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Evaluation of bovine polymorphonuclear leukocyte function
Veterinary Immunology and Immunopathology, 2 (1981) 157--174
157
Elsevier Scientific Publishing Company, Amsterdam -- Printed in Belgium
EVALUATION OF BOVINE POLYMORPHONUCLEARLEUKOCYTE FUNCTION J. A. ROTH and M. L. KAEBERLE Department of Veterinary Microbiology and Preventive Medicine, lowa State U n i v e r s i t y , Ames, IA 50011 (USA) (Accepted 4 November 1980) ABSTRACT Roth, J. A., and Kaeberle, M. L., 1981. Evaluation of bovine polymorphonuclear leukocyte f u n c t i o n . Vet. Immunol. Immunopathol., 2: 157-174. Bovine polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of c a t t l e . Five in v i t r o procedures were u t i l i z e d to evaluate PMN f u n c t i o n : I) Random migration under agarose, 2) Ingestion of 12Sliododeoxyuridine labeled Staphylococcus aureus, 3) Quantitative n i t r o b l u e tetrazolium reduction, 4) Chemiluminescence and 5) l o d i n a t i o n . Normal values for bovine PMNs are reported and i n t e r p r e t a t i o n of results is discussed. The PMN function tests were designed so that a l l 5 procedures may be performed in a short period of time on the same c e l l preparation. This allows f o r the detection and p a r t i a l characterization of a potential PMN dysfunction. INTRODUCTION A major function of the polymorphonuclear leukocyte (PMN) is the phagocytosis and destruction of invading microorganisms,
Therefore the PMN plays an
important role in protecting the animal from microbial i n f e c t i o n .
A congenital
or acquired defect in PMN function w i l l r e s u l t in an enhanced s u s c e p t i b i l i t y to i n f e c t i o n with b a c t e r i a l or fungal pathogens (Baehner, 1972).
When such a
condition arises, i t is desirable to be able to evaluate the phagocytic c a p a b i l i t y of the c i r c u l a t i n g PMNs. The a c t i v i t i e s of the PMN in c o n t r o l l i n g microbial i n f e c t i o n are complex.
In order to adequately evaluate PMN function
a v a r i e t y of in v i t r o tests must be u t i l i z e d concurrently.
Because PMNs are
short l i v e d c e l l s i t is necessary to perform the various function tests w i t h i n a short period of time; therefore each test must be r e l a t i v e l y simple to conduct and require a minimum of laboratory personnel time. This paper reports techniques f o r performing 5 tests that evaluate the function of bovine PMNs. These procedures are r e l a t i v e l y simple to perform. Most of the reagents can be prepared in advance and stored so that a l l of the 0165-2427/81/0000---0000/$ 02.50 © 1981 Elsevier Scientific Publishing Company
158
procedures can be performed the same day the blood is drawn from the animal. The 5 procedures q u a n t i t a t i v e l y evaluate: I ) random migration by PMNs, 2) the a b i l i t y of the PMNs to ingest opsonized p a r t i c l e s , 3)
the burst of
o x i d a t i v e metabolism associated with phagocytosis, and 4)
the myeloperoxidase
catalyzed reaction which is dependent upon the generation of hydrogen peroxide, the presence of myeloperoxidase in the granules, and degranulation.
These
procedures allow f o r the detection and p a r t i a l characterization of a potential PMN dysfunction. MATERIALS AND METHODS PMN preparation Blood was collected from healthy adult c a t t l e i n t o a c i d - c i t r a t e - d e x t r o s e s o l u t i o n (standard formula A) by j u g u l a r venapuncture, and PMNs were isolated using a modification of the method of Carlson and Kaneko (1973). was f i r s t
The blood
centrifuged at I000 X g for 20 minutes, the plasma and buffy coat
layer were aspirated and discarded, and the packed red blood c e l l s were lysed by the addition of two volumes of cold phosphate buffered (0.0132 M, pH 7.2) deionized water.
I s o t o n i c i t y was restored to the red blood cell
lysate a f t e r 45 seconds by the addition of one volume of cold phosphate buffered (0.0132 M, pH 7.2) 2.7% NaCI.
The PMNs present in the red blood cell
lysate were pelleted by c e n t r i f u g a t i o n and washed twice with Hanks balanced s a l t s o l u t i o n without Ca2+ and Mg2+ (HBSS) (Grand Island Biological Co., Grand Island, NY, USA). The cell suspension was then adjusted to a f i n a l concentration of 5.0 X 107 PMNs (neutrophils plus eosinophils) per ml of HBSS. Zymosan preparation Zymosan (Sigma Chemical Co., St. Louis, MO, USA) was suspended in cold Earles balanced s a l t s o l u t i o n with Ca2+ and Mg2+ and phenol red (EBSS) (Grand Island Biological Co., Grand Island, NY, USA) at a concentration of I0 mg/ml by homogenization f o r one minute with a Potter-Elvehjem tissue grinder. Preopsonized zymosan was prepared by adding I00 ml of fresh bovine serum to an equal volume of the zymosan suspension and s t i r r i n g for one hour at room temperature. minutes.
The zymosan was sedimented by c e n t r i f u g a t i o n at 250 × g for I0
Strong c o n g l u t i n a t i o n of the zymosan was reversed by washing the
zymosan p e l l e t twice with 200 ml of 0.01M sodium ethylenediamine-tetraacetic acid (EDTA) with s t i r r i n g for 15 minutes between washes.
The f i n a l p e l l e t was
159
resuspended in I00 ml of EBSS and frozen in aliquots at -20 C.
Prior to use
the preopsonized zymosan was thawed and homogenized with a Potter-Elvehjem tissue grinder.
When other concentrations of preopsonized zymosan were
needed, this preparation was concentrated by c e n t r i f u g a t i o n or d i l u t e d with a d d i t i o n a l EBSS to a r r i v e at the desired concentration. Random migration under agarose Random migration under agarose was evaluated by a modification of the procedure of Nelson, et a l . (1975).
Agar f o r evaluation of random migration
consisted of bicarbonate buffered (0.026 M pH 7.2) Minimum Essential Medium with Earles salts (Grand Island B i o l o g i c a l Co., Grand Island, NY, USA) containing 0.8% agarose (#57035 Gallard Schlessinger, Chemical Mfg. Corp., Carle Place, NY, USA), 10% f e t a l c a l f serum, and I% a n t i b i o t i c antimycotic solution (Grand Island B i o l o g i c a l Co., Grand Island, NY, USA). Five ml of warm agar (48 C) was poured into tissue culture pet r i plates (60 x 15 mm, #1007 Falcon, Oxnard, CA, USA) and the plates were stored at 4 C.
To evaluate
random migration by PMNs, wells 2.0 mm in diameter were made in the agar and were f i l l e d with an a l i q u o t of the PMN suspension (5.0 x 107 PMNs/ml). The migration plates were placed in an incubator with a humidified 5% CO2 atmosphere at 37 C.
Eighteen hours l a t e r the plates were removed from the
incubator, the surface of the agar was flooded with absolute methanol f o r 30 minutes, the methanol was removed and the agar was flooded with 47% formalin f o r an a d d i t i o n a l 30 minutes.
The agar was removed and the cells adhering
to the p e t ri plate were stained with modified Wrights stain.
The area of
migration was determined by tracing an enlarged image produced by a microscope with a drawing attachment, and measuring the area of the image with a planimeter.
The area of the projected image was mathematically converted to
the actual area on the migration plate and the area of the center well was subtracted. Ingestion of Staphylococcus aureus A coagulase p o s i t i v e s t r a i n of Staphylococcus aureus (S. aureus) was isolated from a case of bovine m a s t i t i s . d i r e c t l y onto a blood agar plate.
The milk was quarter streaked
A single colony was transferred to brain
heart infusion broth (BHI) (Difco, D e t r o i t , MI, USA) and incubated at 37°C f o r 18 hours.
Glycerine was added to a f i n a l concentration of 15%, and the
culture was aliquoted and frozen at -70 C.
This stock culture of bacteria
160
was used f o r the remainder of the experiment, Two ml of an overnight culture of S. aureus in BHI was inoculated into a 500 ml Erlenmeyer flask containing 250 ml of BHI with 250 ~Ci of 1251lododeoxyuridine (1251UdR) (New England Nuclear, Boston, MA, USA) and 10-5 M fluorodeoxyuridine (FUdR) (Sigma Chemical Co,, St. Louis, MO, USA). Following incubation fog 18 hours at 37 C, the bacteria were collected by c e n t r i f u g a t i o n , k i l l e d by heating to 60 C f o r 1 hr, and washed twice in 0.015 M phosphate buffered saline s o l u t i o n , pH 7.2 (PBS). The p e l l e t e d bacteria were suspended in PBS to make a stock suspension with a f i n a l concentration t h a t , when d i l u t e d I : I 0 in PBS, had an optical density of 0.4 at 600 nm (1.5 X 109 colony forming units/ml fo r comparably treated l i v e organisms).
The h e a t - k i l l e d 1251-1abeled S. aureus was stored at 4 C and used
over a period of several days. Bovine anti-S, aureus serum was produced by i n j e c t i n g a cow subcutaneously with a washed formalin k i l l e d suspension of S. aureus in PBS on two occasions I0 days apart.
Two weeks a f t e r the l a s t i n j e c t i o n , serum was harvested and
heat i n a c t i v a t e d at 56 C f o r 30 min.
The antibody t i t e r of the serum was
1:160 as determined by a plate a g g l u t i n a t i o n test with h e a t - k i l l e d S. aureus as the antigen. The standard phagocytosis assay was conducted in 12 X 75 mm p l a s t i c tubes containing I00 ~I of 1251UdR labeled S. aureus, 50 ~I of PMN suspension (2.5 X 106 PMNs, bacteria to PMN r a t i o = 60:1), 50 ~I of a I : I 0 d i l u t i o n of bovine anti-S, aureus serum, and 0.3 ml of EBSS. The S. aureus, serum, and EBSS were added to the tubes f i r s t 37 C water bath.
and incubated f o r at least 15 minutes in a
The PMNs were added and the tubes were agitated occasionally
to keep the p a r t i c l e s in suspension.
Exactly I0 minutes a f t e r the addition of
PMNs, 0.5 ml of PBS containing 0.5 unit of lysostaphin (Sigma Chemical Co., St. Louis, MO, USA) was added to each reaction tube and the background tubes. Following incubation at 37 C f o r an additional 30 minutes, 2.0 ml of PBS was added to each tube, and they were centrifuged at 1,250 X g f or I0 minutes. The supernatant f l u i d was aspirated and discarded, and the p e l l e t was washed with an a d d i t i o n a l 2.0 ml of PBS. The f i n a l p e l l e t was placed in a gamma counter to determine the counts per minute (CPM) of r a d i o a c t i v i t y present.
For
each assay a standard tube was set up which contained the standard amount of S. aureus but did not receive any lysostaphin.
A background tube was also set
up which contained a l l reactants except PMNs; i t did receive lysostaphin. All tubes were set up in duplicate and the average value used for c a lc ulat ions . The percent of the S. aureus ingested was calculated using the f ollow ing equation:
161
(CPM in reaction tube) - (CPM in background tube) Percent ingestion = (cPM in s'tandard tube) (CPM in background tube) x I00 The effects of the time allowed f o r phagocytosis and the bacteria to PMN r a t i o were determined by varying one parameter while holding the other parameters constant at the levels used f o r the standard phagocytosis assay. The kinetics of S. aureus ingestion by PMNs was studied by varying the phagocytosis time p r i o r to the addition of lysostaphin from 0 to 30 minutes. The e f f e c t of varying the bacteria to PMN r a t i o was determined by using a constant number of PMNs while varying the number of bacteria. PMN r a t i o s of 15:1, 30:1, 60:1, 120:1 and 240:1 were used.
Bacteria to
The e f f e c t of
cytochalasin B treatment was evaluated by incubating PMNs f o r 20 minutes at 37 C in EBSS containing 20 pg/ml of cytochalasin B (Sigma Chemical Co., St. Louis, MO, USA) and 0.1% dimethyl sulfoxide p r i o r to use in the k i n e t i c study described above. Nitroblue Tetrazolium Reduction Nitroblue tetrazolium (NBT) reduction by PMNs was evaluated by a modification of the procedure of Baehner and Nathan (1968). was prepared by suspending NBT (Grade I I I , USA) in EBSS at a concentration of 2 mg/ml. hour at ambient temperature. a 0.45 pm f i l t e r
The NBT solution
Sigma Chemical Co., St. Louis, MO, The mixture was s t i r r e d for one
Insoluble NBT was removed by f i l t r a t i o n
through
and the solution was stored at 4 C.
Tests were conducted in t r i p l i c a t e in 15 x I00 mm silicon-coated glass test tubes.
The standard reaction mixture contained 0.2 ml of NBT s o l u t i o n ,
5.0 x 106 PMNs, 0.I ml preopsonized zymosan preparation (I0 mg/ml), and s u f f i c i e n t EBSS to bring the t o t a l volume to 1.0 ml. for the determination of resting NBT reduction. amount of preopsonized zymosan was varied.
Zymosan was deleted
In certain experiments the
In other experiments the
preopsonized zymosan was replaced with 0.I ml of serum. All of the reactants, except the PMNs, were added to the tubes and allowed to e q u i l i b r a t e in a water bath at 37 C f o r 15 minutes.
The tubes were l e f t in the water bath, and
the reaction was i n i t i a t e d by adding the PMN suspension. were kept in suspension by periodic shaking.
Cells and p a r t i c l e s
A f t er exactly 5.0 minutes, the
reaction was stopped by adding 5.0 ml of cold ImM N-Ethylmaleimide (Sigma Chemical Co., St. Louis, MO, USA) in saline.
The c ells and p r e c i p i t a t e d
formazan were pelleted by c e n t r i f u g a t i o n at 500 x g f o r I0 minutes.
The super-
natant f l u i d was discarded and the p e l l e t was suspended in 5.0 ml of pyridine. Formazan was extracted by b r i e f sonication followed by heating in a b o i l i n g water bath f o r I0 minutes in a fume hood.
The pyridine-formazan was c l a r i f i e d
162
by c e n t r i f u g a t i o n at 500 x g f o r I0 minutes and the optical density (OD) at 580 nm was immediately determined in a spectrophotometer (Beckman DBG, Beckman Instruments, I n c . , I r v i n e , CA, USA), using a pyridine blank.
The
results are reported as OD/5.0 x 106 PMNs/5 min in 5.0 ml of pyridine. Determination of chemiluminescence Chemiluminescence was measured by a modification of the procedure of Allen et a l . (1972) in a l i q u i d s c i n t i l l a t i o n spectrometer (Model DPM I00, Beckman Instruments, Inc., I r v i n e , CA, USA) at ambient temperature with one p h o t o m u l t i p l i e r tube switched o f f .
The reaction was conducted in 20 ml glass
s c i n t i l l a t i o n v i a l s (Fisher S c i e n t i f i c Co., Pittsburgh, PA, USA). Test v i a l s contained 0.5 ml of preopsonized zymosan solution (0-40 mg/ml) and 1.0 x 107 PMNs in a t o t a l volume of I0.0 ml of bicarbonate buffered (0.026 M, pH 7.2) Geys balanced s a l t s o l u t i o n with Ca2+ and Mg2+ and without phenol red (Grand Island B i o l o g i c a l Co., Grand Island, NY, USA) (GBSS). The zymosan and GBSS were added to the s c i n t i l l a t i o n v i a l ; PMNs were added 1 minute before the v i a l was placed in the counting chamber.
The v i a l was capped, mixed w e l l ,
placed in the counting chamber and l e f t undisturbed f o r the remainder of the experiment. intervals.
Each v i a l was counted f o r 1 minute at approximately I0 minute Counting was continued f o r at least 70 minutes.
A v i a l containing
I0 ml of GBSS with no PMNs or zymosan was counted each cycle to determine the background a c t i v i t y .
The results were graphed as the number of counts per
minute at each I0 minute i n t e r v a l a f t e r the addition of PMNs. Differences in the height and shape of the curves with d i f f e r e n t PMN preparations required the reporting of results as t o t a l net counts per hour (cph).
The
cph were determined by estimating the area under the curve as shown in figure I .
The net cph of a test v i a l was calculated as follows:
net cph = cph of test v i a l - cph of background vial All experiments were conducted in duplicate and the average net cph are reported. Determination of i o d i n a t i o n The i o d i n a t i o n procedure was performed by a modification of the procedure of Klebanoff and Clark (1977).
The standard reaction mixture contained 40
nmole Nal, 0.05 wCi 1251 (Carrier free in 0.I M NaOH, New England Nuclear, Boston, MA, USA), 2.5 x 106 PMNs, and 0.05 ml preopsonized zymosan preparation
163
B
120
C
110.
D
, ~ 100. o
E F
l G "--
60
3O
20
10 0
1~1
21
31
4'1
,51
61
Time(minutes)
Figure I . This f i g u r e i l l u s t r a t e s the method used to estimate the counts per hour. The sum of the areas of the rectangles was determined by the f o l l o w i n g formula: (6) (A) + ( I 0 )
(B) + (I0)
(C) + (I0)
(D) + (I0)
(E) + (I0) (F) + (4) (G)
This is an estimate of the area under the curve f o r the f i r s t and, t h e r e f o r e , of the counts per hour.
60 minutes,
(I0 mg/ml) in a t o t a l volume of 0.5 ml EBSS. Zymosan was deleted f o r the d e t e r m i n a t i o n of r e s t i n g i o d i n a t i o n .
In c e r t a i n experiments the amount of
preopsonized zymosan was v a r i e d ; in other experiments the preopsonized zymosan in the standard r e a c t i o n mixture was replaced with 0.05 ml of unopsonized zymosan (I0 mg/ml) with or w i t h o u t 0.05 ml of serum.
A l l the
components except the PMNs were placed in 12 x 75 mm polystyrene snap cap t e s t tubes (#2058 Falcon, Oxnard, CA, USA) and allowed to e q u i l i b r a t e in a 37 C incubator.
The r e a c t i o n was s t a r t e d by the a d d i t i o n of PMNs, and the
mixture was incubated f o r 20 minutes at 37 C with end over end tumbling approximately 20 times per minute.
The r e a c t i o n was terminated by the
a d d i t i o n of 2.0 ml of cold 10% T r i c h l o r o a c e t i c acid (TCA).
The p r e c i p i t a t e
164
was collected by centrifugation at l,O00 x g for 5 minutes at 4 C and washed twice with 2.0 ml of cold I0% TCA. The counts per minute (CPM) of radioa c t i v i t y remaining in the p r e c i p i t a t e was determined in a Gamma Counter (Autowell I I , Picker Nuclear, North Haven, CT, USA). A blank containing a l l components except serum and leukocytes was run with each experiment and the results subtracted from the experimental values. A standard containing the t o t a l amount of 1251 in the reaction mixture was counted, and the nanomoles of iodide converted to a TCAprecipitable form per lO 7 PMNs per hour were calculated as follows:
(CPM experimental standard -CPMcPM
blank)(40, nmole N a l ) ( i . O lO?:~.~ x lO6 P-~s/PMNs~(60 min~,20 rain/
----
nmole Nal/lO 7 PMNs/hr All reaction tubes were run in duplicate and the average value was used for calculations.
Less than 0.5% of the t o t a l added r a d i o a c t i v i t y was TCA-
precipitable in the blank. RESULTS Leukocyte preparation The PMN i s o l a t i o n procedure performed on 50 separate blood samples from apparently healthy animals yielded the results shown in Table l .
An average
TABLE I Yield and p u r i t y of PMNs isolated from 50 samples of blood from healthy adult c a t t l e (Mean ~ S.D.). Percent PMNs in the PMN y i e l d per ml of Percent recovery of Percent eosinophils
f i n a l preparation whole blood (x lO~) PMNs in the PMN preparation
93.6 l .4 45.8 16.9
+ + ~ ~-
6.9 0.7 15.5 9.0
of 1.4 x 106 PMNs were recovered per ml of'blood; this represented an average recovery of 45.8% of the PMNs present in whole blood.
The f i n a l
PMN preparations contained an average of 76.7% neutrophils, 16.9% eosinophils, and 6.4% mononuclear c e l l s .
165
Random m i g r a t i o n under agarose The mean (~ SEM) area o f random m i g r a t i o n by 16 i n d i v i d u a l preparations of p u r i f i e d PMNs was 34.4 (~ 4.5) mm2 (Table I I ) .
Both e o s i n o p h i l s and
TABLE I I Influence of the presence of MNCs and Con A on the area of random m i g r a t i o n by bovine PMNs. Cells
Area of random m i g r a t i o n (mean + SEM, n = 16) mm2 --
PMNs PMNs + Con A PMNS + MNCs PMNS + MNCs + Con A
34.4 21.8 19.1 11.3
n e u t r o p h i l s were observed to migrate.
+ 7 ¥ #
4.5 3.8 3.3 2.9
When an equal number of autologous
MNCs were added to the PMNs the area of random m i g r a t i o n decreased to 19.1 (~ 3.3) mm2.
The a d d i t i o n of Con A to e i t h e r the p u r i f i e d PMNs or the
PMNs plus MNCs markedly decreased the area of random m i g r a t i o n . S. aureus i n g e s t i o n assay A s i n g l e preparation of 1251-iododeoxyuridine labeled S. aureus was used to evaluate the a b i l i t y
of 48 i n d i v i d u a l PMN preparations to ingest S. aureus.
A 60:1 b a c t e r i a to PMN r a t i o was used and lysostaphin was added a f t e r I0 min. A mean (~ SD) of 52% (~ !2.6%) o f the b a c t e r i a were ingested.
This was an
average of 31.2 organisms ingested per PMN (Table I I I ) . TABLE I I I Mean values f o r the PMN f u n c t i o n tests described here when performed on PMNs obtained from normal animals. PMN f u n c t i o n t e s t
Mean
SD
SEM
(n)
M i g r a t i o n under agarose (mm2) Staph i n g e s t i o n (percent of a 60:1 Bact:PMN r a t i o ) NBT reduction (OD/5.0 x 106 PMNs/ 5 min in 5 ml p y r i d i n e ) Chemiluminescence (cph x 106 ) l o d i n a t i o n (nmole Nal/lO 7 PMNs/hr)
34.4 52.0
17.9 12.6
4.5 1.8
(16) (48)
0.49
0.20
0.03
(56)
5.21 48.3
1.57 18.7
0.25 2.5
(38) (56)
166
The e f f e c t of varying the time allowed f o r phagocytosis to occur before l y s o s t a p h i n was added is shown in Figure 2.
When l y s o s t a p h i n was added at the
700
6050-
Q.
4030-
.o 0 m_
....{ ..... .......
..... f ............ { ............ ...........
it....-'I"
20100
0
5•
10 15 20 T' , , Time Iminutesl
25 '
30
Figure 2. Percent i n g e s t i o n of a suspension of S. aureus by normal PMNs ( ...... ) and by PMNs which were t r e a t e d w i t h Cytochalasin B ( ) when l y s o s t a p h i n was added a t v a r y i n g times f o l l o w i n g the a d d i t i o n of PMNs to the phagocytosis m i x t u r e . A b a c t e r i a to PMN r a t i o o f 60:1 was used. (Mean ~ SEM, n = 4). same time PMNs were added there was an average o f 12.4% uptake of S. aureus. There was an increase in the average percent uptake of S. aureus w i t h time f o r the e n t i r e 30 minute i n c u b a t i o n p e r i o d to a maximum o f 61.1%,
The S.
aureus to PMN r a t i o was 60:1; t h e r e f o r e , each PMN ingested an average of 36.6 organisms during the 30 minute p e r i o d . b a c t e r i a to PMN r a t i o
The e f f e c t of varying the
is r e p o r t e d in Table IV.
As the number of b a c t e r i a
was increased the number o f organisms ingested per PMN also increased; however, the percent uptake remained r e l a t i v e l y PMN r a t i o of 120:1 was reached, significantly 240:1.
constant u n t i l
a b a c t e r i a to
At t h i s p o i n t the percent uptake dropped
w i t h a f u r t h e r marked decrease at a b a c t e r i a to PMN r a t i o of
167
TABLE IV The e f f e c t of increasing the number of bacteria in the phagocytosis mixture on the percent uptake of radiolabeled organisms. Phagocytosis was allowed to procede f o r I0 minutes before lysostaphin was added. Bacteria to PMN r a t i o
Percent uptake*
15:1 30:1 60:1 120:1 240:1
35.1 37.2 40.6 31.1 17.3
+ ¥ ¥ ¥ T
No. of org. ingested/PMN
5.4 4.5 2.9 2.1 1.0
5.3 II.2 24.4 37.3 41.5
*(Mean ~ SEM, n = 4)
When the PMNs were pretreated with cytochalasin B to block ingestion the amount of r a d i o a c t i v i t y associated with the PMNs f o l l o w i n g the phagocytic assay was reduced to background levels (Figure 2).
These results demonstrated
the absence of S. aureus adsorbed to the e x t r a c e l l u l a r surface of the PMN f o l l o w i n g lysostaphin treatment. Nitroblue tetrazolium reduction The mean (~ SD) value obtained fo r NBT reduction by 56 i n d i v i d u a l PMN preparations using a preopsonized zymosan stock solution of I0 mg/ml was 0.49 + 0.20 (OD/5.0 x 106 PMNs/5 min in 5 ml pyridine)(Table I I I ) . The effects of d i f f e r e n t methods of opsonization of zymosan upon NBT reduction by normal PMNs are shown in Table V.
There was very l i t t l e
NBT
TABLE V Influence of the method fo r opsonization of zymosan on NBT reduction and i o d i n a t i o n by bovine PMNs. The amount of formazan produced from NBT by 5.0 x 106 PMNs in 5 minutes are expressed as o p t ic al densities in 5.0 ml of pyridine (Mean + SEM of (n) experiments). NBT reduction Optical Density (580 nm) PMNs (resting) PMNs + zymosan PMNs + serum PMNs + serum + zymosan PMNs + heated serum + zymosan (56 C, 30 min) PMNs + preopsonized zymosan
0.04 0.04 0.02 0.17 0.14
+ 0.003 T0.014 ¥ 0.003 ~ 0.02 ¥ 0.02
0.34 + 0.02
lodination nmole Nal/lO 7 PMNs/hr
(4) (4) (4) (4) (4)
2.2 3.6 2.4 15.9 12.3
+ 0.3 T0.6 ¥ 0.2 ¥ 1.3 ~ 1.7
(50) (50) (50) (50) (29)
(4)
48.3 + 2.5
(56)
168 r e d u c t i o n by " r e s t i n g " PMNs o r by PMNs in the presence of serum or zymosan alone.
Optimal NBT r e d u c t i o n occurred when preopsonized zymosan was used as
the p a r t i c l e
for ingestion.
PMNs s t i m u l a t e d by zymosan in the presence o f
f r e s h serum reduced only a p p r o x i m a t e l y 50% as much NBT as PMNs s t i m u l a t e d by preopsonized zymosan. activity
Heat i n a c t i v a t i o n
d i d not markedly d i m i n i s h the opsonic
o f the serum.
S t i m u l a t i o n o f PMNs w i t h graded amounts o f preopsonized zymosan r e s u l t e d in a p o s i t i v e c o r r e l a t i o n NBT r e d u c t i o n .
between the amount of preopsonized zymosan added and
The NBT reducing c a p a c i t y o f the PMNs was a p p a r e n t l y not
exceeded, even a t high c o n c e n t r a t i o n s o f preopsonized zymosan (Figure 3).
0.5-
8-
E
7-
z 0.4-
~7ol
G
£6" x
x
o. 0.3-
~ 604
LO a
504
~4, 8 0.2-
-+ 3-
o "0 I.--
z~0.1 -
I
v 404 g 304
E ~2-
2oi
o
? 1-
0.0-
O.
"
ol 0 t.o
ld.o
2d0
4do
Concentration of preopsonized zymosan in the stock solution (mg/ml)
Figure 3. I n f l u e n c e o f varying the q u a n t i t i e s o f preopsonized zymosan a v a i l a b l e f o r phagocytosis on NBT r e d u c t i o n ( ~ ) , chemiluminescence (C and i o d i n a t i o n (0=---0) by bovine PMNs. F i f t y m i c r o l i t e r s o f the r e s p e c t i v e stock s o l u t i o n was added to each r e a c t i o n tube.
~),
169
Chemiluminescence T h i r t y - e i g h t determinations of chemiluminescence by normal PMNs using a preopsonized zymosan concentration of I0 mg/ml y ie ld e d a mean (~ SD) of 5.21 (~ 1.57) x 106 cph (Table I I I ) . Figure 3 i l l u s t r a t e s the e f f e c t of varying the quantity of preopsonized zymosan in the reaction mixture. zymosan increased l i g h t emission. conditions employed.
Increasing the amount of preopsonized This did not reach an end point under the
Resting PMNs, in the absence of serum or p a r t i c l e s ,
did not demonstrate any s i g n i f i c a n t chemiluminescence. lodination The mean (~ SD) i o d i n a t i o n value obtained from 56 i n d i v i d u a l PMN preparations stimulated by a preopsonized zymosan stock concentration of I0 mg/ml was 48.3 + 18.7 nmole Nal/lO 7 PMNs/hr. The e f f e c t of varying the quantity of opsonized zymosan a v a i l a b l e f o r phagocytosis is i l l u s t r a t e d in Figure 3.
l o d i n a t i o n increased with increasing q u a n t i t i e s of preopsonized
zymosan. Nearly maximal levels of i o d i n a t i o n were obtained with a preopsonized zymosan preparation of 5 mg/ml; a d d i t i o n a l preopsonized zymosan beyond 5 mg/ml did not markedly increase i o d i n a t i o n .
The effects of d i f f e r e n t
methods of opsonization of zymosan on i o d i n a t i o n by normal PMNs is i l l u s t r a t e d in Table V.
There was very l i t t l e
i o d i n a t i o n by resting PMNs or by PMNs in
the presence of e i t h e r zymosan or serum alone.
Maximal i o d i n a t i o n was
attained in the absence of serum when preopsonized zymosan was used as the ingested p a r t i c l e .
Heat i n a c t i v a t i o n of the serum did not abolish
iodination. DISCUSSION The PMN i s o l a t i o n procedure was rapid, reproducible, and yielded a cell preparation with good b i o l o g i c a l a c t i v i t y and no observable clumping.
In
preliminary experimentation clumping was observed when PMNs were held in suspension in the presence of Ca2+ and Mg2+.
The procedure worked equally
well in animals that were neutropenic; however, the t o t a l y i e l d of PMNs was, of course, reduced.
Carlson and Kaneko (1973) reported a PMN recovery of
90.0% + 28.9% y i e l d i n g a c e l l preparation of 87.8% + 7.0% PMNs. This is a higher percent c e l l recovery but a lower p u r i t y of the PMN preparation than in this report.
Our reduced recovery was probably due to the more l i b e r a l
170
removal of the upper portion of the packed erythrocyte layer before l y s i s . This area contains a mixture of red blood c e l l s , PMNs, and mononuclear c e l l s . By l i b e r a l l y removing the upper portion of the packed red blood c e l l s , the p u r i t y of the PMN preparation can be increased, but many PMNs w i l l be l o s t . The percent of eosinophils in the PMN preparations varied considerably. The cell suspension was adjusted to a standard number of neutrophils plus eosinophils because eosinophils have been demonstrated to be capable of p a r t i c i p a t i n g in a l l of the functions assayed in this report (Simmons and Karnovsky, 1973; Klebanoff, et a l . , 1977). The presence of normal Ficoll-Hypaque p u r i f i e d MNCs markedly reduced the random migration by PMNs, but the mechanism f o r this migration i n h i b i t i o n by unstimulated MNCs is not known. Therefore, when evaluating random migration by PMNs i t is important to p u r i f y the PMNs or at least standardize the number of MNCs present.
The phytomitogen Con A markedly reduced the random migration
by p u r i f i e d PMNs and by PMNs mixed with an equal number of MNC, Whether this i n h i b i t i o n is due to a d i r e c t e f f e c t of Con A on PMNs, or to an i n d i r e c t e f f e c t of a lymphokine released by Con A stimulated MNCs is unknown. The p u r i f i e d PMNs did contain a small proportion of MNCs (Table I) which may have produced s u f f i c i e n t leukocyte migration i n h i b i t i o n factor to account fo r the reduced migration by p u r i f i e d PMNs in the presence of Con A. The method described using 1251-iododeoxyuridine and fluorodeoxyuridine proved to be a simple and convenient method fo r l a b e l i n g S. aureus with a gamma emitting radioisotope.
The heat k i l l e d 1251-iododeoxyuridine labeled
S. aureus may be stored at 4 C and u t i l i z e d over a period of several days; thus saving time and minimizing day to day v a r i a b i l i t y in reagents. The 125 I label was e f f i c i e n t l y released from the S. aureus by lysostaphin digestion. Lysostaphin has been used successfully as an aide f o r evaluating ingestion of S. aureus by PMNs (Verhoef, et a l . , 1977b; Paape, et a l . , 1978).
I t has
been shown to r a p i d l y lyse S. aureus, and i t is not t o x i c f or and does not enter phagocytic c e l l s (Tan, et a l . , 1971; Easomn, et a l . , 1978).
Lysostaphin
is very useful because i t lyses the bacteria which are adherent to the surface of the PMN; this permits d i f f e r e n t i a t i o n from those that are ingested.
When
lysostaphin was added to the phagocytosis mixture at the same time as the PMNs an average of 12.4% of the S. aureus were ingested by the PMNs (Figure I ) . This was apparently due to ingestion that occurred p r i o r to lysostaphininduced l y s i s of the S. aureus in the phagocytosis mixture since blockage of ingestion by PMNs by cytochalasin B treatment resulted in background amounts of r a d i o a c t i v i t y associated with the PMNs. The lag period in lysostaphin action was not f e l t to be important when evaluating the a b i l i t y of PMNs to
171
ingest p a r t i c l e s .
A I0 minute incubation period was chosen f o r the standard
ingestion assay so that the PMNs would not reach t h e i r capacity for ingestion (Figure 2).
This permits determination of the rate of ingestion by PMNs. A
longer incubation period and a higher bacteria to PMN r a t i o would be desirable i f one wished to measure the capacity of ingestion by PMNs. The results in Figure 2 and Table IV indicate that the maximum number of bacteria which can be ingested by a single bovine PMN is approximately 40-45.
This agrees
closely with the calculated maximal uptake of S. aureus by human PMNs as determined by L e i j h , et a l . (1979). The o x i d a t i v e metabolism of the PMN is an important aspect of i t s b a c t e r i c i d a l a c t i v i t y (For a review see Babior, 1978; Klebanoff, 1979).
When
i t receives the proper stimulus an oxidase enzyme on the outer surface of the PMN plasma membrane and on the inner surface of the phagosomal membrane w i l l catalyze the conversion of oxygen to superoxide anion (02).
Superoxide anion
is a highly reactive oxygen moiety which may undergo a spontaneous dismutation to form hydrogen peroxide.
Singlet oxygen (a free radical of oxygen) may be
a short l i v e d intermediary in this dismutation.
Some of the ~ydrogen
peroxide formed may react with a d d i t i o n a l superoxide anion to form hydroxyl radicals.
Superoxide anion, s i n g l e t oxygen, hydrogen peroxide, and the
hydroxyl radical are highly reactive compounds and, when formed inside the phagocytic vacuole, are believed to play an important role in the k i l l i n g of microorganisms by the PMN. NBT is d i r e c t l y reduced by superoxide anion to an insoluble purple formazan (Yost and Fridovich, 1974).
NBT reduction is therefore a measure
of superoxide anion generation by the PMN. Chemiluminescence is also dependent upon the generation of superoxide anion by the PMN, although superoxide anion i t s e l f does not produce l i g h t .
The mechanism f o r l i g h t
production by the PMN is not understood but several possible mechanisms have been proposed (Babior, 1978; Hodgson and Fridovich, 1976).
Goldstein, et a l . ,
(1977) demonstrated that the superoxide anion generating system appears to be associated with the outer surface of the PMN plasma membrane as well as the inner surface of the phagosomal membrane and that superoxide generation may proceed independently of phagocytosis and degranulation.
They found that
superoxide anion recovery in the medium surrounding PMNs was in fact enhanced when the formation of phagocytic vacuoles was i n h i b i t e d by treatment of c ells with cytochalasin B.
This superoxide anion in the medium w i l l reduce
e x t r a c e l l u l a r NBT to formazan and produce chemiluminescence.
NBT reduction
and chemiluminescence therefore appear to be v a l i d measurements of d i f f e r e n t
172
aspects of the o x i d a t i v e metabolism of the PMN but can not be interpreted to be v a l i d measurements of ingestion by PMNs. l o d i n a t i o n is a measure of the a b i l i t y of the PMN to convert inorganic iodide to a t r i c h l o r a c e t i c acid (TCA) p r e c i p i t a b l e (protein-bound) form. The iodide is covalently bound to a suitable acceptor molecule such as the tyrosine residues of protein in the phagocytic vacuole via the action of hydrogen peroxide (H202) and myeloperoxidase.
This system has been found to
e x h i b i t marked t o x i c a c t i v i t y toward bacteria, fungi, and viruses (Belding and Klebanoff, 1979; Simmons and Karnovsky, 1973).
The i o d i n a t i o n reaction is
dependent upon a number of processes (Klebanoff and Clark, 1977).
Hydrogen
peroxide is generated by the action of a membrane bound oxidase enzyme to convert oxygen to superoxide anion which may spontaneously be reduced to form H202. The myeloperoxidase needed is present in the primary granules in the PMN cytoplasm.
This enzyme must be delivered to the phagocytic vacuole or the
e x t r a c e l l u l a r environment by the process of degranulation.
Ingestion of
opsonized p a r t i c l e s w i l l stimulate H202 formation and degranulation in normal PMNs. l o d i n a t i o n has been demonstrated to occur both in the phagocytic vacuole and e x t r a c e l l u l a r l y due to release of myeloperoxidase and H202 into the e x t r a c e l l u l a r environment (Klebanoff and Clark, 1977).
The extent of the
e x t r a c e l l u l a r i o d i n a t i o n may depend upon the conditions under which phagocytosis is conducted.
In an experimental system s i m i l a r to the one described
here, normal i o d i n a t i o n was shown to be dependent upon ingestion (Klebanoff and Clark, 1977).
A depressed i o d i n a t i o n value may be due to lack of
ingestion, a lack of normal o x i d a t i v e metabolism within the PMN, a f a i l u r e of degranulation, a reduced amount of myeloperoxidase in the primary granule, destruction of H202 (by catalase) or interference with the myeloperoxidase catalyzed reaction. t i a t e these defects.
Other experimental procedures are required to d i f f e r e n Because the i o d i n a t i o n reaction is dependent upon a
complex chain of events, i t is a good screening test to detect PMN dysfunction. Optimal NBT reduction and i o d i n a t i o n by PMNs was obtained when preopsonized zymosan was used in a serum free system as the phagocytosable p a r t i c l e (Table V).
In the presence of bovine serum the NBT reduction and i o d i n a t i o n
values were markedly reduced.
This may at l e a st p a r t i a l l y be due to the
presence of conglutinin in bovine serum.
Conglutinin reacts with a mannose
peptide determinant found on the t h i r d component of complement (C3b) and on zymosan. Conglutinin is responsible f o r the powerful clumping of zymosan and of complement coated p a r t i c l e s in bovine serum (Lachmann, 1975). Klebanoff and Clark (1977) reported that heating human serum to 56 C f o r 30 minutes reduced the mean i o d i n a t i o n value f o r human PMNs in the presence of zymosan from 64.1 to 1.8 nmole Nal/lO 7 PMNs/hr, which was equivalent to the
173
level of i o d i n a t i o n by resting PMNs. This demonstrates that only heat l a b i l e factors in human serum are able to opsonize zymosan.
In the bovine system
heating of the serum to 56 C f o r 30 minutes only s l i g h t l y reduced i o d i n a t i o n and NBT reduction by bovine PMNs (Table V).
This indicates that in addition
to a heat l a b i l e opsonin there is also a heat stable opsonin f o r zymosan present in bovine serum.
Klebanoff and
Clark (1977) concluded that when
normal c e l l s and p a t i e n t ' s serum are employed, the i o d i n a t i o n reaction is an i n d i r e c t measure of the opsonic a c t i v i t y of the p a t i e n t ' s serum. They were able to demonstrate decreased opsonic a c t i v i t y f o r zymosan of human serum d e f i c i e n t in the fourth or t h i r d component of complement.
Due to the
presence of c o n g l u t i n i n and a heat stable opsonin f o r zymosan in bovine serum, the i o d i n a t i o n reaction, chemiluminescence, and the NBT reduction test as described here cannot be used to detect opsonic defects in whole bovine serum. The results in Figure 3 indicate that a zymosan preparation of 5 mg/ml yielded nearly optimal values fo r i o d i n a t i o n , however, this level of opsonized zymosan did not give optimal values f o r NBT reduction and chemiluminescence. A concentration of 40 mg of zymosan per ml yielded over 150% more NBT reduction and chemiluminescence but only approximately 10% more i o d i n a t i o n than 5 mg of zymosan per ml.
Chemiluminescence and NBT reduction are
measures of the o x i d a t i v e metabolism of the PMN; therefore, as the opsonized zymosan concentration is increased to 40 mg/ml, there is increased o x i d a t i v e metabolism without a corresponding increase in i o d i n a t i o n .
This suggests
that the o x i d a t i v e metabolism is not the l i m i t i n g f a c t o r in the i o d i n a t i o n reaction by normal bovine PMNs. ACKNOWLEDGEMENTS This work was supported by Grant No. 413-43-08-85-2229 from the Cooperative State Research Service of the United States Department of Agriculture and by formula funds provided by the United States Department of A g r i c u l t u r e , Grant No. 410-23-02.
174
REFERENCES
A l l e n , R. C., Stjernholm, R, C, and Steele, R, H,, 1972, Evidence f o r the generation of an e l e c t r o n i c e x c i t a t i o n state(s) in human polymorphonuclear leukocytes and i t s p a r t i c i p a t i o n in b a c t e r i c i d a l a c t i v i t y , Biochem, and Biophys. Res. Commun. 47:679-684. Babior, B. M., 1978. Oxygen-dependent microbial k i l l i n g by phagocytes, N. Eng. J. Med. 298:721-725. Baehner, R. L., 1972. Disorders of leukocytes leading to recurrent i n f e c t i o n P e d i a t ri c Clinics of North America. 19:935-956. Baehner, R. L. and Nathan, D. G., 1968o Quantitative n i t r o b l u e tetrazolium test in chronic granulomatous disease. N. Eng. J. Med. 278:971-976. Belding, M. E. and Klebanoff, S. J ., 1970. Peroxidase-mediated v i r i c i d a l systems. Science. 167:195-196. Carlson, G. P. and Kaneko, J. J ., 1973. I s o l a t i o n of leukocytes from bovine peripheral blood. Proc. Soc. Exper. B i o l . and Med. 142:853-856. Easmon, C. S. F., Lanyon, H. and Cole, P. J . , 1978. Use of lysostaphin to remove cell-adherent staphylococci during in v i t r o assays of phagocyte function. Br. J. Exp. Path. 59:381-385. Goldstein, I. M., Cerqueira, M., Lind, S. and Kaplan, H. B., 1977. Evidence that the superoxide-generating system of human leukocytes is associated with the c e l l surface. J. Clin. Invest. 59:249-254. Hodgson, E. K. and Fridovich, I . , 1976. The mechanism of the a c t i v i t y dependent luminescence of xanthine oxidase. Arch. Biochem. Biophys. 172:202-205. Klebanoff, S. J., 1979. Oxygen-dependent antimicrobial systems of the neutrophil. In: D. Schlessinger ( E d i t o r ) , Microbiology 1979. American Society for Microbiology, Washington D.C., pp. 87-91. Klebanoff, S. J. and Clark, R. A., 1977. I o d i n a t i o n by human polymorphonuclear leukocytes: a r e - e v a l u a t i o n . J. Lab. Clin. Med. 89:675-686. Klebanoff, S. J . , Durack, D. T., Rosen, H. and Clark, R. A., 1977. Functional studies on human peritoneal eosinophils. I n f e c t . Immun. 17:167-173. Lachmann, P. J., 1975. Complement. In: P. G. H. G e ll, R. R. A. Coombs and P. J. Lachmann ( E d i t o r s ) , C l i n i c a l Aspects of Immunology. Blackwell S c i e n t i f i c Publications, Philadelphia, PA, pp. 323-364. L e i j h , P. C. J . , Van Den Barselaar, M. T., Van Zwet, T. L., DubbeldemanRempt, I. and Van Furth, R., 1979. Kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes. Immunol. 37:453-465~ Nelson, R. D., Quie, P. G. and Simmons, R. L., 1975. Chemotaxis under agarose: a new and simple method f o r measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol. I15:1650-1656. Paape, M. J., Pearson, R. E. and Schultze, W. D., 1978. Variation among cows in the a b i l i t y of milk to support phagocytosis and in the a b i l i t y of polymorphonuclear leukocytes to phagocytose Staphylococcus aureus. Am. J. Vet. Res. 39:1907-1910. Simmons, S. R. and Karnovsky, M. L. 1 9 7 3 . Iodinating a b i l i t y of various leukocytes and t h e i r b a c t e r i c i d a l a c t i v i t y . J. Exp. Med. 138:44-63. Tan, J. S., Watanakunakorn, C. and Phair, J. P., 1971. A modified assay of neutrophil function: Use of lysostaphin to d i f f e r e n t i a t e defective phagocytosis from impaired i n t r a c e l l u l a r k i l l i n g . J. Lab. Clin. Med. 78:316-321. Verhoef, J., Peterson, P. K. and Quie, P. G., 1977. Kinetics of staphylococcal opsonization, attachment, ingestion and k i l l i n g by human polymorphonuclear leukocytes: a q u a n t i t a t i v e assay using [~H] thymidine labeled bacteria. J. Immunol. Methods. 14:303-311. Yost, F. J. and Fridovich, I . , 1974. Superoxide radicals and phagocytosis. Arch. Biochem. Biophys. 161:395-401.
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Elsevier Scientific Publishing Company, Amsterdam -- Printed in Belgium
EVALUATION OF BOVINE POLYMORPHONUCLEARLEUKOCYTE FUNCTION J. A. ROTH and M. L. KAEBERLE Department of Veterinary Microbiology and Preventive Medicine, lowa State U n i v e r s i t y , Ames, IA 50011 (USA) (Accepted 4 November 1980) ABSTRACT Roth, J. A., and Kaeberle, M. L., 1981. Evaluation of bovine polymorphonuclear leukocyte f u n c t i o n . Vet. Immunol. Immunopathol., 2: 157-174. Bovine polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of c a t t l e . Five in v i t r o procedures were u t i l i z e d to evaluate PMN f u n c t i o n : I) Random migration under agarose, 2) Ingestion of 12Sliododeoxyuridine labeled Staphylococcus aureus, 3) Quantitative n i t r o b l u e tetrazolium reduction, 4) Chemiluminescence and 5) l o d i n a t i o n . Normal values for bovine PMNs are reported and i n t e r p r e t a t i o n of results is discussed. The PMN function tests were designed so that a l l 5 procedures may be performed in a short period of time on the same c e l l preparation. This allows f o r the detection and p a r t i a l characterization of a potential PMN dysfunction. INTRODUCTION A major function of the polymorphonuclear leukocyte (PMN) is the phagocytosis and destruction of invading microorganisms,
Therefore the PMN plays an
important role in protecting the animal from microbial i n f e c t i o n .
A congenital
or acquired defect in PMN function w i l l r e s u l t in an enhanced s u s c e p t i b i l i t y to i n f e c t i o n with b a c t e r i a l or fungal pathogens (Baehner, 1972).
When such a
condition arises, i t is desirable to be able to evaluate the phagocytic c a p a b i l i t y of the c i r c u l a t i n g PMNs. The a c t i v i t i e s of the PMN in c o n t r o l l i n g microbial i n f e c t i o n are complex.
In order to adequately evaluate PMN function
a v a r i e t y of in v i t r o tests must be u t i l i z e d concurrently.
Because PMNs are
short l i v e d c e l l s i t is necessary to perform the various function tests w i t h i n a short period of time; therefore each test must be r e l a t i v e l y simple to conduct and require a minimum of laboratory personnel time. This paper reports techniques f o r performing 5 tests that evaluate the function of bovine PMNs. These procedures are r e l a t i v e l y simple to perform. Most of the reagents can be prepared in advance and stored so that a l l of the 0165-2427/81/0000---0000/$ 02.50 © 1981 Elsevier Scientific Publishing Company
158
procedures can be performed the same day the blood is drawn from the animal. The 5 procedures q u a n t i t a t i v e l y evaluate: I ) random migration by PMNs, 2) the a b i l i t y of the PMNs to ingest opsonized p a r t i c l e s , 3)
the burst of
o x i d a t i v e metabolism associated with phagocytosis, and 4)
the myeloperoxidase
catalyzed reaction which is dependent upon the generation of hydrogen peroxide, the presence of myeloperoxidase in the granules, and degranulation.
These
procedures allow f o r the detection and p a r t i a l characterization of a potential PMN dysfunction. MATERIALS AND METHODS PMN preparation Blood was collected from healthy adult c a t t l e i n t o a c i d - c i t r a t e - d e x t r o s e s o l u t i o n (standard formula A) by j u g u l a r venapuncture, and PMNs were isolated using a modification of the method of Carlson and Kaneko (1973). was f i r s t
The blood
centrifuged at I000 X g for 20 minutes, the plasma and buffy coat
layer were aspirated and discarded, and the packed red blood c e l l s were lysed by the addition of two volumes of cold phosphate buffered (0.0132 M, pH 7.2) deionized water.
I s o t o n i c i t y was restored to the red blood cell
lysate a f t e r 45 seconds by the addition of one volume of cold phosphate buffered (0.0132 M, pH 7.2) 2.7% NaCI.
The PMNs present in the red blood cell
lysate were pelleted by c e n t r i f u g a t i o n and washed twice with Hanks balanced s a l t s o l u t i o n without Ca2+ and Mg2+ (HBSS) (Grand Island Biological Co., Grand Island, NY, USA). The cell suspension was then adjusted to a f i n a l concentration of 5.0 X 107 PMNs (neutrophils plus eosinophils) per ml of HBSS. Zymosan preparation Zymosan (Sigma Chemical Co., St. Louis, MO, USA) was suspended in cold Earles balanced s a l t s o l u t i o n with Ca2+ and Mg2+ and phenol red (EBSS) (Grand Island Biological Co., Grand Island, NY, USA) at a concentration of I0 mg/ml by homogenization f o r one minute with a Potter-Elvehjem tissue grinder. Preopsonized zymosan was prepared by adding I00 ml of fresh bovine serum to an equal volume of the zymosan suspension and s t i r r i n g for one hour at room temperature. minutes.
The zymosan was sedimented by c e n t r i f u g a t i o n at 250 × g for I0
Strong c o n g l u t i n a t i o n of the zymosan was reversed by washing the
zymosan p e l l e t twice with 200 ml of 0.01M sodium ethylenediamine-tetraacetic acid (EDTA) with s t i r r i n g for 15 minutes between washes.
The f i n a l p e l l e t was
159
resuspended in I00 ml of EBSS and frozen in aliquots at -20 C.
Prior to use
the preopsonized zymosan was thawed and homogenized with a Potter-Elvehjem tissue grinder.
When other concentrations of preopsonized zymosan were
needed, this preparation was concentrated by c e n t r i f u g a t i o n or d i l u t e d with a d d i t i o n a l EBSS to a r r i v e at the desired concentration. Random migration under agarose Random migration under agarose was evaluated by a modification of the procedure of Nelson, et a l . (1975).
Agar f o r evaluation of random migration
consisted of bicarbonate buffered (0.026 M pH 7.2) Minimum Essential Medium with Earles salts (Grand Island B i o l o g i c a l Co., Grand Island, NY, USA) containing 0.8% agarose (#57035 Gallard Schlessinger, Chemical Mfg. Corp., Carle Place, NY, USA), 10% f e t a l c a l f serum, and I% a n t i b i o t i c antimycotic solution (Grand Island B i o l o g i c a l Co., Grand Island, NY, USA). Five ml of warm agar (48 C) was poured into tissue culture pet r i plates (60 x 15 mm, #1007 Falcon, Oxnard, CA, USA) and the plates were stored at 4 C.
To evaluate
random migration by PMNs, wells 2.0 mm in diameter were made in the agar and were f i l l e d with an a l i q u o t of the PMN suspension (5.0 x 107 PMNs/ml). The migration plates were placed in an incubator with a humidified 5% CO2 atmosphere at 37 C.
Eighteen hours l a t e r the plates were removed from the
incubator, the surface of the agar was flooded with absolute methanol f o r 30 minutes, the methanol was removed and the agar was flooded with 47% formalin f o r an a d d i t i o n a l 30 minutes.
The agar was removed and the cells adhering
to the p e t ri plate were stained with modified Wrights stain.
The area of
migration was determined by tracing an enlarged image produced by a microscope with a drawing attachment, and measuring the area of the image with a planimeter.
The area of the projected image was mathematically converted to
the actual area on the migration plate and the area of the center well was subtracted. Ingestion of Staphylococcus aureus A coagulase p o s i t i v e s t r a i n of Staphylococcus aureus (S. aureus) was isolated from a case of bovine m a s t i t i s . d i r e c t l y onto a blood agar plate.
The milk was quarter streaked
A single colony was transferred to brain
heart infusion broth (BHI) (Difco, D e t r o i t , MI, USA) and incubated at 37°C f o r 18 hours.
Glycerine was added to a f i n a l concentration of 15%, and the
culture was aliquoted and frozen at -70 C.
This stock culture of bacteria
160
was used f o r the remainder of the experiment, Two ml of an overnight culture of S. aureus in BHI was inoculated into a 500 ml Erlenmeyer flask containing 250 ml of BHI with 250 ~Ci of 1251lododeoxyuridine (1251UdR) (New England Nuclear, Boston, MA, USA) and 10-5 M fluorodeoxyuridine (FUdR) (Sigma Chemical Co,, St. Louis, MO, USA). Following incubation fog 18 hours at 37 C, the bacteria were collected by c e n t r i f u g a t i o n , k i l l e d by heating to 60 C f o r 1 hr, and washed twice in 0.015 M phosphate buffered saline s o l u t i o n , pH 7.2 (PBS). The p e l l e t e d bacteria were suspended in PBS to make a stock suspension with a f i n a l concentration t h a t , when d i l u t e d I : I 0 in PBS, had an optical density of 0.4 at 600 nm (1.5 X 109 colony forming units/ml fo r comparably treated l i v e organisms).
The h e a t - k i l l e d 1251-1abeled S. aureus was stored at 4 C and used
over a period of several days. Bovine anti-S, aureus serum was produced by i n j e c t i n g a cow subcutaneously with a washed formalin k i l l e d suspension of S. aureus in PBS on two occasions I0 days apart.
Two weeks a f t e r the l a s t i n j e c t i o n , serum was harvested and
heat i n a c t i v a t e d at 56 C f o r 30 min.
The antibody t i t e r of the serum was
1:160 as determined by a plate a g g l u t i n a t i o n test with h e a t - k i l l e d S. aureus as the antigen. The standard phagocytosis assay was conducted in 12 X 75 mm p l a s t i c tubes containing I00 ~I of 1251UdR labeled S. aureus, 50 ~I of PMN suspension (2.5 X 106 PMNs, bacteria to PMN r a t i o = 60:1), 50 ~I of a I : I 0 d i l u t i o n of bovine anti-S, aureus serum, and 0.3 ml of EBSS. The S. aureus, serum, and EBSS were added to the tubes f i r s t 37 C water bath.
and incubated f o r at least 15 minutes in a
The PMNs were added and the tubes were agitated occasionally
to keep the p a r t i c l e s in suspension.
Exactly I0 minutes a f t e r the addition of
PMNs, 0.5 ml of PBS containing 0.5 unit of lysostaphin (Sigma Chemical Co., St. Louis, MO, USA) was added to each reaction tube and the background tubes. Following incubation at 37 C f o r an additional 30 minutes, 2.0 ml of PBS was added to each tube, and they were centrifuged at 1,250 X g f or I0 minutes. The supernatant f l u i d was aspirated and discarded, and the p e l l e t was washed with an a d d i t i o n a l 2.0 ml of PBS. The f i n a l p e l l e t was placed in a gamma counter to determine the counts per minute (CPM) of r a d i o a c t i v i t y present.
For
each assay a standard tube was set up which contained the standard amount of S. aureus but did not receive any lysostaphin.
A background tube was also set
up which contained a l l reactants except PMNs; i t did receive lysostaphin. All tubes were set up in duplicate and the average value used for c a lc ulat ions . The percent of the S. aureus ingested was calculated using the f ollow ing equation:
161
(CPM in reaction tube) - (CPM in background tube) Percent ingestion = (cPM in s'tandard tube) (CPM in background tube) x I00 The effects of the time allowed f o r phagocytosis and the bacteria to PMN r a t i o were determined by varying one parameter while holding the other parameters constant at the levels used f o r the standard phagocytosis assay. The kinetics of S. aureus ingestion by PMNs was studied by varying the phagocytosis time p r i o r to the addition of lysostaphin from 0 to 30 minutes. The e f f e c t of varying the bacteria to PMN r a t i o was determined by using a constant number of PMNs while varying the number of bacteria. PMN r a t i o s of 15:1, 30:1, 60:1, 120:1 and 240:1 were used.
Bacteria to
The e f f e c t of
cytochalasin B treatment was evaluated by incubating PMNs f o r 20 minutes at 37 C in EBSS containing 20 pg/ml of cytochalasin B (Sigma Chemical Co., St. Louis, MO, USA) and 0.1% dimethyl sulfoxide p r i o r to use in the k i n e t i c study described above. Nitroblue Tetrazolium Reduction Nitroblue tetrazolium (NBT) reduction by PMNs was evaluated by a modification of the procedure of Baehner and Nathan (1968). was prepared by suspending NBT (Grade I I I , USA) in EBSS at a concentration of 2 mg/ml. hour at ambient temperature. a 0.45 pm f i l t e r
The NBT solution
Sigma Chemical Co., St. Louis, MO, The mixture was s t i r r e d for one
Insoluble NBT was removed by f i l t r a t i o n
through
and the solution was stored at 4 C.
Tests were conducted in t r i p l i c a t e in 15 x I00 mm silicon-coated glass test tubes.
The standard reaction mixture contained 0.2 ml of NBT s o l u t i o n ,
5.0 x 106 PMNs, 0.I ml preopsonized zymosan preparation (I0 mg/ml), and s u f f i c i e n t EBSS to bring the t o t a l volume to 1.0 ml. for the determination of resting NBT reduction. amount of preopsonized zymosan was varied.
Zymosan was deleted
In certain experiments the
In other experiments the
preopsonized zymosan was replaced with 0.I ml of serum. All of the reactants, except the PMNs, were added to the tubes and allowed to e q u i l i b r a t e in a water bath at 37 C f o r 15 minutes.
The tubes were l e f t in the water bath, and
the reaction was i n i t i a t e d by adding the PMN suspension. were kept in suspension by periodic shaking.
Cells and p a r t i c l e s
A f t er exactly 5.0 minutes, the
reaction was stopped by adding 5.0 ml of cold ImM N-Ethylmaleimide (Sigma Chemical Co., St. Louis, MO, USA) in saline.
The c ells and p r e c i p i t a t e d
formazan were pelleted by c e n t r i f u g a t i o n at 500 x g f o r I0 minutes.
The super-
natant f l u i d was discarded and the p e l l e t was suspended in 5.0 ml of pyridine. Formazan was extracted by b r i e f sonication followed by heating in a b o i l i n g water bath f o r I0 minutes in a fume hood.
The pyridine-formazan was c l a r i f i e d
162
by c e n t r i f u g a t i o n at 500 x g f o r I0 minutes and the optical density (OD) at 580 nm was immediately determined in a spectrophotometer (Beckman DBG, Beckman Instruments, I n c . , I r v i n e , CA, USA), using a pyridine blank.
The
results are reported as OD/5.0 x 106 PMNs/5 min in 5.0 ml of pyridine. Determination of chemiluminescence Chemiluminescence was measured by a modification of the procedure of Allen et a l . (1972) in a l i q u i d s c i n t i l l a t i o n spectrometer (Model DPM I00, Beckman Instruments, Inc., I r v i n e , CA, USA) at ambient temperature with one p h o t o m u l t i p l i e r tube switched o f f .
The reaction was conducted in 20 ml glass
s c i n t i l l a t i o n v i a l s (Fisher S c i e n t i f i c Co., Pittsburgh, PA, USA). Test v i a l s contained 0.5 ml of preopsonized zymosan solution (0-40 mg/ml) and 1.0 x 107 PMNs in a t o t a l volume of I0.0 ml of bicarbonate buffered (0.026 M, pH 7.2) Geys balanced s a l t s o l u t i o n with Ca2+ and Mg2+ and without phenol red (Grand Island B i o l o g i c a l Co., Grand Island, NY, USA) (GBSS). The zymosan and GBSS were added to the s c i n t i l l a t i o n v i a l ; PMNs were added 1 minute before the v i a l was placed in the counting chamber.
The v i a l was capped, mixed w e l l ,
placed in the counting chamber and l e f t undisturbed f o r the remainder of the experiment. intervals.
Each v i a l was counted f o r 1 minute at approximately I0 minute Counting was continued f o r at least 70 minutes.
A v i a l containing
I0 ml of GBSS with no PMNs or zymosan was counted each cycle to determine the background a c t i v i t y .
The results were graphed as the number of counts per
minute at each I0 minute i n t e r v a l a f t e r the addition of PMNs. Differences in the height and shape of the curves with d i f f e r e n t PMN preparations required the reporting of results as t o t a l net counts per hour (cph).
The
cph were determined by estimating the area under the curve as shown in figure I .
The net cph of a test v i a l was calculated as follows:
net cph = cph of test v i a l - cph of background vial All experiments were conducted in duplicate and the average net cph are reported. Determination of i o d i n a t i o n The i o d i n a t i o n procedure was performed by a modification of the procedure of Klebanoff and Clark (1977).
The standard reaction mixture contained 40
nmole Nal, 0.05 wCi 1251 (Carrier free in 0.I M NaOH, New England Nuclear, Boston, MA, USA), 2.5 x 106 PMNs, and 0.05 ml preopsonized zymosan preparation
163
B
120
C
110.
D
, ~ 100. o
E F
l G "--
60
3O
20
10 0
1~1
21
31
4'1
,51
61
Time(minutes)
Figure I . This f i g u r e i l l u s t r a t e s the method used to estimate the counts per hour. The sum of the areas of the rectangles was determined by the f o l l o w i n g formula: (6) (A) + ( I 0 )
(B) + (I0)
(C) + (I0)
(D) + (I0)
(E) + (I0) (F) + (4) (G)
This is an estimate of the area under the curve f o r the f i r s t and, t h e r e f o r e , of the counts per hour.
60 minutes,
(I0 mg/ml) in a t o t a l volume of 0.5 ml EBSS. Zymosan was deleted f o r the d e t e r m i n a t i o n of r e s t i n g i o d i n a t i o n .
In c e r t a i n experiments the amount of
preopsonized zymosan was v a r i e d ; in other experiments the preopsonized zymosan in the standard r e a c t i o n mixture was replaced with 0.05 ml of unopsonized zymosan (I0 mg/ml) with or w i t h o u t 0.05 ml of serum.
A l l the
components except the PMNs were placed in 12 x 75 mm polystyrene snap cap t e s t tubes (#2058 Falcon, Oxnard, CA, USA) and allowed to e q u i l i b r a t e in a 37 C incubator.
The r e a c t i o n was s t a r t e d by the a d d i t i o n of PMNs, and the
mixture was incubated f o r 20 minutes at 37 C with end over end tumbling approximately 20 times per minute.
The r e a c t i o n was terminated by the
a d d i t i o n of 2.0 ml of cold 10% T r i c h l o r o a c e t i c acid (TCA).
The p r e c i p i t a t e
164
was collected by centrifugation at l,O00 x g for 5 minutes at 4 C and washed twice with 2.0 ml of cold I0% TCA. The counts per minute (CPM) of radioa c t i v i t y remaining in the p r e c i p i t a t e was determined in a Gamma Counter (Autowell I I , Picker Nuclear, North Haven, CT, USA). A blank containing a l l components except serum and leukocytes was run with each experiment and the results subtracted from the experimental values. A standard containing the t o t a l amount of 1251 in the reaction mixture was counted, and the nanomoles of iodide converted to a TCAprecipitable form per lO 7 PMNs per hour were calculated as follows:
(CPM experimental standard -CPMcPM
blank)(40, nmole N a l ) ( i . O lO?:~.~ x lO6 P-~s/PMNs~(60 min~,20 rain/
----
nmole Nal/lO 7 PMNs/hr All reaction tubes were run in duplicate and the average value was used for calculations.
Less than 0.5% of the t o t a l added r a d i o a c t i v i t y was TCA-
precipitable in the blank. RESULTS Leukocyte preparation The PMN i s o l a t i o n procedure performed on 50 separate blood samples from apparently healthy animals yielded the results shown in Table l .
An average
TABLE I Yield and p u r i t y of PMNs isolated from 50 samples of blood from healthy adult c a t t l e (Mean ~ S.D.). Percent PMNs in the PMN y i e l d per ml of Percent recovery of Percent eosinophils
f i n a l preparation whole blood (x lO~) PMNs in the PMN preparation
93.6 l .4 45.8 16.9
+ + ~ ~-
6.9 0.7 15.5 9.0
of 1.4 x 106 PMNs were recovered per ml of'blood; this represented an average recovery of 45.8% of the PMNs present in whole blood.
The f i n a l
PMN preparations contained an average of 76.7% neutrophils, 16.9% eosinophils, and 6.4% mononuclear c e l l s .
165
Random m i g r a t i o n under agarose The mean (~ SEM) area o f random m i g r a t i o n by 16 i n d i v i d u a l preparations of p u r i f i e d PMNs was 34.4 (~ 4.5) mm2 (Table I I ) .
Both e o s i n o p h i l s and
TABLE I I Influence of the presence of MNCs and Con A on the area of random m i g r a t i o n by bovine PMNs. Cells
Area of random m i g r a t i o n (mean + SEM, n = 16) mm2 --
PMNs PMNs + Con A PMNS + MNCs PMNS + MNCs + Con A
34.4 21.8 19.1 11.3
n e u t r o p h i l s were observed to migrate.
+ 7 ¥ #
4.5 3.8 3.3 2.9
When an equal number of autologous
MNCs were added to the PMNs the area of random m i g r a t i o n decreased to 19.1 (~ 3.3) mm2.
The a d d i t i o n of Con A to e i t h e r the p u r i f i e d PMNs or the
PMNs plus MNCs markedly decreased the area of random m i g r a t i o n . S. aureus i n g e s t i o n assay A s i n g l e preparation of 1251-iododeoxyuridine labeled S. aureus was used to evaluate the a b i l i t y
of 48 i n d i v i d u a l PMN preparations to ingest S. aureus.
A 60:1 b a c t e r i a to PMN r a t i o was used and lysostaphin was added a f t e r I0 min. A mean (~ SD) of 52% (~ !2.6%) o f the b a c t e r i a were ingested.
This was an
average of 31.2 organisms ingested per PMN (Table I I I ) . TABLE I I I Mean values f o r the PMN f u n c t i o n tests described here when performed on PMNs obtained from normal animals. PMN f u n c t i o n t e s t
Mean
SD
SEM
(n)
M i g r a t i o n under agarose (mm2) Staph i n g e s t i o n (percent of a 60:1 Bact:PMN r a t i o ) NBT reduction (OD/5.0 x 106 PMNs/ 5 min in 5 ml p y r i d i n e ) Chemiluminescence (cph x 106 ) l o d i n a t i o n (nmole Nal/lO 7 PMNs/hr)
34.4 52.0
17.9 12.6
4.5 1.8
(16) (48)
0.49
0.20
0.03
(56)
5.21 48.3
1.57 18.7
0.25 2.5
(38) (56)
166
The e f f e c t of varying the time allowed f o r phagocytosis to occur before l y s o s t a p h i n was added is shown in Figure 2.
When l y s o s t a p h i n was added at the
700
6050-
Q.
4030-
.o 0 m_
....{ ..... .......
..... f ............ { ............ ...........
it....-'I"
20100
0
5•
10 15 20 T' , , Time Iminutesl
25 '
30
Figure 2. Percent i n g e s t i o n of a suspension of S. aureus by normal PMNs ( ...... ) and by PMNs which were t r e a t e d w i t h Cytochalasin B ( ) when l y s o s t a p h i n was added a t v a r y i n g times f o l l o w i n g the a d d i t i o n of PMNs to the phagocytosis m i x t u r e . A b a c t e r i a to PMN r a t i o o f 60:1 was used. (Mean ~ SEM, n = 4). same time PMNs were added there was an average o f 12.4% uptake of S. aureus. There was an increase in the average percent uptake of S. aureus w i t h time f o r the e n t i r e 30 minute i n c u b a t i o n p e r i o d to a maximum o f 61.1%,
The S.
aureus to PMN r a t i o was 60:1; t h e r e f o r e , each PMN ingested an average of 36.6 organisms during the 30 minute p e r i o d . b a c t e r i a to PMN r a t i o
The e f f e c t of varying the
is r e p o r t e d in Table IV.
As the number of b a c t e r i a
was increased the number o f organisms ingested per PMN also increased; however, the percent uptake remained r e l a t i v e l y PMN r a t i o of 120:1 was reached, significantly 240:1.
constant u n t i l
a b a c t e r i a to
At t h i s p o i n t the percent uptake dropped
w i t h a f u r t h e r marked decrease at a b a c t e r i a to PMN r a t i o of
167
TABLE IV The e f f e c t of increasing the number of bacteria in the phagocytosis mixture on the percent uptake of radiolabeled organisms. Phagocytosis was allowed to procede f o r I0 minutes before lysostaphin was added. Bacteria to PMN r a t i o
Percent uptake*
15:1 30:1 60:1 120:1 240:1
35.1 37.2 40.6 31.1 17.3
+ ¥ ¥ ¥ T
No. of org. ingested/PMN
5.4 4.5 2.9 2.1 1.0
5.3 II.2 24.4 37.3 41.5
*(Mean ~ SEM, n = 4)
When the PMNs were pretreated with cytochalasin B to block ingestion the amount of r a d i o a c t i v i t y associated with the PMNs f o l l o w i n g the phagocytic assay was reduced to background levels (Figure 2).
These results demonstrated
the absence of S. aureus adsorbed to the e x t r a c e l l u l a r surface of the PMN f o l l o w i n g lysostaphin treatment. Nitroblue tetrazolium reduction The mean (~ SD) value obtained fo r NBT reduction by 56 i n d i v i d u a l PMN preparations using a preopsonized zymosan stock solution of I0 mg/ml was 0.49 + 0.20 (OD/5.0 x 106 PMNs/5 min in 5 ml pyridine)(Table I I I ) . The effects of d i f f e r e n t methods of opsonization of zymosan upon NBT reduction by normal PMNs are shown in Table V.
There was very l i t t l e
NBT
TABLE V Influence of the method fo r opsonization of zymosan on NBT reduction and i o d i n a t i o n by bovine PMNs. The amount of formazan produced from NBT by 5.0 x 106 PMNs in 5 minutes are expressed as o p t ic al densities in 5.0 ml of pyridine (Mean + SEM of (n) experiments). NBT reduction Optical Density (580 nm) PMNs (resting) PMNs + zymosan PMNs + serum PMNs + serum + zymosan PMNs + heated serum + zymosan (56 C, 30 min) PMNs + preopsonized zymosan
0.04 0.04 0.02 0.17 0.14
+ 0.003 T0.014 ¥ 0.003 ~ 0.02 ¥ 0.02
0.34 + 0.02
lodination nmole Nal/lO 7 PMNs/hr
(4) (4) (4) (4) (4)
2.2 3.6 2.4 15.9 12.3
+ 0.3 T0.6 ¥ 0.2 ¥ 1.3 ~ 1.7
(50) (50) (50) (50) (29)
(4)
48.3 + 2.5
(56)
168 r e d u c t i o n by " r e s t i n g " PMNs o r by PMNs in the presence of serum or zymosan alone.
Optimal NBT r e d u c t i o n occurred when preopsonized zymosan was used as
the p a r t i c l e
for ingestion.
PMNs s t i m u l a t e d by zymosan in the presence o f
f r e s h serum reduced only a p p r o x i m a t e l y 50% as much NBT as PMNs s t i m u l a t e d by preopsonized zymosan. activity
Heat i n a c t i v a t i o n
d i d not markedly d i m i n i s h the opsonic
o f the serum.
S t i m u l a t i o n o f PMNs w i t h graded amounts o f preopsonized zymosan r e s u l t e d in a p o s i t i v e c o r r e l a t i o n NBT r e d u c t i o n .
between the amount of preopsonized zymosan added and
The NBT reducing c a p a c i t y o f the PMNs was a p p a r e n t l y not
exceeded, even a t high c o n c e n t r a t i o n s o f preopsonized zymosan (Figure 3).
0.5-
8-
E
7-
z 0.4-
~7ol
G
£6" x
x
o. 0.3-
~ 604
LO a
504
~4, 8 0.2-
-+ 3-
o "0 I.--
z~0.1 -
I
v 404 g 304
E ~2-
2oi
o
? 1-
0.0-
O.
"
ol 0 t.o
ld.o
2d0
4do
Concentration of preopsonized zymosan in the stock solution (mg/ml)
Figure 3. I n f l u e n c e o f varying the q u a n t i t i e s o f preopsonized zymosan a v a i l a b l e f o r phagocytosis on NBT r e d u c t i o n ( ~ ) , chemiluminescence (C and i o d i n a t i o n (0=---0) by bovine PMNs. F i f t y m i c r o l i t e r s o f the r e s p e c t i v e stock s o l u t i o n was added to each r e a c t i o n tube.
~),
169
Chemiluminescence T h i r t y - e i g h t determinations of chemiluminescence by normal PMNs using a preopsonized zymosan concentration of I0 mg/ml y ie ld e d a mean (~ SD) of 5.21 (~ 1.57) x 106 cph (Table I I I ) . Figure 3 i l l u s t r a t e s the e f f e c t of varying the quantity of preopsonized zymosan in the reaction mixture. zymosan increased l i g h t emission. conditions employed.
Increasing the amount of preopsonized This did not reach an end point under the
Resting PMNs, in the absence of serum or p a r t i c l e s ,
did not demonstrate any s i g n i f i c a n t chemiluminescence. lodination The mean (~ SD) i o d i n a t i o n value obtained from 56 i n d i v i d u a l PMN preparations stimulated by a preopsonized zymosan stock concentration of I0 mg/ml was 48.3 + 18.7 nmole Nal/lO 7 PMNs/hr. The e f f e c t of varying the quantity of opsonized zymosan a v a i l a b l e f o r phagocytosis is i l l u s t r a t e d in Figure 3.
l o d i n a t i o n increased with increasing q u a n t i t i e s of preopsonized
zymosan. Nearly maximal levels of i o d i n a t i o n were obtained with a preopsonized zymosan preparation of 5 mg/ml; a d d i t i o n a l preopsonized zymosan beyond 5 mg/ml did not markedly increase i o d i n a t i o n .
The effects of d i f f e r e n t
methods of opsonization of zymosan on i o d i n a t i o n by normal PMNs is i l l u s t r a t e d in Table V.
There was very l i t t l e
i o d i n a t i o n by resting PMNs or by PMNs in
the presence of e i t h e r zymosan or serum alone.
Maximal i o d i n a t i o n was
attained in the absence of serum when preopsonized zymosan was used as the ingested p a r t i c l e .
Heat i n a c t i v a t i o n of the serum did not abolish
iodination. DISCUSSION The PMN i s o l a t i o n procedure was rapid, reproducible, and yielded a cell preparation with good b i o l o g i c a l a c t i v i t y and no observable clumping.
In
preliminary experimentation clumping was observed when PMNs were held in suspension in the presence of Ca2+ and Mg2+.
The procedure worked equally
well in animals that were neutropenic; however, the t o t a l y i e l d of PMNs was, of course, reduced.
Carlson and Kaneko (1973) reported a PMN recovery of
90.0% + 28.9% y i e l d i n g a c e l l preparation of 87.8% + 7.0% PMNs. This is a higher percent c e l l recovery but a lower p u r i t y of the PMN preparation than in this report.
Our reduced recovery was probably due to the more l i b e r a l
170
removal of the upper portion of the packed erythrocyte layer before l y s i s . This area contains a mixture of red blood c e l l s , PMNs, and mononuclear c e l l s . By l i b e r a l l y removing the upper portion of the packed red blood c e l l s , the p u r i t y of the PMN preparation can be increased, but many PMNs w i l l be l o s t . The percent of eosinophils in the PMN preparations varied considerably. The cell suspension was adjusted to a standard number of neutrophils plus eosinophils because eosinophils have been demonstrated to be capable of p a r t i c i p a t i n g in a l l of the functions assayed in this report (Simmons and Karnovsky, 1973; Klebanoff, et a l . , 1977). The presence of normal Ficoll-Hypaque p u r i f i e d MNCs markedly reduced the random migration by PMNs, but the mechanism f o r this migration i n h i b i t i o n by unstimulated MNCs is not known. Therefore, when evaluating random migration by PMNs i t is important to p u r i f y the PMNs or at least standardize the number of MNCs present.
The phytomitogen Con A markedly reduced the random migration
by p u r i f i e d PMNs and by PMNs mixed with an equal number of MNC, Whether this i n h i b i t i o n is due to a d i r e c t e f f e c t of Con A on PMNs, or to an i n d i r e c t e f f e c t of a lymphokine released by Con A stimulated MNCs is unknown. The p u r i f i e d PMNs did contain a small proportion of MNCs (Table I) which may have produced s u f f i c i e n t leukocyte migration i n h i b i t i o n factor to account fo r the reduced migration by p u r i f i e d PMNs in the presence of Con A. The method described using 1251-iododeoxyuridine and fluorodeoxyuridine proved to be a simple and convenient method fo r l a b e l i n g S. aureus with a gamma emitting radioisotope.
The heat k i l l e d 1251-iododeoxyuridine labeled
S. aureus may be stored at 4 C and u t i l i z e d over a period of several days; thus saving time and minimizing day to day v a r i a b i l i t y in reagents. The 125 I label was e f f i c i e n t l y released from the S. aureus by lysostaphin digestion. Lysostaphin has been used successfully as an aide f o r evaluating ingestion of S. aureus by PMNs (Verhoef, et a l . , 1977b; Paape, et a l . , 1978).
I t has
been shown to r a p i d l y lyse S. aureus, and i t is not t o x i c f or and does not enter phagocytic c e l l s (Tan, et a l . , 1971; Easomn, et a l . , 1978).
Lysostaphin
is very useful because i t lyses the bacteria which are adherent to the surface of the PMN; this permits d i f f e r e n t i a t i o n from those that are ingested.
When
lysostaphin was added to the phagocytosis mixture at the same time as the PMNs an average of 12.4% of the S. aureus were ingested by the PMNs (Figure I ) . This was apparently due to ingestion that occurred p r i o r to lysostaphininduced l y s i s of the S. aureus in the phagocytosis mixture since blockage of ingestion by PMNs by cytochalasin B treatment resulted in background amounts of r a d i o a c t i v i t y associated with the PMNs. The lag period in lysostaphin action was not f e l t to be important when evaluating the a b i l i t y of PMNs to
171
ingest p a r t i c l e s .
A I0 minute incubation period was chosen f o r the standard
ingestion assay so that the PMNs would not reach t h e i r capacity for ingestion (Figure 2).
This permits determination of the rate of ingestion by PMNs. A
longer incubation period and a higher bacteria to PMN r a t i o would be desirable i f one wished to measure the capacity of ingestion by PMNs. The results in Figure 2 and Table IV indicate that the maximum number of bacteria which can be ingested by a single bovine PMN is approximately 40-45.
This agrees
closely with the calculated maximal uptake of S. aureus by human PMNs as determined by L e i j h , et a l . (1979). The o x i d a t i v e metabolism of the PMN is an important aspect of i t s b a c t e r i c i d a l a c t i v i t y (For a review see Babior, 1978; Klebanoff, 1979).
When
i t receives the proper stimulus an oxidase enzyme on the outer surface of the PMN plasma membrane and on the inner surface of the phagosomal membrane w i l l catalyze the conversion of oxygen to superoxide anion (02).
Superoxide anion
is a highly reactive oxygen moiety which may undergo a spontaneous dismutation to form hydrogen peroxide.
Singlet oxygen (a free radical of oxygen) may be
a short l i v e d intermediary in this dismutation.
Some of the ~ydrogen
peroxide formed may react with a d d i t i o n a l superoxide anion to form hydroxyl radicals.
Superoxide anion, s i n g l e t oxygen, hydrogen peroxide, and the
hydroxyl radical are highly reactive compounds and, when formed inside the phagocytic vacuole, are believed to play an important role in the k i l l i n g of microorganisms by the PMN. NBT is d i r e c t l y reduced by superoxide anion to an insoluble purple formazan (Yost and Fridovich, 1974).
NBT reduction is therefore a measure
of superoxide anion generation by the PMN. Chemiluminescence is also dependent upon the generation of superoxide anion by the PMN, although superoxide anion i t s e l f does not produce l i g h t .
The mechanism f o r l i g h t
production by the PMN is not understood but several possible mechanisms have been proposed (Babior, 1978; Hodgson and Fridovich, 1976).
Goldstein, et a l . ,
(1977) demonstrated that the superoxide anion generating system appears to be associated with the outer surface of the PMN plasma membrane as well as the inner surface of the phagosomal membrane and that superoxide generation may proceed independently of phagocytosis and degranulation.
They found that
superoxide anion recovery in the medium surrounding PMNs was in fact enhanced when the formation of phagocytic vacuoles was i n h i b i t e d by treatment of c ells with cytochalasin B.
This superoxide anion in the medium w i l l reduce
e x t r a c e l l u l a r NBT to formazan and produce chemiluminescence.
NBT reduction
and chemiluminescence therefore appear to be v a l i d measurements of d i f f e r e n t
172
aspects of the o x i d a t i v e metabolism of the PMN but can not be interpreted to be v a l i d measurements of ingestion by PMNs. l o d i n a t i o n is a measure of the a b i l i t y of the PMN to convert inorganic iodide to a t r i c h l o r a c e t i c acid (TCA) p r e c i p i t a b l e (protein-bound) form. The iodide is covalently bound to a suitable acceptor molecule such as the tyrosine residues of protein in the phagocytic vacuole via the action of hydrogen peroxide (H202) and myeloperoxidase.
This system has been found to
e x h i b i t marked t o x i c a c t i v i t y toward bacteria, fungi, and viruses (Belding and Klebanoff, 1979; Simmons and Karnovsky, 1973).
The i o d i n a t i o n reaction is
dependent upon a number of processes (Klebanoff and Clark, 1977).
Hydrogen
peroxide is generated by the action of a membrane bound oxidase enzyme to convert oxygen to superoxide anion which may spontaneously be reduced to form H202. The myeloperoxidase needed is present in the primary granules in the PMN cytoplasm.
This enzyme must be delivered to the phagocytic vacuole or the
e x t r a c e l l u l a r environment by the process of degranulation.
Ingestion of
opsonized p a r t i c l e s w i l l stimulate H202 formation and degranulation in normal PMNs. l o d i n a t i o n has been demonstrated to occur both in the phagocytic vacuole and e x t r a c e l l u l a r l y due to release of myeloperoxidase and H202 into the e x t r a c e l l u l a r environment (Klebanoff and Clark, 1977).
The extent of the
e x t r a c e l l u l a r i o d i n a t i o n may depend upon the conditions under which phagocytosis is conducted.
In an experimental system s i m i l a r to the one described
here, normal i o d i n a t i o n was shown to be dependent upon ingestion (Klebanoff and Clark, 1977).
A depressed i o d i n a t i o n value may be due to lack of
ingestion, a lack of normal o x i d a t i v e metabolism within the PMN, a f a i l u r e of degranulation, a reduced amount of myeloperoxidase in the primary granule, destruction of H202 (by catalase) or interference with the myeloperoxidase catalyzed reaction. t i a t e these defects.
Other experimental procedures are required to d i f f e r e n Because the i o d i n a t i o n reaction is dependent upon a
complex chain of events, i t is a good screening test to detect PMN dysfunction. Optimal NBT reduction and i o d i n a t i o n by PMNs was obtained when preopsonized zymosan was used in a serum free system as the phagocytosable p a r t i c l e (Table V).
In the presence of bovine serum the NBT reduction and i o d i n a t i o n
values were markedly reduced.
This may at l e a st p a r t i a l l y be due to the
presence of conglutinin in bovine serum.
Conglutinin reacts with a mannose
peptide determinant found on the t h i r d component of complement (C3b) and on zymosan. Conglutinin is responsible f o r the powerful clumping of zymosan and of complement coated p a r t i c l e s in bovine serum (Lachmann, 1975). Klebanoff and Clark (1977) reported that heating human serum to 56 C f o r 30 minutes reduced the mean i o d i n a t i o n value f o r human PMNs in the presence of zymosan from 64.1 to 1.8 nmole Nal/lO 7 PMNs/hr, which was equivalent to the
173
level of i o d i n a t i o n by resting PMNs. This demonstrates that only heat l a b i l e factors in human serum are able to opsonize zymosan.
In the bovine system
heating of the serum to 56 C f o r 30 minutes only s l i g h t l y reduced i o d i n a t i o n and NBT reduction by bovine PMNs (Table V).
This indicates that in addition
to a heat l a b i l e opsonin there is also a heat stable opsonin f o r zymosan present in bovine serum.
Klebanoff and
Clark (1977) concluded that when
normal c e l l s and p a t i e n t ' s serum are employed, the i o d i n a t i o n reaction is an i n d i r e c t measure of the opsonic a c t i v i t y of the p a t i e n t ' s serum. They were able to demonstrate decreased opsonic a c t i v i t y f o r zymosan of human serum d e f i c i e n t in the fourth or t h i r d component of complement.
Due to the
presence of c o n g l u t i n i n and a heat stable opsonin f o r zymosan in bovine serum, the i o d i n a t i o n reaction, chemiluminescence, and the NBT reduction test as described here cannot be used to detect opsonic defects in whole bovine serum. The results in Figure 3 indicate that a zymosan preparation of 5 mg/ml yielded nearly optimal values fo r i o d i n a t i o n , however, this level of opsonized zymosan did not give optimal values f o r NBT reduction and chemiluminescence. A concentration of 40 mg of zymosan per ml yielded over 150% more NBT reduction and chemiluminescence but only approximately 10% more i o d i n a t i o n than 5 mg of zymosan per ml.
Chemiluminescence and NBT reduction are
measures of the o x i d a t i v e metabolism of the PMN; therefore, as the opsonized zymosan concentration is increased to 40 mg/ml, there is increased o x i d a t i v e metabolism without a corresponding increase in i o d i n a t i o n .
This suggests
that the o x i d a t i v e metabolism is not the l i m i t i n g f a c t o r in the i o d i n a t i o n reaction by normal bovine PMNs. ACKNOWLEDGEMENTS This work was supported by Grant No. 413-43-08-85-2229 from the Cooperative State Research Service of the United States Department of Agriculture and by formula funds provided by the United States Department of A g r i c u l t u r e , Grant No. 410-23-02.
174
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