Alteration of Polymorphonuclear Neutrophil Leukocyte Response to

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 22, 2018 | https://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch015

15 Alteration of Polymorphonuclear Neutrophil Leukocyte Response to Shear Stress Exposure In Vitro: Effects of Prostaglandin E and Dipyridamole Derivative RA-233 1

1

DOUGLAS J. STOCKWELL and LARRY V. McINTIRE

Rice University, Biomedical Engineering Laboratory, Department of Chemical Engineering, Houston, TX 77001 R. RUSSELL MARTIN Baylor College of Medicine, Department of Internal Medicine, Houston, TX 77030

The effects of in vitro fluid mechanical trauma on buffered suspensions of polymorphonuclear neutrophil leukocytes (PMNs) were studied with and without preincubation with the platelet antiaggregating agents RA-233 (at 1 × 10 M) and prostaglandin E (at 3 × 10 M). Total PMN counts were reduced by exposure to shear stress of 100 and 300 dyn/cm for 10 min. The antiplatelet drugs diminished this reduction at the higher shear stress. Enzyme release, cell adhesion, and aggregation produced by mechanical trauma were all decreased by preincubation with the drugs, but the diminished generation of chemiluminescence following phagocytosis of opsonized zymosan by stressed PMNs was not affected. These results suggest a preservational effect of these "antiplatelet" agents on PMNs exposed to fluid mechanical trauma. -6

-6

1

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ncreased m a r g i n a t i o n o f leukocytes i n t h e c i r c u l a t i o n o f the l u n g t h r o u g h adhesion to t h e p u l m o n a r y e n d o t h e l i u m a n d formation o f platelet m i c r o aggregates have b e e n postulated as i m p o r t a n t components i n the etiology o f 1

Author to whom correspondence should be addressed. 0065-239#82/0199-0209$06.00/0 ® 1982 American Chemical Society Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS

p u l m o n a r y a n d cardiovascular complications a c c o m p a n y i n g surgical p r o cedures r e q u i r i n g e x t e n d e d extracorporeal c i r c u l a t i o n . I n previous reports o f the effects o f fluid m e c h a n i c a l trauma, l o w to moderate levels o f shear stress (100 to 300 dyn/cm ) p r o d u c e d a l t e r e d p o l y m o r p h o n u c l e a r n e u t r o p h i l leuko2

cyte ( P M N ) m o r p h o l o g y a n d f u n c t i o n . Shear-related changes i n c l u d e d i n creased a d h e s i o n , aggregation a n d c e l l lysis, moderate decreases i n c h e m i l u m i n e s c e n c e a c c o m p a n y i n g phagocytosis, a n d substantial loss of lysosomal Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 22, 2018 | https://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch015

e n z y m e s (1-4).

T h e s e alterations, i f they occur w i t h m e c h a n i c a l t r a u m a

a c c o m p a n y i n g e x t e n d e d extracorporeal c i r c u l a t i o n , may c o n t r i b u t e to the pathogenesis of post-perfusion reactions i n the lungs. A n t i p l a t e l e t therapy, i n c l u d i n g d i p y r i d a m o l e (Persantin) a n d its derivatives a n d prostaglandins, has b e e n effective i n r e d u c i n g microaggregate form a t i o n d u r i n g h e m o p e r f u s i o n (5-7). T h i s result p r o m p t e d us to investigate the action o f antiplatelet drugs o n the leukocyte responses to i n v i t r o shear stress. T h i s c h a p t e r demonstrates that a derivative of d i p y r i d a m o l e , R A - 2 3 3 , a n d prostaglandin E

{

( P G E O have the capacity to preserve P M N integrity

f o l l o w i n g exposure to l o w levels of shear stress i n v i t r o . T h e use of these two drugs s i m u l t a n e o u s l y is suggested b y t h e i r c o m p l e m e n t a r y actions i n raising intracellular c y c l i c adenosine m o n o p h o s p h a t e .

Experimental Heparinized (10 units/mL) blood was obtained from normal volunteers after written consent was given according to the declaration of Helsinki. A portion of blood was retained to determine white blood cell counts (Coulter, Model ZBI), hematocrit values, and leukocyte alkaline phosphatase (LAP) levels. Leukocytes were separated from the blood by sedimentation in heparinized dextran, as described previously (8). Leukocyte-rich supernatant was collected and washed twice in sterile distilled water for 30 s, then restored to the isotonic state with an equal volume of 0.30M saline. The cell suspension was then layered on 5-mL volumes of Ficoll-Hypaque white cell separation fluid (Ficoll; Sodium Hypaque, Winthrop) in 15-mL conical-tip centrifuge tubes centrifuged at 400 x g for 20 min. The white blood cell pellets obtained in this manner were more than 95% granulocytes and were consistently more than 90% viable when tested by trypan blue exclusion. These leukocytes were suspended in phosphate-buffered saline containing 150 mg/100 mL glucose (PBS/G) at concentrations of 2 x 10 PMN/mL. PMN suspensions were used without further delay in incubations with drugs and in shearing experiments, as determined by the individual studies performed. Incubation and Drugs. The RA-233 employed in this study was obtained from Wilbur Benson, Boehringer-Ingelheim, of Stamford, Connecticut. P G E i was provided by Joel Moake of the University of Texas School of Medicine at Houston. Cell suspensions were maintained at room temperature until addition of antiplatelet agents, at which time both drug-exposed suspensions and control suspensions were incubated at 37°C. Test samples contained 1 X 10" M RA-233 and 3 X 10" M P G E while control samples received equal volumes of sterile saline. Both control and test suspensions were incubated for 10 min in a water bath at 37°C, then were sheared for 10 min in the Rice University ROM-8 viscometer which was maintained at 37 ± 1°C with warmed forced air. 6

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Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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Application of Shear Stress. The Rice University ROM-8 viscometer has been described previously (9). This apparatus permits volumes of 8 mL of fluid to undergo uniform shear stress exposure at readily quantifiable levels. For the present experi ments, all surfaces coming into contact with leukocyte suspensions were coated with silicone (Siliclad), which had been demonstrated earlier to minimize or eliminate surface-mediated effects on PMNs (2). The surface-to-volume ratio in the viscometer could be varied by a factor of three using different bobs. Effectively, the fluid volume was varied at nearly constant surface area. Increasing the surface-to-volume ratio increased the accessibility of the surface to cellular elements in the sheared fluid. Shear stress levels were 100 and 300 dyn/cm for the 10-min exposure, which had been documented previously to produce functional alterations in PMNs. Control samples were placed into the viscometer for 10 min, but were not subjected to rotational shear stress. After exposure to the viscometer, cell suspensions were as sayed without further delay as described in the next section. P M N Assays. Leukocytes in suspension were quantitated by electronic particle counting to determine the changes induced by mechanical trauma. Values obtained after incubation with drugs or with saline were the baseline values to which post shear values were related. The assay for chemiluminescence has been described previously (4). Briefly, this procedure consists of measuring the light emitted following phagocytosis of opsonized zymosan particles, employing a model LS-100C Beckman liquid scintillation counter. Peak values for chemiluminescence were proportional to the total accumulated light emission over the 30-min test period, so tests were reported as peak chemilumines cence responses. Measurement of β-glucuronidase released in the surrounding medium was per formed using a modified Sigma assay as described previously (2). Alkaline phos phatase released from PMNs was assayed using the technique of DeChatelet and Cooper (10), employing a modification of Sigma assay Number 104 (Sigma Co.). A semiquantitative assessment of LAP activity was performed using a cytochemical assay (Sigma Co.). Azo dye staining of alkaline phosphatase-containing granules permitted grading of enzyme reactions from 0 to 4+. Values were reported as the total score for 100 cells evaluated microscopically. Measurement of PMN adhesion was performed using serum-coated glass slides at 37 C. Two, four-chambered Lab-Tek slides were filled with 200 μL of autologous serum per chamber, and were placed in a 37°C incubator for 15 min to warm the slide and to preincubate the glass surface with the serum. A volume of 40 μL of PMN leukocyte suspension was added to each test chamber, and the suspension was distrib uted evenly by gentle rocking and swirling for 30 s. Slides were replaced in the incubator on a flat surface for 25 min. The slides were then removed, and each chamber was centered on the stage of an inverted light microscope, where five medium-powered (400x) microscopic fields were counted for PMN leukocytes in contact with the glass. The serum was poured off gently, and the chambers were washed by slowly refilling and pouring off PBS/G heated to 37°C. Chambers were refilled with 200 μL of PBS/G and recounted for the adherent cells. The percentage of adherent cells was calculated by dividing the counts after washing with the counts before washing X 100. Tests of PMN adherence were always performed in duplicate. 2

C,

Results T h e total n u m b e r of leukocytes was c o u n t e d on a l l samples after each step i n the i n c u b a t i o n a n d shearing p r o t o c o l . T h e electronic particle counts


212


are p r e s e n t e d as percentages of the counts i n the initial suspension, p r i o r to the 10-min incubations at 37°C ( F i g u r e 1). I n the c o n t r o l specimens w h i c h w e r e not exposed to t h e d r u g a n d w h i c h w e r e p l a c e d i n the v i s c o m e t e r c h a m b e r but not sheared, the initial 1 0 - m i n i n c u b a t i o n at 37°C p r o d u c e d a d r o p to 92 ± 5 % (mean ± S E M ) i n the e l e c t r o n i c particle count w i t h a f u r t h e r decrease to 89 ± 6% after 20 m i n at 37°C. I n contrast to this gradual progres


sive aggregation, suspensions r e c e i v i n g the two antiplatelet agents d e m o n strated a m a r k e d transient aggregation, w i t h the e l e c t r o n i c particle count d r o p p i n g to 82 ± 7% of the c o n t r o l after 10 m i n , a n d subsequently r i s i n g to 92 ± 8 % after 20 m i n . T h e basis of the transient aggregation a n d disaggre gation observed w i t h P M N s i n c u b a t e d w i t h drugs is not w e l l u n d e r s t o o d . S h e a r i n g c o n t r o l P M N suspensions (not exposed to drugs) for 10 m i n at 100 and 300 d y n / c m p r o d u c e d a 25.5 ± 7 % 2

and 53.2 ± 3 . 5 % d r o p , r e -

σ

Before -Incubated Incubation 10 min.

On ly 20min.

- Incubated β S h e a r e d - Unincubated 100dynes 200dynes 300dynes 40 90 cm cm2 cm (minutes) 2

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Figure 1. Bar graphs showing changes in electronic particle count in PMN suspensions with and without drugs (1 x 10~ M RA-233 and 3 x 10~ M PGEj). All incubations and shearing were done at 37°C. The unincubated samples were maintained at room temperature for the time specified. The 200 dyn/cm columns show no error bars as they represent the results of a single day's runs. Key: •, without drug (control) and •, with drug. 6

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spectively, i n the e l e c t r o n i c particle count. I n contrast, samples r e c e i v i n g the antiplatelet drugs decreased 30.8 ± 6% f o l l o w i n g shear at 100 dyn/cm , b u t 2

d i d not d r o p significantly farther after the h i g h e r shear stress exposure (36.8 ± 2.8%). T h i s partial preservation of leukocyte counts after shearing at 300 dyn/cm c o r r e s p o n d e d to the r e d u c e d n u m b e r s of l y s e d and aggregated 2

cells i n m i c r o s c o p i c slides of the d r u g - t r e a t e d suspensions, c o m p a r e d to


sheared samples not exposed to drugs. A d h e s i o n of P M N s to serum-coated glass slides f o l l o w i n g shear stress exposure is p r e s e n t e d i n F i g u r e 2. Increased P M N adhesion was n o t e d after shear at 100 and 300 dyn/cm , b o t h for d r u g - t r e a t e d a n d untreated suspen 2

sions.

F o r platen-exposed

controls,

adhesion was r e d u c e d significantly

(13 ± 7%) i n d r u g - t r e a t e d samples c o m p a r e d to u n i n c u b a t e d suspensions (30 ± 9.5%). T h e d r u g - t r e a t e d leukocytes had lower levels of adherence fol l o w i n g exposure to shear stresses of 100 d y n / c m (21 ± 9%), c o m p a r e d to 2

42 ± 1 1 % for c o n t r o l samples. T h e slight apparent r e d u c t i o n i n adherence after exposure to 300 dyn/cm p r o b a b l y reflected selection of a less adherent 2

population of cells available for the assay, t h r o u g h selective loss of substantial n u m b e r s of adhesive-sheared P M N s to aggregation. B y c o m b i n i n g the adhe sion assay w i t h the particle counts and m i c r o s c o p i c assessments of aggregates in suspension, we are able to estimate that m o r e than 6 0 % of the P M N s

ΙΟΟι

co

ι

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1

80-

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Figure 2. Adhesion to serum-coated glass slides of sheared and unsheared leukocytes was reduced significantly by 1 x 10~ M RA-233 and 3 x 10~ M PGEi (±SEM). Only a less adherent population of cells remained to be tested at shear stresses greater than 300 dyn/cm because cell aggregates were removed during processing. Keu: ·, without drug (control) and A, with drug. 6

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r e m a i n i n g intact after 300 d y n / c m d e m o n s t r a t e d properties o f adhesion. 2

T h i s observation corresponds to those made p r e v i o u s l y at s i m i l a r shear stresses u s i n g a n y l o n fiber filtration t e c h n i q u e for m e a s u r i n g adhesion ( i ) . T h e filtration assay removes b o t h c e l l aggregates and i n d i v i d u a l adhesive PMNs. Phagocytosis o f o p s o n i z e d z y m o s a n particles p r o d u c e d c h e m i l u m i n e s cence i n P M N s , w h i c h was r e d u c e d by shear stress exposures o f 100 and 300 Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 22, 2018 | https://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch015

dyn/cm

2

( F i g u r e 3). Peak

chemiluminescence

levels w e r e

r e d u c e d to

74.4 ± 10% o f u n s h e a r e d c o n t r o l values b y exposure to 100 d y n / c m , and to 2

67 ± 5 % o f c o n t r o l values after exposure to 300 d y n / c m . F o l l o w i n g p r e 2

incubation w i t h P G E j and R A - 2 3 3 , s i m i l a r decreases i n c h e m i l u m i n e s c e n c e w i t h shear exposure o c c u r r e d . H o w e v e r , although not shown i n t h e g r a p h , the c e l l suspensions r e c e i v i n g shear stress t e n d e d to achieve peak values o f c h e m i l u m i n e s c e n c e somewhat m o r e slowly than the unsheared sample. Release

o f t h e lysosomal e n z y m e β-glucuronidase

into m e d i a sur

r o u n d i n g P M N s was s t u d i e d w i t h a n d w i t h o u t p r e i n c u b a t i o n w i t h drugs ( F i g u r e 4). T h i s lysosomal e n z y m e is c o n t a i n e d p r i m a r i l y i n the a z u r o p h i l i c granules of P M N s , a n d m a y b e a m a r k e r o f the active release o f these granules, and p r e s u m a b l y o f other e n z y m a t i c contents. Release may occur following lysis o f cells, o r as a result o f s u b l y t i c trauma. A 10-min exposure to 100 d y n / c m o f shear stress p r o d u c e d release o f 23 ± 4 % o f t h e total 2

PHAGOCYTOSIS - CHEMILUMINESCENCE

5 3

*

OLJ

0

.

I

100

200

I

300

SHEAR STRESS, dynes/cm

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Figure 3. Leukocyte peak chemiluminescence, with 1 X 10~ M RA-233 and 3 x 10~ M PGEi showing only small preservational effects at 100 dyn/cm , diminishing at higher shear stresses (sheared in PBS/G at 37°C for 10 min). Key: ·, without drug (control) and A , with drug. 6

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STOCKWELL ET AL.

100 σ α> 2? 80 h

co


I Ο

cr Ο _J I

300

SHEAR STRESS, dynes/cm Figure 4. Leukocyte β-glucuronidase activity released into surrounding media after shearing in PBS/G at 37°C for 10 min (±SEM). Significant preservation of activity occurred with 1 X 10~ M RA-233 and 3 x 10~ M PGEi at all stress levels. Key: ·, without drug (control) and A , with drug. 6

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β-glucuronidase content o f P M N s , w h i l e release o f 76 ± 5 % o f the e n z y m e o c c u r r e d at 300 dyn/cm . M a r k e d l y lower levels of e n z y m e release o c c u r r e d 2

w h e n cells w e r e i n c u b a t e d and sheared i n the presence of P G E j and R A - 2 3 3 . D r u g - t r e a t e d cells released o n l y 8 ± 4 % o f total β-glucuronidase after 100 dyn/cm , and 22 ± 6 % after 300 dyn/cm . M i n i m a l release of only 9 % of the 2

2

e n z y m e o c c u r r e d w h e n incubations w e r e p e r f o r m e d for 20 m i n at 37°C w i t h o u t shearing, w i t h s i m i l a r degrees o f release for b o t h drug-treated and control suspensions. T h e effects of m e c h a n i c a l shear stress o n release of L A P were m e a s u r e d c o l o r i m e t r i c a l l y i n supernatant fluids after shear, a n d c y t o c h e m i c a l l y o n b l o o d films. L A P is c o n t a i n e d p r i n c i p a l l y i n a different set o f granules (the specific granules) than t h e a c i d hydrolases, such as β-glucuronidase. W h e n placed i n the v i s c o m e t e r b u t not sheared, b o t h u n d r u g g e d and d r u g g e d suspensions e x h i b i t e d a 17% loss o f L A P activity. D a t a at 200 d y n / c m w e r e 2

l i m i t e d to the results o f a single e x p e r i m e n t , w h i c h revealed slightly less release (31%) o f L A P i n supernatants f r o m drug-treated cells c o m p a r e d to 3 8 % L A P release i n supernatants o f untreated P M N s . A t the h i g h e r shear stress l e v e l , 3 5 % of the L A P was released f r o m drug-treated samples, w h i c h was substantially less than t h e 66 ± 9 % release o c c u r r i n g i n supernatants o f untreated P M N s ( F i g u r e 5). T h e c y t o c h e m i c a l assay for alkaline phosphatase activity is s e m i q u a n t i tative, and the absolute results w e r e not expected to c o r r e s p o n d to the direct measurement o f e n z y m e activity i n supernatants s u r r o u n d i n g sheared


216



LEUKOCYTE

ALKALINE

PHOSPHATASE

300

SHEAR STRESS, dynes/cm Figure 5. LAP activity released into surrounding media after shearing in PBS/G at 37°C for 10 min. Partial preservation of unreleased activity with 1 x 10~ M RA-233 and 3 x I 0 ~ M P G E , (presented as % LAP not released to facilitate comparison with Figure 6). Key: ·, without drug (control) and A , with drug. 6

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P M N s . U s i n g this m i c r o s c o p i c assay o f e n z y m e activity, shear stress o f 100 dyn/cm p r o d u c e d a 40 ± 8 % d r o p i n L A P , w i t h a modest increase to 5 0 % loss 2

w i t h i n the intact cells r e m a i n i n g after 300 dyn/cm of shear stress ( F i g u r e 6). 2

In P M N leukocytes i n c u b a t e d w i t h the two drugs, o n l y a 17 ± 9 % decrease o c c u r r e d after 100 d y n / c m

2

a n d a 33 ± 10% decrease after 300 dyn/cm . 2

U n s h e a r e d samples that w e r e exposed to the v i s c o m e t e r surface for 10 m i n showed m i n i m a l changes i n c y t o c h e m i c a l values for L A P , decreases b e i n g about 5 % o f the total activity i n P M N s that w e r e not exposed to the visc o m e t e r surface.

Discussion As the c l i n i c a l use of b l o o d - p u m p i n g devices has increased, a n d surgical p r o c e d u r e s i n v o l v i n g e x t e n d e d extracorporeal c i r c u l a t i o n have b e c o m e w i d e s p r e a d , the effects of these p r o c e d u r e s o n f o r m e d elements of the b l o o d have b e c o m e c l i n i c a l l y i m p o r t a n t . A m o n g the better s t u d i e d t r a u m a - i n d u c e d effects o n b l o o d is that platelet microaggregates may f o r m , sometimes i n conjunction w i t h small c l u m p s o f P M N s , followed b y sequestering o f these elements i n the microvasculature (particularly that o f the lung). T h e release of lysosomal e n z y m e s a n d vasoactive substances f r o m platelets a n d P M N s


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may p r o d u c e increased vascular p e r m e a b i l i t y to plasma c o m p o n e n t s , and i f these abnormalities persist u n c h e c k e d , c l i n i c a l degeneration a n d cardiop u l m o n a r y failure may occur. C r a d d o c k et al. (11-14) d o c u m e n t e d that activation o f the c o m p l e m e n t system l e a d i n g to increased P M N adhesion may occur d u r i n g hemodialysis and h e m o p e r f u s i o n . A n a d d i t i o n a l m e c h a n i s m that may b e i m p o r t a n t i n the


pathogenesis o f p e r f u s i o n abnormalities is the c o n t r i b u t i o n o f the effects o f trauma f r o m b u l k shear stress, w h i c h is c o n s i d e r e d i n this study. Shear stress levels e n c o u n t e r e d d u r i n g hemodialysis and use o f several c o m m o n b l o o d p u m p s range f r o m 10 to 250 d y n / c m for p e r i o d i c exposures 2

of b e t w e e n 1 s and 1 m i n . T h e s e levels are w e l l w i t h i n the range o f shear stress exposures used i n this investigation. I n a d d i t i o n , e x t r e m e l y h i g h shear stresses (4000 dyn/cm ) o f short (millisecond) d u r a t i o n m a y occur i n the 2

presence o f m e c h a n i c a l valves, i n c l u d i n g artificial heart valves (15, 16). E a r l i e r w o r k i n this laboratory and others d e m o n s t r a t e d i n v i t r o dysfunction o f b o t h platelets and leukocytes f o l l o w i n g application o f l o w - l e v e l shear stress (1-4). T h e effects of m e c h a n i c a l t r a u m a can b e a t t r i b u t e d to b u l k shear stress effects rather than surface-mediated ones, since c h a n g i n g the surface-to-volume ratio b y a factor o f three d i d not alter the leukocyte o r platelet response to a g i v e n shear stress intensity and exposure d u r a t i o n (2). Platelet microaggregate f o r m a t i o n and adhesion alterations o c c u r r e d followi n g shear stress exposure i n t h e range o f 150 d y n / c m for 2 m i n , w h i l e 2

Ol

I 0

I

I

100

200

I 300

SHEAR STRESS, dynes/cm

2

Figure 6. Loss of cytochemically assayed LAP activity after shearing in PBS/G at 37°C for 10 min. Partial preservation of activity with 1 x 10~ M RA-233 and 3 x 10~ M P G E , at all stress levels. The points at 200 dyn/cm represent a single day's run. Key: ·, without drug (control) and A , with drug. 6

6


2

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leukocytes showed increased adhesion, alteration i n phagocytosis and chemotaxis, e n z y m e release, a n d decreased hexose monophosphate shunt activity after 150 d y n / c m for 10 m i n (2-4). T h e d e v e l o p m e n t a n d discovery of agents that affect platelet aggregation (antiplatelet agents) have c o n t r i b u t e d to the c l i n i c a l c o n t r o l of this aspect of post-perfusion l u n g c o m p l i c a t i o n s . O n e of the supposed actions of these agents is o n the c y c l i c n u c l e o t i d e system. D i p y r i d a m o l e (Persantin) a n d its derivative R A - 2 3 3 f u n c t i o n , at least i n part, as phosphodiesterase i n h i b i t o r s , c o n t r i b u t i n g to the elevation of i n t r a c e l l u l a r cyclic adenosine monophosphate ( c A M P ) b y r e d u c i n g its degradation to A M P . O n the other h a n d , P G E is k n o w n to stimulate adenylate cyclase, i n c r e a s i n g p r o d u c t i o n of c A M P b y that m e m b r a n e - b o u n d e n z y m e . W h e t h e r the i n vivo effects of either of these antiplatelet agents are m e d i a t e d b y t h e i r action o n intracellular c A M P remains to be s h o w n . T h e c o m p l e m e n t a r y actions of the two c h e m i c a l agents, however, make t h e m a l o g i c a l choice for the present study.


2

2

T h e action of P G E i and R A - 2 3 3 u p o n granulocytes f o l l o w i n g shear stress trauma can o n l y be s u r m i s e d i n c o m p l e t e l y f r o m the present i n v i t r o study. W h a t e v e r interactive effects these agents have w i t h b l o o d proteins, c o m p l e m e n t , or other f o r m e d elements of the b l o o d have b e e n m i n i m i z e d to investigate t h e i r m o d e of action u p o n granulocyte f u n c t i o n . G r a n u l o c y t e release of lysosomal e n z y m e s does involve m u l t i p l e and c o m p l e x processes. H o w e v e r , P G E j and R A - 2 3 3 appear to have a definite preservational effect u p o n this process, d u e i n part, to r e d u c t i o n of c e l l lysis following shear stress exposure. Increases i n adhesion r e s u l t i n g f r o m shearing P M N leukocytes also were greatly r e d u c e d by p r e i n c u b a t i o n w i t h the antiplatelet agents i n a m a n n e r similar to the effect of these agents u p o n platelet response to shear stress (17). T h i s s i m i l a r i t y suggests possibly related actions u p o n P M N leukocyte adhesion d u r i n g antiplatelet therapy after i n vivo c a r d i o p u l m o n a r y b y pass. T h e fact that no effect of these agents u p o n c h e m i l u m i n e s c e n c e res u l t i n g from P M N leukocyte phagocytosis was f o u n d may reflect the lack of relationship b e t w e e n P M N c h e m i l u m i n e s c e n c e and the cyclic n u c l e o t i d e system, or may be due to peculiarities w i t h i n the assay. T h e c h e m i l u m i n e s cence assay is p e r f o r m e d on samples w i t h the same adjusted P M N count. T h u s , even t h o u g h the activity p e r intact P M N is not changed b y d r u g incubation, the fact that P M N counts are h i g h e r after shear stress (300 dyn/cm ) i n the d r u g - i n c u b a t e d samples i m p l i e s that the total phagocytic ability of the treated P M N suspensions w i l l be h i g h e r . P M N leukocytes have been shown to have r e d u c e d efficacy i n c o m b a t i n g bacterial sepsis f o l l o w i n g prostaglandin therapy, despite the lack of evidence of effects of elevated intracellular c A M P u p o n P M N leukocyte phagocytosis after either m e c h a n i cal stress or i n f l a m m a t i o n (18). 2

T h e partial preservation of leukocyte n u m b e r s , f o l l o w i n g shearing of the suspension, i n the presence of P G E ! and R A - 2 3 3 is most p r o b a b l y d u e to the c o m b i n e d effects of r e d u c e d adhesion and aggregation and m i n i m i z a t i o n of


15.

STOCKWELL ET AL.


219

c e l l lysis, as n o t e d u p o n m i c r o s c o p i c examination o f slides made w i t h the suspensions. A l t h o u g h t h e sharp d r o p i n the w h i t e b l o o d c e l l count d u r i n g c a r d i o p u l m o n a r y bypass a n d subsequent r e b o u n d p r o l i f e r a t i o n o f i m m a t u r e granulocytes m i g h t not b e d e t r i m e n t a l o r pathological, preservation o f gran ulocytes b y the p r e v e n t i o n o f c e l l lysis a n d release can be c o n s i d e r e d signifi cant if this action occurs i n v i v o . T h e r e f o r e , the action o f these antiplatelet


agents may partly i n v o l v e o r b e s u p p l e m e n t e d b y a m e l i o r a t i o n o f leukoc yte functional changes r e s u l t i n g f r o m fluid m e c h a n i c a l trauma.

Acknowledgments T h i s research was s u p p o r t e d b y N I H G r a n t s H L 17437 a n d H L 18686. C o m p u t a t i o n a l assistance was p r o v i d e d b y the C l i n f o Project f u n d e d b y the D i v i s i o n o f Research Resources o f N I H u n d e r G r a n t R R - 0 0 3 5 0 . T h e authors thank M a r g a r e t P u t m a n for h e r t e c h n i c a l assistance i n this project.

Literature Cited 1. Dewitz, T. S.; Hung, T. C.; Martin, R. R.; McIntire, L. V. J. Lab. Clin. Med. 1977, 90, 728-736. 2. Dewitz, T.S.;McIntire,L. V.; Martin, R. R.; Sybers, H. D. Blood Cells 1979, 5, 499-510. 3. Dewitz, T. S.; Martin, R. R.; Sous, R. T.; Heliums, J. D.;McIntire,L. V. Microvasc. Res. 1978, 16, 263-271. 4. Dewitz, T.S.;McIntire,L. V.; Martin, R. R. Artif. Organs 1980, 4, 311-316. 5. Woods, N. F.; Weston, M. J. "The Use of Antiplatelet Agents During Hemo dialysis," Dial. Transplant. 1979 8, 958-960. 6. Addonizio, V. P.; Strauss, J. F.; Macarak, E. J.; Colman, R. W.; Edmunds, L. H. Surgery 1978 83, 619-624. 7. Richardson, P. D.; Galletti, P. M.; Born, G. V. R. Trans. Am. Soc. Artif. Intern. Organs 1976, 22, 22-28. 8. Martin, R. R.; Warr, G.; Gouch, R.; Knight, V. J. Lab. Clin. Med. 1973, 81, 520-526. 9. McCallum, R. N.; Lynch, E. C.; Heliums, J. D.; Alfrey, C. P. "Rheology of Biological Systems"; Thomas: New York, 1973; p. 70. 10. DeChatelet, L. R.; Cooper, M. R. Biochem. Med. 1970, 4, 61-68. 11. Craddock, P. R.; Fehr, J.; Dalmasso, A. P.; Brigham, K. L.; Jacob, H. S. J. Clin. Invest. 1977, 59, 879-888. 12. Craddock, P. R.; Fehr, J.; Bringham, K. L.; Kronenberg, R. S.; Jacob, H. S. New Eng. J. Med. 1977, 296, 769-773. 13. Craddock, P. R.; Hammerschmidt, D. E.; Moldow, C. F.; Yamada, O.; Jacob, H. S. Semin. Hematol. 1979, 16, 140-147. 14. O'Flaherty, J. T.; Craddock, P. R.; Jacob, H. S. Blood 1978, 51, 731-739. 15. Roschke, E. J.; Harrison, E. C.; Blankenhorn, D. H.Proc.28th Ann. Conf. Eng. Med. Biol. 1975, 17, 269. 16. Roschke, E. J.; Harrison, E. C. "Fluid Shear Stress in Prosthetic Heart Valves," J. Biomech. 1977, 10, 299-308. 17. Hardwick, Α., personal communication. 18. Deporter, D. Α.; Dieppe, P. Α.; Glatt, M.; Willoughby, D. A. J. Pathol. 1977, 21, 129. RECEIVED for review January 16, 1981. ACCEPTED May 1, 1981.


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