Diminished Polymorphonuclear Leukocyte Adherence

FUNCTIONDEPENDENTONRELEASEOF CYCLIC AMP

BY ENDOTHELIALCELLS AFTER

STIMULATION OF, -RECEPTORSBY EPINEPHRINE

LAURENCEA. BOXER, JOHNM. ALLEN, and ROBERTL. BAEHNER, with the technicalassistance of VICKTORIA AMICK, Department of Pediatrics, Division of PediatricHematologylOncology, James Whitcomb Riley Hospital for Children,Indianapolis, Indiana 46223

A B S T RA C T To investigate the biochemical andcellular basis for the rise in polymorphonuclear leuko-cyte (PMN) count during epinephrine administration,PMN from subjects receiving epinephrine werestudied for their capacity to adhere to nylon woolfibers and endothelial cell monolayers. After adminis-tration of epinephrine, the PMNcount increased by80% at 5 min, and isolated PMNadherence to nylonfibers fell from a base line of 44±2-18+3%. In contrast,when subjects were infused with the 13-antagonistpropanolol before receiving epinephrine, the PMNcount failed to rise and PMNadherence was normal.Exposure of PMNendothelial cell monolayers to 0.1,uM epinephrine led to diminished PMNadherencethat could be blocked by 10 uM propanolol but notby 10 ,uM phentolamine. Sera obtained from sub-jects 5 min after receiving epinephrine or from super-nates derived from endothelial cell monolayers ex-posed to 90 nMepinephrine inhibited PMNadherenceto nylon fibers. Addition of anticyclic AMPantiserabut not anticyclic guanosine monophosphate antisera tothe postepinephrine sera or to the postepinephrinesupernate derived from the endothelial cell mono-layers abolished their inhibitory effect of PMNad-herence to nylon fibers. In contrast, direct exposureof PMN to epinephrine failed to affect their ad-herent properties. Because it has been previouslyshown that endothelial cells contain 18-receptors andrespond to catecholamines by raising their intracellularconcentrations of cyclic AMP, and that PMNadherenceis attenuated by cyclic AMP, it would appear thatdiminished PMN adherence after epinephrine ad-ministration is mediated through endothelial cell

,3-receptor activity, which in turn impairs PMNmar-gination in vivo and could account for the rise in cir-culating PMN.

INTRODUCTION

The fight or fright reaction incites the release of epi-nephrine and norepinephrine from the adrenal medullaresulting in tachycardia, bronchodilation, peripheralvasoconstriction, lymphocytosis, and neutrophilia.Polymorphonuclear neutrophil leukocytes (PMN)llabeled with diisopropyl [32P]fluorophosphate, whenreturned to their donor's circulation, distribute equallyinto a circulation and a marginating pool. Athensand co-workers (1) rapidly infused epinephrine intofive human subjects and observed a rise in the bloodPMNconcentration to about twice the control levelswhile recovering an average of 72% of the donordiisopropyl [32P]fluorophosphate PMN, whereas, beforeinfusion, only 50% of the cells were retrieved (1, 2).Although PMN are known to reversibly adhere tovascular endothelium in vivo (3, 4), the cellularfactors regulating cell adherence to vascular endo-thelium (5-7) and to artificial surfaces in vitroare incompletely understood (8, 9). In an attemptto measure cell interaction with artificial surfaces, anin vitro assay has been developed that measures theadhesiveness between PMNand nylon fibers and re-cently has been shown to reflect similar adhesive prop-erties between PMN and endothelial monolayers(5). Using both techniques for measuring PMNad-hesion, we investigated the biochemical and cellularbasis for impaired PMNadhesion to endothelium in

Received for publication 3 October 1979 and in revised 1 Abbreviations used in this paper: GMP, guanosine mono-form 24 April 1980. phosphate; PMN, polymorphonuclear leukocyte.

268 J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. * 0021-9738/80/0810268107 $1.00Volume 66 August 1980 268-274

vitro after epinephrine exposure. We found thatdiminished PMNadherence in vitro after epinephrineadministration is mediated through the release of cyclicAMPby endothelial cells, which in turn could accountfor the rise in circulating PMNin vivo during epineph-rine administration.

METHODS

In viv) studies. After obtaining informed consent, adultvolunteers received 0.5-0.6 mg of epinephrine subcutane-ously; 20 cm3 of peripheral blood was obtained at 0, 5, 15,and 30 min for total white counts and differentials as wellas for PMNadherence studies. In some studies, the samesubjects were given an initial dose intravenously of 5 mg ofpropanolol followed by a constant infusion of propanolol0.08 mg/min for 45 min, at which time epinephrine was ad-ministered subcutaneously (10). To ascertain whether serafrom subjects receiving epinephrine would affect PMNad-herence, postepinephrine sera were recovered at varyingtimes and freshly obtained purified PMNat 1 x 107 cells/mlwere incubated for 5 min in 1 ml of the sera before passageover nylon fiber columns or before incubation with endo-thelial cell monolayers. The postepinephrine sera was main-tained at 4°C before use with PMN. In some studies, asindicated in the legends to the tables, the sera was incubated5 min with either anticyclic AMPor anticyclic guanosinemonophosphate (GMP) antiserum.

PMNadherenice. PMNadherence to nylon wool fibers wasassayed by a modification (8, 9) of the method of MacGregoret al. (11). The columns were packed with 70 mgof nylon woolto a height of 15 mmabove the constriction of the pipette.Suspensions of PMNand erythrocytes were derived from aFicoll-Hypaque gradient (12) and washed once in 0.15 MNaCl. 1 ml suspension of the PMNin human AB-negativeserum was incubated with various drugs for some experimentsfor 5 min before passage of the PMNover the columns.The suspensions of PMNcontaining between 0.8 and 2.0X 10-7 cells/mI were applied to the top of triplicate columnsand allowed to flow through by gravity at 37°C. Compari-son of pre- and postcolumn PMNcounts permitted calcula-tion of the percent of PMNadhering to the columns. Resultsof the triplicate columns were averaged for each blood speci-men and expressed as the percent PMNadherence. Thevariability of PMNadherence to the nylon fibers in the threecolumns did not exceed 10% beyond the meani.

Cultures of human endothelial cells were prepared fromhuman umbilical veins according to the modification ofCzervionke et al. (13) of the method of Jaffe et al. (14).Cells were suspended in medium 199, with Earle's salts(M-199): 9.87 g/liter medium 199 powder, 2 mML-glutamiine,0.05g/liter neomycin sulfate, 0.0025 g/liter fungizone (ampho-tericin B), and 2.2 g/liter NaHCO3, pH 7.4 (M-199 with 20%human serum in a ratio of 8 parts M-199:2 parts humanserum). The cells were seeded in 35 x 10 mim petri dishesand incubated in a 5% CO2 atmosphere at 37°C. After 24 h,the mnonolayers were washed with fresh M-199 with 20%human serum and then relayered vith 2.5 ml of the samiiemedium. Confluent primary endothelial moniolayers, con-taining 0.7-0.8 x 106 cells, were used for adherenice studies4-7 d after seeding. Cell counts were determined with ahemocytometer after suspension of the cells with 0.25%trypsin-0.05% trisodium EDTA in phosphate-bufferedsaline. Purified PMN were prepared by Ficoll-Hypaquedensity sedimentation using the technique of Boyum (12).After separation, the PMNwere washed twice in Krebs-

Ringer phosphate, pH 7.4, and then suspended in normalAB-negative human serum at a final concentration of 0.8-2.0x 107 PMN/ml. After removal of the M-199 from the tissuecultures, the plates were washed twice with Dulbecco'sphosphate-buffered saline, pH 7.4, and then 1 ml of PMNsuspension was added to triplicate plates just covering the35-mm diameter surfaces (5). The tissue culture-PMN overlaywas incubated at 37°C in a 5% CO2 atmosphere for varyingperiods of time as indicated in the legends to the tables; thesuspension was then aspirated. Comparison of the PMNcounts before and after incubation permitted calculationof the percentage of PMNadhering to the monolayers. Inexperiments evaluating the effect of drugs on the tissueculture-PMN overlay, the drugs were added directly to theexperimental plates at concentrations indicated in the tables.A gentle agitation followed to assure adequate mixing. Inother experiments, supernates derived from stimulated endo-thelial cell culture monolayers were incubated with freshlyobtained 1 x 107 PMN/ml for varying periods of time beforedetermination of subsequent PMN adherence to eithernylon fiber columns or fresh endothelial cell monolayers. Asindicated in the legends to the tables, anticyclic nucleotideantisera were added to the supernates for 5 min at 37°Cbefore determination of PMNadherence. Immunofluores-cence microscopy was used to demonstrate that antihemo-philiac factor was present in the cells leading to theiridentity as endothelial cells (14, 15).

Reagetnts. Powdered medium 199 (with Earle's salts, with-out HaHCO3), L-glutamine, neomycin sulfate solution, andfungizone (amphotericin B) were purchased from GibcoLaboratories, Grand Island Biological Co., Grand Island,N. Y. Culture flasks, petri dishes, and plastic centrifugetubes were purchased from Falcon Labware, Div. of Becton,Dickinson & Co., Oxford, Calif. Phentolamine, EDTA,and epinephrine were obtained from Sigma Chemical Co., St.Louis, NIo. Injectable epinephrine was purchased fromParke, Davis, & Co., Detroit, Mich.; propanolol hydro-chloride (Inderal) inijectable from Ayerst Laboratories, NewYork and Sigmna Chemical Co.

Statistics. Statistical significance was determined by com-paring drug effects with the control using the t test.

RESULTS

After the subcutaneous injection of 0.5-0.6 mg ofaqueous epinephrine, the absolute PMNcount rose at5 min and remained elevated throughout the 30 minof study (Fig. 1). Purified PMNobtained at various timepoints showed an initial adherence of 44±2% to nylonwool fibers before epinephrine administration and thenPMNadherence fell throughout the length of the study(Table I). The dose of epinephrine was sufficient toincrease the cardiac rate by 15-30 beats/min withconcomitanit widening of the pulse pressure. Systolicpressures rose by as much as 15 miml Hg, whereasdiastolic pressures fell by 10 mmHg. If the same sub-jects were treated initially with propainolol, a 8-adrenergic blocking agent, followed by epinephrine,the pulse rate decreased slightly and the diastolic bloodpressure rose slightly. On the other hand, the absolutePMNcount increased by no >23% at 15 min afterepinephrine administration (Fig. 1). Under the lattercircumstances, purified PMNobtained at various time

Imtipaired Polyymorphon uclear Adherenc e by CIJclic AAMP 9269

TABLE ITime Course of PMNAdherence to Nylon Fibers after

Epinephrine Administration or Administrationof Propanolol and Epinephrine

Propanolol thenTinie Epinephrine alone epinephrine

min %

0 44+2 44+35 18±3 41±2

15 23±11 40±330 26± 12 42±4

Values in the second column represent the mean±SD PMNadherence to nylon wool fibers of triplicate determinationsof six separate experiments on three control subjects. Valuesin the third column represent duplicate determinations ofthree separate experiments of PMNadherence after initialproponolol infusion to the same three subjects and then epi-nephrine administration.

herence to the greatest extent. This effect was pre-served by maintaining the PMNfor 1 h at 4°C beforerewarming the cells to 37°C for 5 min and then passingthe cells over the nylon fiber columns. Under suchconditions, PMNadherence was 26±5%. The effect ofsera on PMNadherence obtained 5 min after epi-

0 5 15 30

TIME (minutes)

FIGURE 1 The relative rise in the peripheral blood PMNcount in three separate subjects after the subcutaneous ad-ministration of 0.5 mg of aqueous epinephrine is shown inthe upper panel and represented by mean±SD. The rela-tive rise in peripheral blood PMN count subsequent topropanolol infusion in the same subjects followed by epineph-rine injection is shown in the lower panel. The remainder ofPMNcounts in the lower panel are expressed relative to thezero time in the subjects treated with propanolol andepinephrine.

points maintained normal adherence to nylon woolfibers throughout the duration of the study (Table I).

Purified PMNwere incubated for 5 min at 370C withvarying concentrations of epinephrine ranging be-tween 10 to 0.1 mMto establish whether the ef-fect of epinephrine on PMNadherence to nylon fi-bers was mediated directly or indirectly through a

soluble factor. As indicated in Table II, epinephrinefailed to directly affect PMNadherence to nylon fibers.In contrast, sera obtained from the subjects receivingepinephrine diminished adherence of freshly obtainedPMNfrom normal subjects. The sera obtained 10 minafter epinephrine administration attenuated PMNad-

TABLE IIEffect of Drugs and Postepinephrine Sera on PMN

Adherence to Nylon Fibers

AdherenceTreatment (meantSD) P value

None 47+3 (6)Epinephrine

0.1 mM 45±2(6) NS1,uM 44±2(6) NS90 mM 46+2(6) NS

Postepinephrine sera0 min 46±4 (9)

Anticyclic AMP 46±4 (3) NSAnticyclic GMP 50±5(3) NS

5 min 32±5 (3) <0.005Anticyclic AMP 53+6 (3) NSAnticyclic GMP 27+5 (3) <0.005

10 min 27+2 (3) <0.00515 min 34+7 (3) <0.0130 min 38±6 (3) <0.02

The number in parentheses represents the number of indi-vidual studies performed in triplicate. The PMNwere incu-bated with each of the drugs or with the postepinephrinesera, obtained at the indicated times from the subjects, for5 min before passage over the nylon fiber columns. In someexperiments a 1:50 dilution of either anticyclic AMPor anti-cyclic GMPantiserum was added to the postepinephrine sera.

270 L. A. Boxer, J. M. Allen, and R. L. Baehner

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TABLE IIIEffect of Endothelial Cell Monolayer Supernate

on PMNAdherence to Nylon Fibers

AdherenceTreatment (mean-SD) P values

None 48±44(11)Anticyclic AMP 57+9 (3) NSAnticyclic GMP 45+1 (3) NS

Epinephrine0.9 ,uM 19+6 (3) <0.001

Anticyclic AMP 47±8 (3) NSAnticyclic GMP 22±2 (3) NS

90 nM 37±3 (3) <0.0019 nM 49±2(3) NS

Isoproterenol0.1 ,uM 11± 1 (3) <0.00110 nM 29±7(3) <0.0011 nM 44±5(3) NS

The number in parentheses represents the number of indi-vidual endothelial cell monolayers derived from single um-bilical cords. The monolayers were covered with 1.5 ml offresh human serum to which the drugs were added for 5 minat 37°C. After addition of the drugs, the supernates were re-covered and added to fresh PMNfor 5 min before passage ofthe PMNover the nylon fiber columns. In some experiments,a 1:50 dilution of either anticyclic AMPor anticyclic GMPantiserum was added to postepinephrine supernate.

nephrine administration was completely abrogated byanticyclic AMPantiserum but not by anticyclic GMPantiserum. Exposure of PMNto the respective anti-sera alone lacked effect on adherence of the cells tonylon fibers.

Because epinephrine failed to directly affect PMNbut postepinephrine sera produced a marked impair-

ment in PMNadherence to nylon fibers, human endo-thelial cells were considered a potential source for theelaboration of the soluble factor modifying PMNad-herence. As noted in Table III, supernates derivedfrom endothelial cell monolayers previously exposed toepinephrine for 5 min significantly impaired adherenceof freshly obtained PMNto nylon fibers at concentrationsachievable in vivo. Similarly, exposure of endothelialcell monolayers to the f3-agonists epinephrine or iso-proterenol resulted in supernates that impaired PMNadherence to nylon fibers. Because f-agonists areknown to raise intracellular levels of cyclic AMP inendothelial cells as well as plasma levels in vivo, westudied the influence of anticyclic nucleotide antiserain supernates derived from endothelial cells previ-ously exposed to epinephrine. Anticyclic AMP, but notanticyclic GMP, completely abolished the effect of thepostepinephrine supernate on impairing PMN ad-herence to nylon fibers. Neither antiserum had any ef-fect on supemates derived from unstimulated endo-thelial cells. Similar to the temporal effect of seraobtained from subjects exposed to epinephrine, super-nate derived from endothelial cell monolayer stimu-lated by epinephrine impaired freshly obtained PMNadherence to nylon fibers (Table IV). The effect of thepostepinephrine supernate was maximal at 5 min andlost by 30 min. The preincubation of the endothelialcell monolayers with propanolol before the additionepinephrine abolished the inhibitory effect of thesupernate derived from the endothelial cell mono-layers.

The addition of 0.9 uM epinephrine directly toendothelial cell monolayers previously incubated for10 min with PMNled to decreased adherence of PMNto the endothelial cells. Addition of 10 ,M of the,3-antagonist propanolol to the endothelial cell pre-vented the effect of epinephrine on decreasing PMN

TABLE IVTime Course ofStimulated Endothelial Cell Monolayers Supernates on PMNAdherence to Nylon Fibers

Adherence(mean±SD)

Time (min)Stimulant 0 5 10 15 30

90 nM epinephrine 47±4 (3) 20±3 (3) 26+3 (3) 32±2 (3) 45±1 (3)P<0.001 P<0.001 P<0.001 NS

10 ,uM propanolol, then 47±4 (3) 46±5 (3) 53±1 (3) 45±5 (3) 46±2 (3)90 nM epinephrine NS NS NS NS

The number in parentheses represents the number of individual endothelial cell monolayers derivedfrom single umbilical cords. The monolayers were covered with 1.0 ml of fresh human serum to whichpropanolol was added for 2 min followed by epinephrine for 5 min. After incubation with the drugsat the indicated time, the supemates were recovered and added to fresh PMNfor 5 min at 37°C beforepassage of the PMNover nylon fiber columns.

Impaired Polymorphonuclear Adherence by Cyclic AMP 271

adherence (no addition, 44±3%; 0.9 ,M epinephrine,30±5%; 10 ,uM propanolal followed by 0.9 ,uM epi-nephrine, 42±8%). On the other hand, the addition of10 ,tM of the a-antagonist, phentolamine, failed toabrogate the effect of epinephrine (10 ,uM phentol-amine followed by 0.9 ,iM epinephrine, on decreasingPMN adherence 34±4%). Neither propanolol norphentolamine by themselves had any effect on PMNadherence to endothelial cell monolayers.

To further substantiate whether the findings ob-served with the nylon fiber columns paralleled thoseobtained with the endothelial cell monolayers, freshlyobtained PMNwere incubated for 5 min with eitherpostepinephrine sera obtained from subjects or post-epinephrine supernates derived from endothelial cellmonolayers; the subsequent adherence of the treatedPMNto new endothelial cell monolayers, containing10 ,UM propanolol to block the effect epinephrine, wasmonitored. As noted in Table V, both postepinephrineconditions impaired PMNadherence similar to that ob-served with the nylon fibers. Incubation of PMNthem-

TABLE VEffect of Postepinephrine Sera, Postepinephrine Supernates,

and Epinephrine on PMNAdherence toEndothelial Cell Monolayers

AdherenceTreatment (meantSD) P value

In vivoNone 46±4 (3)Postepinephrine sera 30±9(3) <0.01

In vitroNone 46±3 (3)Postepinephrine supernate 30±4 (3) <0.01Saline, wash 42±1 (3)Epinephrine, wash* 18±6 (3) <0.005

The number in parentheses represents the number of indi-vidual endothelial cell monolayers derived from single um-bilical cords. The PMNfrom three different subjects wereincubated for 5 min at 37°C with postepinephrine sera ob-tained 5 min after the administration of 0.5 mg epinephrinesubcutaneous, or the PMNwere incubated for 5 min at 37°Cwith postepinephrine supemates obtained 5 min after expo-sure of endothelial cell monolayers to 90 nM epinephrine.The PMNsuspended in either the postepinephrine sera orsupernate were added to fresh endothelial cell monolayersfor 10 min and then the number of adherent PMNdetermined.* Endothelial cell monolayers were exposed to 0.9 ,uM epi-nephrine or 10 gl saline suspended in 1.0 ml fresh sera for5 min at 37°C. The supemate was removed; phosphate-buf-fered saline, pH 7.4, was added to the cell suspension andthen removed; fresh PMNsuspended in 1.0 ml fresh serumwere added for 10 min before determination of PMNadherence.

selves with 90 nM epinephrine for 5 min followed bya wash in phosphate-buffered saline and resuspen-sion of the PMNin normal sera failed to attenuate PMNadherence to the endothelial cell monolayers (data notshown). On the other hand, if the endothelial cellmonolayers were directly incubated with epinephrinefor 5 min in the presence of serum, washed with phos-phate-buffered saline and a fresh PMNsuspension inserum added, PMNadherence to the stimulated mono-layer was significantly decreased (Table V). Similarly,PMNdirectly obtained from subjects 5 min after ad-ministration of epinephrine manifested attenuated ad-herence to endothelial cell monolayers (46±3% pre-epinephrine compared with 29±3% postepinephrine).

DISCUSSION

The effects of epinephrine on causing a rise in theperipheral PMNcount were first noted over 70 yr ago(16). These observations have been confirmed by manyworkers (2, 17); some have suggested that the sourceof the additional PMNin the bloodstream arises fromthe release of PMNfrom the marginating pool (1, 2).The development of techniques for establishing in vitrocultures of human endothelial cells (13, 14) allowedthe study of the interactiQn of PMNwith endothelialcells, leading to an understanding of some of themechanisms involved in PMNadherence, which likelyreflects PMNmargination in vivo (5).

The role traditionally assigned to the endothelialcell was that of a rather passive lining of the lumen ofthe vessels. Recent findings, however, suggest a muchmore active physiological role for the endothelialcomponent of the vessel wall. Bevan and co-workers(18) have suggested that adrenergic receptors may bepresent in the tunica intima of rabbit aorta, and thatinternal endothelial cells may play a role in regulatingchanges in vascular diameter induced by blood-bornvasoactive agents. Buonassisi and Venter (19) studiedendothelium cultured from rabbit aorta and found acatecholamine-stimulated cyclic AMP response thatcould be fully blocked by the 8-adrenergic antagonistpropanolol. Similarly, Herbst and his associates (20)found that norepinephrine increased cyclic AMPfor-mation in an incubated suspension of brain micro-vessels that could be abolished with propanolol.

In the present study, the ability to culture humanendothelial cell monolayers provided the means toinvestigate some factors regulating PMNendothelialcell interaction. Supernates derived from endothelialcells exposed to the a-agonist epinephrine and iso-proteronal-impaired PMNadhere to both nylon fibersand fresh endothelial cell monolayers. Employing theformer assay to monitor PMNadherence, the effect ofthe postepinephrine supernate was directly linked tothe length of exposure and dose of epinephrine, and

272 L. A. Boxer, J. M. Allen, and R. L. Baehner

was observed at a concentration of epinephrine achiev-able in vivo (21-23). Furthermore, the response ofthe endothelial cell monolayers to epinephrine wasdependent upon the activity of their /8 rather thana-receptors.

Paralleling the in vitro observations, sera re-covered from subjects receiving epinephrine at-tenuated PMN adherence to both endothelial cellmonolayers or to nylon fiber columns, similar to find-ings of others obtained with postepinephrine plasmaby employing the latter technique (24). The inhibitoryactivity found in the postepinephrine sera diminishedover a 30-min interval. Administration of propanololto subjects at concentrations known to prevent the risein plasma cyclic AMP(10) prevented the increase inPMN counts as well as led to retention of normaladherent properties after epinephrine injection. Directexposure of PMNto epinephrine in vitro failed to affecttheir adherent properties, which likely relates to theinability of epinephrine to substantially raise intra-cellular cyclic AMPlevels (24). Exposure of the PMN-endothelial cell overlay to epinephrine led to dimin-ished PMNadherence. Furthermore, once PMNen-countered postepinephrine sera, their attenuatedadherent properties could be preserved by maintainingthe cells at ice bath temperature for 1 h, which parallelsthe time and temperature conditions required for re-covering and purification of PMNfrom the blood ofsubjects administered epinephrine.

It would appear from the results of both the in vitroand in vivo studies employing the epinephrine an-tagonist, propanolol, that the soluble inhibitory factor iscyclic AMP. To confirm this hypothesis, both the post-epinephrine sera and the supernate derived from endo-thelial cell monolayers were incubated with anticyclicnucleotide antibodies. It was only in the presence ofanticyclic AMPantisera but not anticyclic GMPanti-sera that the inhibitory effect of PMNadherence wascompletely abrogated. The observation that cyclic AMPfound in the postepinephrine sera in this study is cap-able of impairing PMNadherence parallels the in vitroobservation by others that cyclic AMPadded to PMNwill attenuate their adherence to both nylon fibersand endothelial cell monolayers (5). These findingsindicate that,8-receptors on endothelial cells are stimu-lated by epinephrine to release cyclic AMP, which inturn could attenuate PMNadherence to endothelialvasculature thereby leading to a rise in circulating PMNduring epinephrine administration.

ACKNOWLEDGMENTS

We would like to thank the nursing staff of Saint Vincent'sHospital for providing the umbilical cords and Dr. John Hoakand associates of the University of Iowa for their help andencouragement.

This work was supported by a grant from the James Whit-

comb Riley Memorial Foundation. This work was done duringthe tenure of an Established Investigatorship (AmericanHeart Association) by Dr. Boxer.

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