Effects of polymorphonuclear leukocytes on endothelial cell growth

L-01.

HRO?1BU'-;IS RE55EARC.H Printed in Great Britain

pp. 397-50;: Pergamon Press,

12,

1.978 Ltd.

R.E'FIXTS OF POLYMCRPHONUCLHARLEXKOCYTBS ON HNDOTHELIALCELL GROWTH

Hussain I. Saba, Robert C. Hartmann and Sabiha R. Saba Departmentsof Medicine and Pathology University of South Florida, College of Medicine and Veterans AdministrationHospital, Tampa, Florida USA

('Received 20.10.1977: Accepted

in revised form 20.11.1977. &Editor M.I. Barnhart) /"

ABSTRACT Polymorphonuclearleukocytes and their lysosomal constituents from rabbit and human sources were exsmined for their influence upon the growth of endothelial cells obtained from human umbilical cords. The endothelialcells were cultured in the presence of freeze thawed extracts of whole RcIN-leukocytes, freeze thawed and acid extract8 of P&&leukocyte lyeosomes and lysosomal cationic proteins (LCP). Growth stimulatoryas well as growth inhibitory activitieswere found in the lysosomal constituentsof PMNleukocytes.

INTRODUCTION The concept that endothelialcells might play an important role in thrombosisand hemostasis is not novel (1,2). This concept, however, has failed to attain widespread attention until recently. Within the last decade the endothelislcell's role in hemoatasis has been reemphasizedby the work of Spaet (3), Ashford (4) and a number of other workers (5,6). One problem in the study of endothelialcells has been the previous inability to obtain a sufficient number of these cells for in vitro studies. Recently, this difficulty appears to have been overcome, and endothelial cells can now be grown in sufficient numbers in tissue cultures for in vitro studies (T-12). This new breakthrough in endothelialcell research has led to discovery of a number of possible direct roles of these cells in hemostasis (13). Jaffe et al (4) demonstratedthe synthesis of factor VIII antigen and von Willebrand factor activity in these cells (15). Saba and Mason (16) have found an inhibitor of platelet wegation and other functions in endothelial cells. Finally, endothelisl cell injury has been implicated in the inflammatoryresponse (6,17-19). The roles of P+R?-leukocytes and their lysosomsl constituentsin inflammatory responses have been well delineated (20-22). A number of activities relevant to thrombosis and fibrinolyeiahave been found also in these cells 397

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(23-26). It is also known that in certain parts of the vascular system circulating PMN-leukocytesare present in close proximity to the endothelial lining. This population of PM&leukocytes comprises the so-cdlled =mulocyte Pool (27). The endothelialcell's role as the physical barrier between blood, its soluble and formed elements, and the tissue spaces is critical. Abnormalities of structure and function of endothelialcells can be related to diseases of blood vessels includingmicrosngiopathy(28) and thromboangitis(29). Since continuity of the vascular endotheliumis essential for survival of the organism, it is important to elucidate those factor(s) that influence the integrity, function, and growth of endothelialcells. Surprisingly,not much informationis available in regard to factors that influence the proliferationand growth of endothelialcells. Gospodarowicz (30)found that a fibroblasticgrowth stimulatingfactor obtained from the pituitary gland produced a significantstimulationof growth of endothelial cells in culture. Saba and Mason (31) have tested the effects of platelets end certain platelet componentsupon endothelislcell @ow-th and reported the presence of an endothelial cell growth stimulatingactivity in these cells. Effects of PM&leukocytes on the growth of endotheliumhave not been reported. The present study demonstratesthe influencesof PM&leukocytes and their lysosomal constituentsupon the mitotic activity of human endothelial cells in tissue culture. MATERIALS~ME!THODS Isolation of Rabbit end Human PM&Leukocytes PMN-Leukocytesfrom rabbits were obtained by inducing sterile peritoneal exudates with 0.25% glycogen in saline containing 68,000 U. Penicillin and 94 mg Streptomycinper L. Twelve to fourteen hours after intraperitoneal injection, the exudates were hervested, pooled together in ice-chilledflasks and the cells isolated by differentialcentrifugationby a method previously described (25). Human PMN-leukocyteswere obtained from patients with acute ;rJ ~~;$~logenous leukemia, in whom the white cell count exceeded . The human PMN-leukocyteswere separated from blood collected in EDNA anticoagulantby use of a Ficoll-hypaquegradient (32); a discreet band of PMN-leukocyteswas collected. The isolated PMN-leukocytesfrom both rabbit and human sources were then washed twice and processed to obtain various fractions. Ninety-five percent of these cells were recognized to be polymorphonuclesrleukocytes. Whole PMN-leukocyteextract was obtained by freezing and thawing (Fl')six times a cell susnension in Tris-bufferednoxmal saline, pH 7.4. The frozen and thawed extract was then centrifugedat 8OOOxg. The soluble supernatant fraction was collected and filter sterilized through a Millipore 0.224 filter (MilliporeCorp., Bedford, Mass.) for use in tissue culture experiments. PMN-leukocytelysosomes were obtained by homogenizationof human or rabbit granulooytesin 0.34 M sucrose according to the method describedby Cohn and Hirsch (33).

Preparation of I?'soluble fraction from PM&leukocyte lysosomes was obtainei by freezing and thawing a suspension of lysosomes in isotonic Pris-buffered saline. The suspensionwas then centrifugedat 8OOOxg. The resulting soluble supernatsntfraction, referred to as the FT extract, was then filter sterilized with a Yillipore 0.22~ filter and used in cell cultllrestudies. Acid extract of PMN-leukocytelysosomes was obtained by extractingwhole lysosones with 0.2N &SO4 for 45 minutes. The extract was then centrifugedat 83OOxg to obtain a supernatantthat was dialyzed first against O.OlN-HCl and then against Tris-bufferedsaline pH 7.4. This dialyzed acid extract was filter sterilizedwith a 0.22 p filter for use in cell culture experiments. The lysosomal cationic protein (LCP) fraction was obtained from a 2096vol/vol ethanol precipitationof the acid extract of lysosoaes by a method previously described (34). The 2C% ethanol extract was then dialyzed against Tris buffered saline pH 7.4 previously filtered for sterility and used in the zell culture studies. Isolation of Enndothelial Cells Endothelial cells were isolated from fresh human umbilical cords following the establishedmethod previously described (8,16,31).The umbilical vein was cannulated and filled with 0.2% collagenase (WorthingtonBiochemical Corp., Freehold, New Jersey) in modified Tyrode's solution (Ml's)(16). After 20 minutes of incubation at room temperature,the collagenasesolution containing the separated endothelidl cells was collected. The cell suspensionwas centrifuged,washed, and resuspended in tissue culture medium. Before implanting,the endothelial cells were checked for viability by the Erythrocin B (31)uptake test; at least 9596of the cells were viable. Method of Demonstratingthe Effect of PMN-LeukocyteFractions on the GroLFth of Endothelial Cells Cultures were started by seeding ?ndothelialcells at low cell density (rJl.OxlO4 cells/cm2) in polystyrene culture flasks (Falcon Plastics, Los Angeles, Ca.), with a growth area of 25 cm2. At approximately24 to 36 hours post seeding, log phase growth was achieved. The endothelialcell cultures were then divided into control and experimentalgroups each consisting of a minimum of two flasks. The old medium was decanted, and 4.0 ml of fresh tissue culture medium was added to each control and test flask. The control flasks then received 1.0 ml.of Tris-bufferedsaline at pH 7.4. The test flasks received the same volume of one of the various PMN-leukocytefractions. Test and control flasks were incubated at 37OC in a mixture of 95% air - 5% co2. The cultures were observed daily. Following four to five days of growth, when confluency in either the test or control group flasks was apparent, the experimentswere termimted, and the average cell density in each flask determined. For each determination,duplicatemonolayer cultures were washed twice with modified Tyrode's solution, and the cells harvested using 0.25% try-&n Detached cells were collected supplementedwith 0.05% EIYTAin norm&saline. in 10% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.) in MTS and counted using's hemocytometer. Triplicate counts consisting of 24 white cell squares were obtained for both the control end test cell populations. To determine the significanceof the cell count (mean cell number/cm2 + S.E.) the Students' t test (two-tailed)was employed. This was done to determine whether the difference between a control mean cell density and a test mean cell density was significantstatistically. Significancewas established only when a 2 value exceeded the 95% confidence limit (35).

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RESULTS

The Effect of the Soluble Fraction of ET BIN-Leukocyteson Endothelid Cell Growth A growth stimulatoryresponse of endothelial cells to the soluble FJJ extract of PM&leukocytes was observed. The stimulatoryresponse increased as the protein concentrationof this fraction was increased in the culture medium. Results are shown in Fig. 1. With 80~ of protein per ml the effect was insignificant. However, when 180 end $+O,ug of:protein were added per ml of tissue culture medium, the cell density increased to 7% and 22% of control

FIG. 1 Effect of the soluble fraction from l?l? extract of whole PMI?leukocytes on endothelid cell growtth: The abscissa represents the concentrationof this fraction perml of culture media. The ordinate shows the percentage change in cell growth. Percentage of stimulation (+) and inhibition (-) was calculated against the control group as 100% growth. Mean percentage change of growth 2 standard error for each protein conoentration represents at least four experiments.

Effects of the Soluble Fraction of FT Lysosomes from PNN-Leukocyteson En&thelisl Cell Growth Growth stimulation of endothelisl cells aleo was seen when ET extracts of lysosomes were used. However, in contrast to the soluble FJ?fraction of whole PMN-leukocytes,the stimulatory response of endothelial cells decreased progressivelywith higher concentrationsof the soluble BT lysoeomal fraction. As shown in Fig. 2, when 80-, 180 w, and 340~ of this fraction per ml of tissue culture medium was used, cell growth was 1396,9j6,and3.546 greater than each respective control.

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The effects of soluble Fl!fraction on PKN-leukocytelysosomes on endothelialceI1 growth: The abscissa represents the protein con centration per ml of culture media in the test flasks. The ordinate represents the percentage of change ingrowth. Percentage of stimulation (+) and inhihition (-) was calculated against the control flask as lCC$ growth. Mean percentage change of@;mwth~stsndi& error for each protein concentration representsat least four experiments.

Effects of an Acid Extract of Lysosomes on FndothelialCell Growth When different protein concentrationsof the acid extract of PMN-leukocyte lgsosomes were tested for effects on endothelial cell growth, the pattern was similar to that found in studies with FT extracts of lysosomes. Astimulatory growth effect was seen with the lower concentrationsof this fraction tested, and this stimulationprogressivelydecreased as the protein concentraWith 60 ,ng tion of the fraction was increased in the culture medium (Fig. 3).

FIG.

3

The effects of an acid extract of

\Ii

PNN-leukocytelysosomes on endcthelial cell grow-& The abscissa represents the concentrationof protein in micrograms per milliliter of culture media. The ordinate representspercentage of change in growth. Percentage of stimulation (+) and inhibition (-) was calculatedagainst the control flask as 10% growth. Mean percentage change of growth + standard error for each pmtein concentration represents at least four experiments.

of the acid extract protein used per ml of tissue culture medium, a 2196stimulatory effect was noted. However, with 100 m per ml, stimulationfell to 1896%; with 160 w per ml, stimulationwas only 12%; and with 36o,~g, a significant inhibition (-2196)was noted. The same pattern is depicted also with human lysoeomal fractions. As shown in Table 1, when acid extract protein from human PM&lysosomes was increased from 60 to 100~ per ml, cell growth fell from 2246to 12% comparing the respective controls.

PHS'S L ENDOTHELIAL

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Effect of Human PMN-LysosomeFractions on the Growth of EklothelialCells

I

Test Agent

Protein :oncentration [n Micrograms

Rndothelial Cell Density Cells/cm2 x 104 * Test

Nuubrr of In Growth Experiments

Control

cid Extract of

Lysosomal Cationic Protein: ,(LCP)

60

8.32

6.83

+22

3

100

3.77

3.38

+12

3

60

4.04

4.65

-14

100

5.16

6.22

-17

160

4.65

5.27

-24

* = Mean Values

The Effect of the Lysosomal Cationic Protein Fraction on EndothelialCell Growth When the lysosomal cationic protein fraction was used in the culture medium, a signifioantinhibitory effect of endothelialcell growth was observed at all concentrationstested. No stimulationof endothelialcell growth was observed with a wide range of protein concentrations. Indeed, inhibition progressivelyincreased as the lysosomal cationic proteinsfraction (LCP) concentrationwas increased in the growth medium. With protein concentrations of 60, 100, and 160 m per ml of rabbit LCP fraction, growth was inhibited by 696,1596,and 22% respectively (Fig. 4). When the protein concentration was increased from 160 to 340 mg per ml of culture medium, little increase in inhibitionwas observed. The same pattern of inhihitionwas observed when the humen LCP fraction was used in the endothelialcell cultures (Table 1).

py-

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I;

effects of '?ysosomalcationic pl'oteinsfrom PRN-leukocytescn endothelial ceil growth: The abscissa represents the concentration of lysosomal cationic protein in micrograms per milliliter ol‘ culture media. Tne ordinate represents the percentage of chsne in growth. Percentage of inhibition was calculated against the control flask as lOC$ growth. Mew percentage change of growth + standard error for each protein concentration represents at least four experiments.

The

PROTEIN

CONCENTRATION

IN MlCRCXiRAMS

DISCUSSION The results of these studies indicate that PEN-leukocytesinfluence the growth of endothelialcells in several different ways. The FT soluble extracts of PMN-leukocytescaused significant stimulationof cell growth. The amount of stimulationof endothelial~~11 growth was directly proportionalto the amount of protein in FT extracts of whole PM&leukocytes. When lysosonal extracts were used, a significantamount 'of growth stimulationoccurred at low protein concentration(80~ of FT extract of lysosomes);this same amount (80~3) of FI extract of whole PKN-leukocytesdid not produce any significant stimulation. The cell growth stimulatorypattern, with an even better response, was seen when acid extracts of lysosomes were used. The above findings indicate that PMN-leukocytescontain a growth stimulating factor for endothelialcells that appears to be localized in the PEJ leukocyte lysosomes. This is evident when stimulatoryeffects of weight-perweight protein values of extracts from whole leukocytes are compared with those of lysosomal extracts. 80 ug of protein from whole leukocyte extract produced no si@rificant stimulation (-2q6),but this amount of protein from the lysosomal extract exhibited significantgrowth stimulation (+ls) effect. On the other hand, higher concentrationsof either 3°For acid lysosomal extracts caused a progressive inhibition in endothelial cell growth. This inhibition indicates either a nonspecific effect of higher concentrationsof protein or the presence of a specific inhibitory factor in the lysosomes of PMN-leukocytes. The latter seems to be the more likely possibility. This is indicated by the finding that a more purified fraction of lysosomes,the lysosomal cationic proteins(LCP),produced inhibition of growth at significantlyiower protein concentrationsthan occurred with FT extracts of either PMN-leukocytes or its lysosomes. Indeed, no stimulatoryactivity was demonstratedover a wide-range of protein concentrationof this cationic protein fraction from

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lysosomes. Therefore, it appears that lysosomes of PM?-leukocytespossess both stimulatoryand inhibitory activities for endothelialcell growth. The growth stimulatoryactivity appears to be localized in the lysosomes of INN-leukocytes;its biochemical nature remains to be defined. The inhibitory activity, however, appears to be localized in the cationic proteins fraction of lysosomes, a fraction that contains no detectable stimulatoryeffects. It should be pointed out that in our present study we have not been able to use the normal human polymorphonuclesrleukocytes. This is due to the difficulty in obtaining the normal human donors for leukopheresisin order to collect sufficientamounts of leukocytes for these studies. The pattern of inhibitory and stimulatoryactivities in human leukemic polymorphonuclearleukocyteson endothelialcells is, however, similar to those in rabbit granulocy-tes. PMN-leukocytesand their extracts have been investigatedin vitro by others for their effect upon the growth of cell lines other than endothelial cells. Kovacs, et al (36) added the innoculum of canine blood granulocytes to soft agsr cultures of autologous bone marrow hematopoieticcells. They demonstratedthe presence of an activity in the FWN-leukocytesthat inhibited the colony formation rate of hematopoieticcells from bone marrow. Shadduck (37) recently studied FT extracts of native as well as peritoneal exudate gcanulocytesfor their capacity to stimulate colony formation of mice bone marrow cells. These studies indicated the presence of both stimulatoryand inhibitoryactivities for colony growth in the leukocyte extracts. The stimulatory activity from granulocyteswas only minimal, whereas the inhibitory activity was.found in the FI extract of granulocytesas well as in mononuclear macrophage cells. Shadduck further indicated that the inhibitor in the granulocy-teswas a small molecular weight protein. It is not clear at this stage whether the stimulator/inhibitoractivities in gxnulocy-tesdescribed by these workers are similar to those demonstratedby us to influence the growth of endothelialcells. It is interestingto note, however, that as in the work reported by Shadduck, the activity in grenulocytesthat inhibits endothelial cell growth appears to be present mainly in the low molecular weight lysosomal cationic proteins fraction (3~~38). PMN-leukocytesand their lysosomes have not been studied so far for their influenceupon the growth and function of endothelial cells. However, the adherence of PMN-leukocytesto the endothelial cells has been observed and reported by many workers and has been thought to be a manifestationof the inflammatoryresponse at the microvascular level. Plorey (39) reported sticking of white cells on inflamed vascular endothelium. Allison (40) observed the rolling action of granulocyteson the surface of endotheliumwhere blood flow was reduced. Fry (41) reported attachment of grsnulocytesto the endothelium. S&mid-Schoenbein, et al (42) studied the interaction of vascular endotheliumand leukocytes. Their observations indicate that PM&leukocytes come in close contact with endothelial cells under certain conditions. Nevertheless, it has remained undeterminedwhether leukocytes do influence directly the function of endothelialcells. Our studies indicate that DIN-leukocytes and their lysosomal constituentsdo influence growth of endothelialcells. Granulocytesappear to stimulate as well as inhibit the growth of endothelial cells in vitro. In view of the close relationshipbetween PMN-leukocytesand endothelial cells in the microcirculation,it is tempting to postulate that substances from these cells might be involved not only in control of their growth but also in other aspects of endothelialcell function. Furthermore,pathologic states associated with leukocyte disorders might produce alteration of endothelial cell function and vascular integrity.

i’Ol.lL!,?;O.?

This work was supported 3:; the Veterans Adinir-istration Medical Researzh Service, Tsznpa, Florida; the Anerican Cancer Society Grant Co the Oniversity of South Florida, Tanpa, Florida; and by Research Grant XL 18012 from B.I.S. %ne authors wish to achowledge their anpreciatioz t.oMr. Craig ';. .L?derson, for his technical assistance in this project.

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