 accumulation in

the upper well of the chamber (Fig 3). In the absence of

granulocytes and under conditions in which granulocyte

migration was inhibited, accumulation of TxB2 was also

minimal. The combined data for serum (three hours,

6.0 ± 2.7 ng/mL) and for zymosan-activated plasma in the

lower well (three hours, 4.0 ± 0.4 ng/mL) is shown in Fig 3.

DISCUSSION

Using both scanning and transmission electron micro-

scopy, we have shown previously that the endothelial layer of

bovine pulmonary artery intimal explants is continuous and

“tight” junctions between endothelial cells are maintained.’4

Additionally, we have shown,9 using such explants mounted

in chemotaxis chambers and 5’Cr-Iabeled granulocytes, that

granulocyte chemotaxis across an endothelial cell layer

reached a plateau between the second and third hours of

incubation, when approximately 50% of the label was in the

explant. Unstimulated or random granulocyte migration was

lower and continued to increase to approximately 35% over a

three-hour incubation period. Inhibition of granulocyte

migration (granulocytes and zymosan-activated plasma in

the upper well) revealed a plateau from one hour, when

approximately 20% of the label was in the explant. Removal

of the endothelium by gentle scraping resulted in a rate of

granulocyte chemotaxis similar to that seen into the intact

explant.9

Granulocyte chemotaxis and prostacyclin produc-

lion. The present study followed the time course of both

prostacyclin and thromboxane production during granulo-

cyte chemotaxis toward zymosan-activated plasma and ran-

dom migration across an intact endothelial cell layer of

pulmonary artery intimal explants. Granulocyte chemotaxis

and, to a lesser extent, unstimulated granulocyte migration

led to striking accumulations of prostacyclin, but not throm-

boxane, in the upper well of the chemotaxis chamber after

two to three hours of incubation. Under conditions in which

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1382 MEYRICK ET AL

granulocytes were stimulated but migration was inhibited by

placing zymosan-activated plasma in the upper well, prosta-

cyclin production was similar to that seen with random

migration. In the absence of granulocytes (ie, with the

explant alone) only a small accumulation of prostacyclin was

seen over the three-hour period of study. In the absence of

endothelium, but in the presence of granulocyte migration,

prostacyclin accumulation was minimal. These findings mdi-

cate that passage of granulocytes across the endothelial cell

layer is followed by a marked increase in prostacyclin

accumulation and that the endothelial cells are the major

source of prostacyclin.

Under conditions of granulocyte chemotaxis, the present

study has shown a marked increase in prostacyclin produc-

tion occurring between the second and third hours of incuba-

tion, a time at which we have shown previously, using

51Cr-labeled granulocytes, that chemotaxis of granulocytes

toward zymosan-activated plasma has reached a plateau,9

and additionally, by electron microscopy, that the majority

of granulocytes are through the endothelial cell layer and its

basal lamina.8 Thus, increased production of prostacyclin is a

late event in granulocyte chemotaxis across an endothelial

layer, occurring mainly after migration has occurred. This

finding correlates well with our observations in sheep given

endotoxin. In these experiments, migration of granulocytes

into the interstitium occurs as early as 30 minutes following

the start ofendotoxin infusion5 and prior to the peak increase

in prostacyclin concentrations in lung lymph, which occurs

between 60 and 90 minutes.2’4

Zymosan-activated plasma is known to cause activation of

granulocytes both in vivo and in vitro. Our results with

zymosan-activated plasma in the upper well show that gran-

ulocyte activation and interaction of activated granulocytes

with the endothelial surface does cause a moderate increase

in prostacyclin production, but the increase is strikingly

higher when chemotaxis of granulocytes occurs. Thus, maxi-

mum generation of prostacyclin requires that granulocytes

migrate through an endothelial cell layer, since adherence of

activated granulocytes to the endothelial surface is a less

potent stimulus for prostacyclin production than chemotaxis

and maximum concentrations of prostacyclin occur late in

the chemotactic process.

From in vitro studies, it is known that the major source of

prostacyclin is the endothelial cell.’�’7 Harlan and Calla-

han’8 have recently shown that granulocytes stimulated with

phorbol myristate acetate cause cultured bovine endothelial

cells to release prostacyclin and that this release is H2O2-

dependent. Our findings also show that stimulated granulo-

cytes cause increased production of prostacyclin by the

endothelial cell. However, following granulocyte migration

through the endothelial cell layer (chemotaxis), prostacyclin

production is markedly enhanced. Thus, our studies indicate

that for such large amounts of prostacyclin to be released,

granulocytes must achieve an abluminal position.

In vitro experiments have shown previously that both the

aortic and pulmonary artery walls stripped of endothelium

can produce modest amounts of prostacyclin.’9 Thus, it is

possible that the endothelial cell is not the sole source of

prostacyclin but that some other cell type in the subendothe-

hum or arterial media contributes to its production. Addi-

tionally, it has been demonstrated, using immunofluores-

cence staining, that levels of prostacyclin synthase are

similar throughout the entire intima and media of vessel

walls.2#{176}However, our results with scraped explants strongly

suggest that the increased levels of prostacyclin in these

studies were derived principally from endothelial cells. Fur-

thermore, since the amount of prostacyclin that accumulated

was greater following chemotaxis than following random

migration, the number of granulocytes that obtain an ablu-

minal position may determine the amount of prostacyclin

produced.

Mechanism of increased prostacyclin production. The

mechanism for increased production of prostacyclin is not

certain. Endothelial cells grown in culture when stimulated

by thrombin, bradykinin, and histamine’6’21’22 produce

increased amounts of prostacyclin. It is possible, but not

likely, that such mediators were present in our preparations.

The increase in prostacyclin production in the present study

far exceeds the values reported in those earlier studies even

when the endothelial cell number (the endothelial cell num-

ber on the exposed surface of an intimal explant of 8 mm

diameter is approximately 500,000 cells) is taken into

account. Additionally, the timing of the marked increase in

prostacyclin metabolite is much later than that reported in

those studies. Thus, presence of mediators that stimulate

prostacyclin production could contribute to, but would not

seem to account entirely for, the increased prostacyclin

production seen following granulocyte chemotaxis. The pres-

ent data also confirm the findings of Goldsmith et al,23 who

demonstrated that prostacyclin production by ex vivo arterial

segments is markedly greater than that of the endothelial

monolayers.

The relative lateness of the increase in prostacyclin seen in

the present study may be explained by the availability of

arachidonate. It may be that granulocytes caused release of

arachidonate either from themselves or from other cells when

they burrowed through the endothelium, subendothelium or

media of the explants that were utilized by the abluminal

aspect of the endothelium. Alternatively, it may be that

granulocytes caused release ofarachidonate from endothelial

cell membranes, but only on contact with the abluminal

surface of endothelial cells. Under normal in vivo conditions,

a glycocalyx covers the luminal surface of endothelial cells

and could act as a barrier to granulocyte-endothelial interac-

tions, whereas a glycocalyx is not found on the abluminal

surface and thus interactions that trigger arachidonate

release could occur more readily.

The consequences of the increase in prostacyclin produc-

tion are also obscure. Migration of granulocytes through an

endothelial layer occurs in the absence of structural evidence

of endothelial cell damage but may still cause subtle pertur-

bations in endothelial metabolism and, certainly, changes in

cell shape. Release of the vasodilator prostacyclin, after

migration has occurred, could hasten the return of the

endothelial layer to its normal configuration. Alternatively,

the principal physiologic effect of prostacyclin release could

be an effect on microvessels distal to the site of prostacyclin

production.

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PROSTACYCLIN AND GRANULOCYTE MIGRATION 1383

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ACKNOWLEDGMENT

The authors thank i. Morrissey and L. Stone (Morrissey Meatsand Provisions, Nashville, Tenn) for supplying bovine blood and

pulmonary artery.

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