A single centrifugation step method for the simultaneous separation of different leukocytes with special reference to Basophilic leukocytes

Journal of Immunological Methods, 57 (1983) 1-7 Elsevier Biomedical Press

1

A Single Centrifugation Step Method for the Simultaneous Separation of Different Leukocytes with Special Reference to Basophilic Leukocytes H . F . K a u f f m a n , P . R . L e v e r i n g a n d K. D e Vries Department of Allergology, Clinic for Internal Medicine, State University Hospital, Groningen, The Netherlands (Received 22 March 1982, accepted 5 August 1982)

A simple method of separating mononuclear leukocytes, basophils, neutrophils and an eosinophil fraction from h u m a n blood is described, using discontinuous Percoll gradient centrifugation. Mononuclear cells were found in the upper layer (band 1) at a density of ~ 1.072. The second band with a density of ~< 1.082 contained most of the basophilic leukocytes (av. 70% and 8-20% of the differential cell count) and a minor part of the mononuclear cells (av. 8%). In the third band, density ~< 1.1, 82-91% of the polymorphonuclear cells (PMN) were found, consisting mainly of neutrophils. 35-70% of the eosinophilic P M N cells are located in band four (density > 1.1). The function of the basophil-enriched fraction was tested by degranulation with specific antigen. Key words: basophilic leukocytes - - leukocyte separation method - - discontinuous gradient cell separation

Introduction

Measurement of function of different cell types is often used in studies on the pathogenesis of different diseases. Special attention has been paid to the study of basophils, mononuclear cells, eosinophils and neutrophils in the studies on bronchial asthma. Procedures for the simultaneous purification of these different cell types from human blood are needed. Many purification procedures described for certain cell types are laborious and often exclude the purification of one or more other cell types. The use of a new commercially available gradient medium, Percoll, and its advantages for cell purification have already been described for different cell types (Ulmer and Flad, 1979; G~rtner, 1980; Segal et al., 1980; Hjorth et al., 1981). However, little is known about the separation and simultaneous purification of basophilic leukocytes (G~rtner, 1980) and no functional studies have been reported. The present study describes a simple and rapid method for simultaneous purification of intact basophils, mononuclear cells, PMN cells and a platelet-rich eosinophilic fraction. Special attention was paid to the purification and the functional integrity of the basophilic fraction. The functional integrity of t h e basophilic leukocytes was tested with cells of allergic individuals by degranulation with specific allergen. 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

Materials and Methods

Ten millilitres venous blood were taken in syringes containing 1.5 ng EDTA ( N a 2 ) / m l blood from healthy volunteers; for the basophil degranulation test (BDT), 20 ml of blood were used. Five parts of blood were mixed with one part of 6% dextran (Dextran T-500, Pharmacia Fine Chemicals, Uppsala) in 3% glucose and 0.15 M NaCI and left at 37°C for 30-40 min for sedimentation of the red cells. The dextran plasma layer containing the white blood cells was collected and centrifuged for 10 min at 150 × g and 4°C. Most of the plasma was removed and the cells were resuspended in a residual 1 ml volume of plasma. These preparations are referred to as leukocyte concentrates.

Discontinuous density gradient centrifugation A stock solution of Percoll (Pharmacia, Uppsala) was prepared by mixing 9 parts of Percoll with 1 part of 1.5 M NaCI (density 1.123 g / m l ) and stored at - 2 0 ° C . The Percoll solution was adjusted to a p H of 7.4 by addition of 1 M HC1. NaC1 solution (0.15 M, p H 7.4) was added to give mixtures of densities 1.100, 1.082 and 1.072 g / m l , using the formula:

vx=v

.

( n o - 0) (p-p×)

° . . . . .

Vx = volume of NaC1 solution; Vo = volume of Percoll stock solution; Po = density of Percoll (1.130 g/ml); Px = density of 0.15 M NaC1 (1.058 g/ml); p = desired density. Preliminary studies in our laboratory with different gradient steps showed that basophils are optimally recovered at a density of 1.082 while the degree of purification is optimal for retention of monocytes and lymphocytes in the upper layer at a density of 1.072. Gradients were formed by carefully pipetting 1.5 ml volumes of decreasing densities successively into polypropylene tubes (17 × 100 ml Falcon 2017) with plastic pipettes (Falcon 7506F). The leukocyte concentrates (1 ml) were layered on top of the gradients and centrifuged in a swing-out rotor at 400 × g for 20 rain at 4°C using a Minifuge 2 centrifuge (Heraeus Christ). Acceleration and stopping of the run were both performed carefully to avoid disturbing the gradient. With larger amounts of cells (20-40 ml of blood), 4 ml volumes of Percoll solution of identical densities were layered one over the other and 1- - 2 ml (av.) leukocyte concentrates/20 ml blood layered on top. The cells concentrated at the interface and bottom of the tube were collected into polystyrene tubes (Falcon 2095), by means of a peristaltic p u m p (LKB 2120 Varioperpex pump, LKB, Bromma). The cells were washed once with 10 ml minimal essential medium (MEM, Serva, Heidelberg) at 4°C.

Cell counting, differentiation and viability The washed cells were resuspended in 0.5 ml of autologous plasma before counting in a Coulter counter (Coulter Electronics, Harpenden Herts).

Cytocentrifuge smears were prepared from each fraction and from the leukocyte concentrates, and differential counts were done after staining with MayGrunwald/Giemsa. Viability of the cells as tested by trypan blue exclusion was excellent, and exceeded 98%. The functional integrity of the purified basophils was tested by the basophil degranulation test (BDT) based on a staining procedure as described by Benveniste (1981). The BDT was carried out with cells obtained from patients who had been previously examined for allergy to house dust mite by skin testing with D. pteronissinus. The cells of band 2 (1.082 ~< p < 1.072) were washed once in MEM, washed again in Tris-A buffer at 4°C (NaC1 120 mM, KC1 5 mM, Tris 25 mM, pH 7.4, human serum albumin 0.03%) and resuspended in 3.6 ml Tris ACM (Tris-A buffer with 1 mM Ca 2+ and 1 mM Mg z+) at room temperature. Aliquots (0.5 ml) of cell suspension were added to polystyrene tubes containing 0.1 ml house dust mite extract (D. pteronissinus, Diephuis Laboratories, Groningen) in Tris-ACM buffer of various concentrations ranging from 0.005 ng to 500 n g / m l with 10-fold increments. Blank tubes contained cells and buffer without antigen. The cells were incubated for 30 min at 37°C in a water bath. Degranulation was stopped by placing the tubes in ice, followed by centrifugation for 10 min at 600 × g and 4°C. The cells were resuspended in 0.25 ml 0.1 M EDTA (Na2), 0.9% NaC1 and stained with Alcian blue as described by Gilbert and Ornstein (1975). Basophils were counted in a Fuchs-Rosenthal haemocytometer (3.2 mm 3). The purification procedure gave basophil counts in the blanks exceeding 100 cells/chamber. Basophil counts are expressed as percentages of the total cell count.

Results

Density gradient centrifugation as described above separates cells from blood into 4 separate cell layers. One band is at the top of the low density Percoll layer (density = 1.072 g/ml), two at the interfaces of the Percoll layers with densities 1.072/1.082 g / m l and 1.082/1.100 g / m l respectively, and a fourth band at the bottom of the tube contains cells with densities > 1.100 g/ml. Table I shows the distribution and recoveries of leukocytes from the same healthy individual on 11 different occasions. The lowest density cells (band 1) contained virtually only mononuclear cells (99.7%) and very few basophils (0.3%). The second band consisted of mononuclear cells (82.3%), basophils (15.4%) and polymorphonuclears (2.1%). The basophils in this second band were enriched more than 20 times and accounted for 69.3% of the total basophil count. The third band contained mainly polymorphonuclear cells (97.5%) with small numbers of basophils and mononuclear cells. The higher density cells at the bottom of the tube contained many platelets and 7.6% of the total cell count. These cells appeared to be mainly polymorphonuclears (98.9%) and included varying proportions (35-70%) of eosinophilic leukocytes. The low standard deviation of the differential counts (between 0.1 and 3.9%) indicates that the reproducibility of the procedure is high. In Table II the distribution and recoveries are shown for 1 healthy normal and 3 allergic patients with asthma with

d<1.072 1.072l.l~

1 2 3 4

38.9±7.9 4.1±1.4 45.6±1.9 7.6±1.8

Overall recovery a

50.5

99.7±0.1 82.3±3.9 2.3±0.7 1.1±0.9

a %

100

86.3±11.9 7.6±2.9 2.4±0.9 0.2±0.2 0.7

0.3±0.1 15.4±3.1 0.1+ 0.1 0

D

D b

R~

Basophils

Mononuclear cells

of total amount. b D = differential count as % of total cells in the band. c R = differential recovery as % of cells applied to the gradient.

Be~re separation -

Density ( g / m l )

Band

100

12.3±5.9 69.3±12.8 7.0±7.2 0

R

48.8

0 2.1±1.3 97.5±0.8 98.9±0.9

D

100

0 0.1±0.1 81.9±6.5 13.9±3.0

R

Polymorphonuclear cells

D I F F E R E N T I A L COUNTS A N D RECOVERIES OF L E U K O C Y T E S 1N T H E 4 BANDS O F CELLS F RO M 1 I N D I V I D U A L A F T E R 11 I N D E P E N D E N T SEPARATIONS ON D I S C O N T I N U O U S P E R C O L L D E N S I T Y G R A D I E N T S

TABLE I

1.072 < d < 1.082

1.082 < d < 1.100

d > 1.100

2

3

4

1 2 3 4

1 2 3 4

1 2 3 4

1 2 3 4

1 2 3 4

Ind. no. d

7.1 5.4 14.8 7.3

45.1 41.2 63.9 48.3

4.1 3.8 2.7 4.6

40.4 47.2 21.6 38.8

45.3 54.6 24.0 44.8

2.0 5.0 0 0

2.2 1.2 0.2 0.4

81.2 77.9 75.6 83.6

99.8 99.8 99.2 99.5

D b

0.3 0.5 0 0

2.2 0.9 0.5 0.4

7.3 5.4 8.5 8.6

89.0 86.2 89.3 86.1

R ~

0.9 1.0 0.3 0.5

0 0 0 0

0.2 0.2 0 0

17.0 20.8 8.0 8.6

0.2 0.3 0.3 0.4

D

0 0 0 0

10.0 8.3 0 0

77.4 76.8 72.0 79.1

9.0 11.8 21.6 21.0

R

DENSITY

Basophils

PERCOLL

M o n o n u c l e a r cells

BY D I S C O N T I N U O U S

Overall recovery a

a % o f total a m o u n t . b D = differential c o u n t as % of total cells in the b a n d . R = differential r e c o v e r y as % o f cells a p p l i e d to the g r a d i e n t . d Ind. no.: 1 = h e a l t h y n o r m a l ; 2 - 4 = allergic p a t i e n t s with a s t h m a .

Before separation

d < 1.072

Density (g/ml)

OF LEUKOCYTES FROM 4 INDIVIDUALS AS D E S C R I B E D IN T A B L E I

l

Band

SEPARATION CONDITIONS

T A B L E II

0 0 0.5 0.1

53.8 44.4 75.7 54.7

98.0 95.0 100.0 100.0

97.6 98.6 99.8 99.6

1.8 1.4 16.4 5.8

D

12.9 ! 1.6 19.6 13.3

81.8 91.5 84.2 88.0

0.1 0.1 0.6 0.5

0 0 0.1 0.1

R

CENTRIFUGATION.

Polymorphn u c l e a r cells

GRADIENT

_~ 8

4'

21

i

C

i

i

510"~' 5103

i

i

5.102 51(31

i

i

5

50

/Jg house dust rnite/ml

~.

Fig. I. Degranulation of basophilic leukocytes purified by discontinuous Percoll gradient centrifugation (band 2). Cells of three allergic individuals ( * *, • • , zx zx) and one healthy control (©-O) tested at increasing house dust mite allergen concentrations. Cells counted under the microscope after staining with Alcian blue. Conditions as described under Materials and Methods.

different ratios of cell populations. The leukocytes in the 4 bands show similar distribution patterns with 17--27-fold enrichment of basophils in band 2 and recoveries between 72 and 79%. The differential counts of the cells in bands 1 and 3 (mononuclear and polymorphonuclear cells, respectively) reflect the different numbers of these cells found before separation. The functional integrity of the basophils in band 2, from 3 allergic individuals and one non-allergic control was tested by the basophil degranulation test. As shown in Fig. l, the basophilic fractions (band 2 cells) of the allergic individuals show a decrease in amount of Alcian blue stainable cells when incubated with increasing amounts of house dust mite allergen. By contrast, the basophil count of the healthy control did not change at different allergen concentrations. Thus the basophils retain their functional integrity with respect to allergen responsiveness after being submitted to the separation procedure.

Discussion

This study describes a simple and rapid method for separation of different cell types (mononuclear cells, basophils and polymorphonuclear cells) involved in the pathophysiology of bronchial asthma. The eosinophilic leukocytes were mainly found in the high density cell fraction (band 4). This is in accordance with the studies of G~irtner (1980) who used continuous gradient separation on Percoll. The recovery of this cell type may possibly be optimized by slightly decreasing the density of the higher density Percoll

layer (p = 1.100), but this was not tested in this study. The simultaneous separation of other cell types makes it possible to study different cell characteristics in one blood sample, purified under identical conditions with viabilities of 98% or more. The more than 20-fold enrichment of basophils attained in the second band, is greater than previously described for one-step centrifugation methods ( B ~ u m , 1968). In general, the other cells in this band are mostly mononuclear cells, although the number of polymorphonuclear cells tends to increase when larger quantities of these latter cells are present before separation (individuals 3 and 4, Table II). It was shown that the basophil cell fraction of allergic individuals responds to house dust mite allergen in a dose-dependent manner. The study of basophil degranulation by staining techniques has been introduced recently but the numbers counted were relatively small (Benveniste, 1981) excluding patients with low basophil counts. The basophil count of band 2 preparations, which exceed 100 cells per Fuchs-Rosenthal chamber, should lead to more accurate antigen dose response curves and will also be useful when the basophil count in the blood is low. It is important to note that in accordance with the observations of Pruzansky et al. (1980), partially degranulated cells were never observed at the different house dust mite concentrations used. This indicates that in the basophil population there are cells with different sensitivities towards the allergen, total degranulation of some cell groups occurring at a given allergen concentration while other cells remain unaffected. Preliminary experiments with both the mononuclear cell fraction (band 1) and polymorphonuclear cells (band 3) showed functional activities in the isoproterenol-stimulated cAMP response and in zymosan-stimulated beta-glucuronidase release, respectively, indicating that these cells are also functionally intact with respect to parameters studied in bronchial asthma (May et al., 1970; Busse and Sosman, 1977). This finding is in agreement with that of Hjorth et al. (1981) with granulocytes which had also been isolated by Percoll gradient centrifugation.

Acknowledgements We thank Mr. S. Van der Heide and Mr. H. Meurs for discussions and reading the manuscript, and Mr. H. Lange for his expert technical assistance.

References Benveniste, J., 1981, Clin. Allergy 11, 1. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 1. Busse, W.W. and Sosman, 1977, J. Clin. Invest. 59, 1080. G~irtner, I., 1980, Immunology 40, 133. Gilbert, H.S. and L. Ornstein, 1975, Blood 46, 279. Hjorth, R., A.K. Johnson and P. Vretblad, 1981, J. Immunol. Methods 43, 95. May, Ch.D., M. Lyman, R. Alberto and J. Cheng, 1970, J. Allergy 46, 12. Pruzansky, J.J., C.R. Zeis and R. Patterson, 1980, J. Immunol. 40; 411. Segal, A.W., A. Fortunato and T. Herd, 1980, J. lmmunol. Methods 32, 209. Ulmer, A.J. and H.D. Flad, 1979, J. Immunol. Methods 30, 1.