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A new assay for leukocyte chemotaxis using cell retrieval, electronic particle counting and flow cytometry
Journal of Immunological Methods, 49 ( 1 :;82) 215 --233 Elsevier Biomedical Press
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A NEW A S S A Y F O R L E U K O C Y T E C H E M O T A X I S U S I N G C E L L RETRIEVAL, ELECTRONIC PARTICLE COUNTING AND FLOW CYTOMETRY
IVAR AMUND GRIMSTAD and HAAKON B. BENESTAD Institute of Physiology, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway
(Received 4 May 1981, accepted 18 September 1981)
A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.
Key words: chemotaxis --leukocyte isolation --flow cytometry --automated cytology
INTRODUCTION Several assays have b e e n i n v e n t e d t o s t u d y crawling m o v e m e n t s o f cells. T h e results o b t a i n e d w i t h these m e t h o d s m a y e l u c i d a t e basic p r o c e s s e s o f cell m o t i l i t y . T h e y m a y also serve practical p u r p o s e s , since d e f e c t s in, f o r e x a m p l e , l e u k o c y t e m o t i l i t y dispose to disease. T h e l o c o m o t i o n o f single cells m a y b e o b s e r v e d m i c r o s c o p i c a l l y (Gail a n d B o o n e , 1 9 7 0 ; Z i g m o n d , 1 9 7 8 ) . O t h e r in vitro m e t h o d s have b e e n designed t o m e a s u r e t h e n e t d i s p l a c e m e n t o f g r e a t e r n u m b e r s o f cells: s o m e are b a s e d o n m i c r o s c o p i c a l d e t e c t i o n o f cells a d h e r e n t t o m i c r o p o r e m e m b r a n e s , a f t e r having m i g r a t e d i n t o o r t h r o u g h t h e m . T h e r e s p o n s e can b e m e a s u r e d b y assessing t h e n u m b e r o f cells t h a t have m o v e d a c e r t a i n d i s t a n c e ( B o y d e n , 1 9 6 2 ) , or t h e distance travelled b y t h e leading cells ( Z i g m o n d a n d Hirsch, 1 9 7 3 ) , or b y a c o m b i n e d a p p r o a c h e s t i m a t i n g m e a n cell d i s p l a c e m e n t ( M a d e r a z o a n d W o r o n i c k , 1 9 7 8 ) . T h e n u m b e r o f cells t h a t have m i g r a t e d a p r e d e t e r m i n e d distance can also b e c o u n t e d r a d i o c h e m i c a l l y (Gallin e t al., 1 9 7 3 ) . O t h e r m e t h o d s d e p e n d o n similar m i c r o s c o p i c m e a s u r e m e n t s o n cells crawling u n d e r agarose in dishes (Cutler, 1 9 7 4 ; Orr a n d Ward, 1 9 7 8 ) or along 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press
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(Ketchel and Favour, 1955) or out of (Clausen, 1971) capillary tubes. Micropore membrane techniques are the most sensitive ones in assessing leukocyte chemotaxis (Kreutzer et al., 1978). However, all variants so far described render difficult further study of cells introduced to the systems, and involve either extensive handling of the cells (e.g. radiolabeling) before migration starts or tedious microscopy. The assay described here avoids these drawbacks, as well as the subjectivity of the observer. It allows extensive cell migration; it is sensitive, precise, and easy to standardize; and it gives more reproducible results than other methods. The easy retrieval of cells that have migrated may allow studies, previously n o t feasible, on motile cells other than leukocytes as well e.g., neoplastic cells capable of invasion and metastasis. M A T E R I A L S AND M E T H O D S
Cell migration chambers Chambers of the new type (Fig. 1) were made in the following way: punched-out 13 mm diam. micropore membranes of various types were heatsealed (Benestad and Reikvam, 1975) onto acrylic plastic cups designed for this purpose. The depth of a resulting cell inoculum c o m p a r t m e n t is 4 mm and its volume ~315 pl. Alternatively, membranes can be glued onto the acrylic parts (Benestad, 1970), although this was not done in the present study. Hundreds of such compartments may be assembled during one working day. Absence of leaks was verified by the 'air escape test', blowing
Fig. 1. Left: 3 cell m i g r a t i o n c h a m b e r s o f the n e w t y p e . In an a d d i t i o n a l cell i n o c u l u m c o m p a r t m e n t , the filling orifice can be seen. Right: 2 assembled cell migration c h a m b e r s . D i f f e r e n t fluid v o l u m e s can be a c c o m m o d a t e d in the cell retrieval c o m p a r t m e n t s , because the p o s i t i o n o f the cell i n o c u l u m c o m p a r t m e n t can be varied.
217 air through the 1.3 mm diam. filling orifice in the b o t t o m of the cup into the c o m p a r t m e n t under water. A stainless steel rod with a conical tip was used to plug the orifice after filling the inoculum compartment with the cell suspension to be tested. The rod also served to position this c o m p a r t m e n t in the vial constituting the cell retrieval compartment. This was the cut off lower 20 mm of a low pressure polyethylene 'polyvial' (Zinsser, F.R.G.). These had fiat bottoms, internal diam. 14 mm, and were usually filled with 540 pl (in some experiments as little as 150 pl) prewarmed (37°C) medium with or without chemotactic factor before insertion of the cell inoculum compartment. It is easy to avoid trapping of air bubbles below the membrane. The closing caps for the retrieval compartments were made of acetal plastic and had neoprene rubber o-rings to tighten both against the wail of the vial and the central rod. However, keeping the inoculum compartment just below the cap, while positioning the latter, allowed air to escape around a narrow portion of the metal rod before this was pushed down. The fluid surfaces in the two compartments were at the same level during incubation. Before use, all parts of the cell migration chambers were kept at 80°C dry heat overnight (Benestad, 1970). After use the cell inoculum compartments were kept in 2 mol/1 NaOH overnight. This treatment dissolved cellulose membranes and detached polycarbonate ones. After thorough washing, all components except the membranes could be re-used, apparently almost indefinitely, making this new assay very economical.
Micropore rnembranes We routinely used Sartorius SM 11324 cellulose nitrate membranes with average 5 pm pore diam. (Sartorius-Membranfilter GmbH, F.R.G.). Mixed cellulose acetate and nitrate membranes (Millipore Corp., U.S.A.) and polycarbonate membranes (Nuclepore Corp., U.S.A.) were also tested. For economical reasons and to reduce the notorious filter heterogeneity (Maderazo and Woronick, 1978; Ward, 1978), 13 mm diam. disks were punched o u t from large filter sheets of one fabrication lot. Both filter sides had been inspected beforehand through a stereo microscope, so that the Sartorius membrane could be sealed to the plastic cup with the smoother side turning outwards, since this arrangement gave markedly higher and more reproducible migratory responses.
Leukocyte preparation Heparinized blood (20 IU/ml) was obtained, after informed consent, by venipuncture of healthy laboratory personnel and medical students of b o t h sexes, 20--45 years old. Blood was similarly drawn from a few selected patients. We avoided prolonged venous stasis and delayed mixing with the anticoagulant, and kept the cells at ~0°C whenever the procedures did not
218 require room temperature or 37°C. Routinely, erythrocytes were removed by sedimentation, using BOyum's 2-phase Isopaque-methylcellulose technique (BOyum, 1968), at 37°C. Removal of the majority of the red cells took 15--30 min, and a supernatant containing a non-selected sample (usually >70%) of the blood leukocytes was obtained, ahnost free from contamination with separation fluid ingredients. The majority of the blood platelets remained in the leukocyte fraction, but most of them ~.~ould be removed by the following 2 centrifugations. Finally, the leukocytes were suspended, usually at - 5 × 106 neutrophils per ml, in a medium hereafter called 1% HSA. This was Medium 199 (GIBCO, U.S.A., Cat. no. 235) with 25 mmol/1 HEPES buffer, supplemented with 1% (w/v) human serum albumin (HSA), Cohn fraction V (Sigma, U.S.A.) and ampicillin 200 #l/ml (Doktacillin, Astra, Sweden). Preliminary experiments showed that the antibiotic did not affect leukocyte migration. The osmolality of the medium was adjusted to 290 mosM/kg, the pit to 7.20, and it was sterile filtered through a 0.22 #m porosity membrane. In some experiments red blood cells were sedimented by mixing 1 part of 6% (w/v) dextran in 0.9% NaCI (Macrodex, Pharmacia, Sweden) with 10 parts of blood. On other occasions almost pure suspensions of either granulocytes or mononuclear leukocytes were obtained by subjecting the leukocyte fraction of the Isopaque-methylcellulose separation to the FicollIsopaque (Lymphoprep, Nyegaard & Co. A/S, Norway) density separation technique (BCyum, 1976). Contaminating red cells were lysed in some experiments, either by exposing the cells twice to cold, pure water for 10 sec before restoration of isotonicity, or by treating them for 5 rain at room temperature with 0.75V~ NH4CI in 0.02 mol/1 Tris-HC1 buffer at pH 7.35. Chamber inoculation and incubation The chamber membranes were moistened in 1% HSA for seconds. Before filling with cell suspension, the inoculum compartments were placed in a special rack, so that the wetted membranes did n o t touch ground. The cell suspension (240 t~l) was supplied from a 10 ml Gilette disposable syringe (Gilette Ltd., U.K.) through a 25-gauge 10 mm long hypodermic needle by means of a Hamilton PB 600-10 dispenser (Hamilton Bonaduz AG, Switzerland). At least 6 replicate chambers were routinely prepared. The inoculum compartments were suspended in retrieval compartment vials, and the chamber assemblies were ordinarily incubated in air at 37°C for 120 min. The pH remained constant for much longer incubation periods. Cell harvesting procedure This was designed to disaggregate possible cell clumps and detach all cells that had migrated completely through the membrane and were adhering to its outside or to the walls of the cell retrieval c o m p a r t m e n t (i.e., the plastic vial). Addition of 60 tll of a 2.7% (w/v) Na2-EDTA solution (final conc. 0.27% or ~7.3 mmol/l) to chelate Ca 2~ and Mg 2~ and brief shaking with a
219 whirlmixer after a few minutes proved to be sufficient. Samples from the inoculum suspension had been taken for smearing and h e m o c y t o m e t r i c or electronic counting, as detailed elsewhere (Benestad, 1970). In preliminary experiments only, smears were also made of the cell suspensions in the retrieval compartments. At least 500 cells were counted in the h e m o c y t o m e t e r and 400 cells in a smear. Zaponin (Coulter Electronics Ltd., U.K.), 40 pl to 10--16 ml saline with 0.035% NAN3, were used to lyse red cells before counting with a Coulter Counter Model D Industrial (Coulter Electronics Ltd. or a Celloscope 101 (Linson Instruments, L. Ljungberg and Co., Sweden). In some control experiments, Triton X-100 (Sigma) was used as a lysing agent. The results were expressed as the percentage of inoculated neutrophils that had emigrated and were retrieved from the polyethylene vials. During a 2 h period granulocytes were virtually the only cells to cross the filter; differential counting of this population was therefore omitted after the pilot experiments. Very rarely, the cell count for one of the replicates could be much higher than for the others. If, after retesting, the cell inoculum compartment disclosed leaks, the high count was disregarded.
Conventional Boyden chambers. Preparation of membranes for microscopy Neuro Probe blind well chemotaxis chambers (Nuclepore Corp., U.S.A.) were used to compare the new m e t h o d with an established one (Boyden, 1962). The upper compartments of the conventional chambers were closed with a thick tape, so that both types of chamber could be incubated under identical conditions. The conventional chambers were prepared in triplicate. Fixation, staining, clearing and microscopical examination of cells on membranes from the conventional and in some cases also the new chambers were done according to standard techniques (Maderazo and Woronick, 1978). Chemotactic attractants Fresh serum was prepared from defibrinated blood. It was diluted and supplemented with 200 pg/ml ampicillin before use. Zymosan A (Sigma) activated serum (Brade et al., 1973) was obtained by incubating 5 mg/ml of the yeast polysaccharide in fresh serum for 30 min at 37°C in a shaking water bath, thus activating the complement system via the alternative pathway. Zymosan was removed by centrifugation, and ampicillin, 200 pg/ml, was added. Escherichia coli culture filtrate (E. coli CF) was obtained by culturing E. coli HF 4704 Thy, first for 4 h at 37°C in Davis and Mingioli (1950) medium, modified by the addition of 10 g/1 Casamino acids (Difco, U.S.A.). The suspension was then diluted 25 × with Medium 199 and agitated at 37°C for 18 h. Next, the bacteria were pelleted (5000 × g, 10 min, 4 ° C) and the supernatant modified corresponding to the recipe for 1% HSA. Small portions were frozen, only once, to --70°C and could be kept at this temperature for several months without detectable loss of chemotactic activity.
220 The synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was dissolved at 10 --2 mol/1 in d h n e t h y l sulfoxide (DMSO). These reagents were both supplied by Sigma. At the m a x i m u m effective concentration of FMLP, 10 -s mol/1, the DMSO did not affect leukocyte migration, as shown by adding it to chemotactic factors soluble in aqueous media.
Flow cytometry (FCM) In some experiments leukocytes were analysed with FCM before and after emigration in the new chambers, using a model FC 200/FC 4800A-50 Cytofluorograf e~ (Ortho Instruments, U.S.A.). Leukocytes present in a hypotonic ( - 1 6 0 mosM, pH 7.3) solution of acridine orange (~4 pg/ml, Sigma) get greenly fluorescent nuclei, redly fluorescent cytoplasmatic RNA, and -- if p h a g o c y t e s - - r e d l y fluorescent granules. Granulocytes show more intense red fluorescence than do monocytes. As a cell flows through the exciting blue laser light, the simultaneous emission of green and red light can be monitored separately by the apparatus. On this basis every cell is represented as a small dot on a scatterplot. By setting windows on this oscilloscope screen, differential counts can be obtained by FCM, which simultaneously records total cell concentrations (Adams and Kamentsky, 1974). The staining was checked with fluorescence microscopy and the FCM results controlled with the conventional techniques. Propidium iodide staining to obtain DNA histograms has been described elsewhere (Benestad and StrCm-Gundersen, 1982). Na2S~Cr04 labeling of blood cells The label (Institut for Energiteknikk, N-2007 Kjeller, Norway) was added to a suspension of Isopaque-methylcellulose separated blood cells in 1% HSA (7 X 106 cells/ml), at a concentration of 7 pCi/ml. The cell suspension was incubated for 1 h at 37 ° C, diluted with cold saline and washed twice. Fluorescein isothiocyanate (FITC) labeled E. coli phagocytosis assay FITC, isomer 1 (Sigma) and E. eoli HF 4704 Thy were used, essentially following the procedure described by Gelfand et al. (1976). Leukocytes taken before or after chamber emigration were allowed to settle and adhere onto thoroughly cleaned coverslips for 30 min, in a humified atmosphere of 5% CO2 and 95% air. Next, non-adherent cells were gently washed off. FITClabeled bacteria were deposited on the coverslips, incubation continued for 20 rain, and phagocytosis terminated by a 4°C wash. The coverslips were finally examined by alternate phase contrast and fluorescence microscopy. Membrane damage to leukocytes This was assessed by counting stained cells in a h e m o c y t o m e t e r after adding trypan blue to a concentration of 0.08%.
221 Assessing the time course o f concentration gradient extinction Two radiolabeled compounds with molecular weights close to those of various chemotactic substances were used; the extracellular space marker SlCr-EDTA (The Radiochemical Centre, Amersham, U.K.; MW 362) and 12sIparathyroid h o r m o n e (~2sI-PTH; MW 9500, positively charged at pH 7.2-7.4, and repurified by chromatography the day before use). Either 240 pl or 720 pl volumes of markers in medium were added to the retrieval compartments, omitting the HSA to avoid problems of protein binding. After incubation at 37°C, the contents of both compartments were aspirated and 100 pl samples were counted in a Packard Auto-Gamma Scintillation spectrometer, Model 5220, adjusted so that the 2 isotopes could be counted simultaneously. S ta tis tics Histogram and boxplot representation of our own data and of those given by Baisero (1973) indicated that they were not normally distributed. Nonparametric statistical methods were therefore used. Median values with 75% confidence intervals {corresponding to the range of triplicates) are given unless otherwise stated. Assays based on microscopical examination of membranes are rarely run with more than 3 parallel chambers, and we therefore chose this number for such a method. With the new assay it was easily feasible to use a greater number of parallel chambers, and this was consistently done, also in direct comparisons with the microscopy assay. The rationale was to include all relevant features of both assays in the comparisons. Twosided Wilcoxon or Wilcoxon-Van Elteren tests (Van Elteren, 1960) were used to assess the significance of differences between experiments or groups of experiments, respectively. RESULTS Retrieval of emigrated cells The main feature of the new assay is the counting of cells free in suspension rather than adherent to filters. Therefore, it must be shown i.a. that virtually all emigrated cells can be retrieved for counting. This was done in 2 different ways: by routine harvesting, the cell suspensions were removed from 18 retrieval compartments, which were then carefully rinsed with saline. Thorough flushing of these compartments with a 0.1% v/v Triton X-100/saline mixture, which under these conditions lyses the plasma membranes, did not release detectable numbers of nuclei, as assessed by electronic particle counting. Other chambers were subjected to routine harvesting and a saline rinse. Fixation, staining, and examination with light microscopy did n o t reveal any cells on the lower surface of the membranes or on split retrieval compartment vials. In separate experiments, some chambers were disassembled before the retrieval compartment vials were subjected to the routine harvesting proce-
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Fig. 2. Top curve: this was o b t a i n e d w i t h t h e f l u o r e s c e i n a t e d latex m i c r o b e a d s used for a p p a r a t u s a d j u s t m e n t s . The c o e f f i c i e n t o f variation (CV) o f the main peak was 4.5%. Middle curves: these r e p r e s e n t t w o parallel s a m p l e s o f i n o c u l u m , c o n t a i n i n g all classes o f l e u k o c y t e s . The CVs o f the main peaks were 5.5% and 5.6%. B o t t o m curves: these were o b t a i n e d b y a n a l y z i n g n e u t r o p h i l s that h a d e m i g r a t e d t o w a r d s 1% HSA (left peak) and 25c£ E. coli CF (right peak). The CVs were 3.9% and 3.4%, respectively. Fig. 3. Time course o f n e u t r o p h i l d i r e c t i o n a l e m i g r a t i o n t h r o u g h t 4 0 p m thick Sartorius c e l l u l o s e n i t r a t e m e m b r a n e s o f various average pore sizes: 3 p m (' J), 5 t/m (,)), and 8 p m ( . ) . The e x p e r i m e n t s h o w n was r e p r e s e n t a t i v e for a total n u m b e r o f 4 e x p e r i m e n t s . Results are e x p r e s s e d as m e d i a n values and 75% c o n f i d e n c e intervals, as described in Materials and M e t h o d s .
dure. When cells on the outer membrane surfaces were counted and the counts added to those obtained for the rest of the retrieval compartments, the total counts were n o t exceeded by those for parallel chamber assemblies treated in the routine way. This shows that the standard harvesting procedure did n o t liberate significant numbers of leukocytes or their nuclei from the interior of the membrane.
Characterization of emigrated cells Microscopy of cell suspensions showed that clumping was negligible, and of smears that cell m o r p h o l o g y was apparently unchanged. More than 97% of the emigrated cells were neutrophils, after both random and directional 2 h emigration. Trypan blue was excluded by 1>98% of inoculum cells, emigrated cells, and cells incubated for 2 h with the chemoattractant mostly
223
used, i.e. 25% E. coli CF. Similarly, FCM histograms of propidium iodide stained cells (Fig. 2) showed that neither cell aggregation was a problem (insignificant numbers of particles with tetraploid amounts of DNA, or more), nor 'broken nuclei' (sub-diploid amounts of DNA) were detected. Phagocytosis of FITC-labeled E. coli was assessed for neutrophils from 2 donors. For each of them 1000 cells were scored, 500 taken from the inoculum and 500 from the emigrated cells. Taken together, inoculum neutrophils contained an average of 1.90 and emigrated neutrophils 1.92 bacteria per cell. Membrane pore size
Unexpectedly, neutrophils migrated more rapidly through 5 pm than through 8 pm Sartorius membranes at all incubation times tested (Fig. 3). This was also the case with other fabrication lots. Migration was slower
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Fig. 4. Time course of gradient extinction. A mixture of the ~'-ray emitting compounds S I C r . E D T A a n d 12sI-PTH was filled into the cell retrieval c o m p a r t m e n t s ( c p m starting side) of cell-free cell m i g r a t i o n c h a m b e r s . A f t e r various periods at 37°C, samples were taken f r o m b o t h t h e cell c h a m b e r s a n d retrieval c o m p a r t m e n t s a n d t h e i r radioactivities m e a s u r e d . S o m e of the curves are labeled, since for clarity t h e e x p e r i m e n t a l points of t h e s e curves have b e e n o m i t t e d : 2 4 0 a n d 720 are t h e pl fluid volumes in the retrieval c o m p a r t m e n t s . S denotes Sartorius cellulose n i t r a t e m e m b r a n e s ; N, N u c l e p o r e polycarbonate m e m b r a n e s ; - 12sI-PTH; . . . . . . , S l C r - E D T A ; e, 2 4 0 S ; 0, 7 2 0 S ; A, 240 N; A, 7 2 0 N. Each e x p e r i m e n t a l p o i n t r e p r e s e n t s 1 c h a m b e r . In a 10 h e x p e r i m e n t , t h e results for t h a t p e r i o d were r e p r o d u c e d .
224 t h r o u g h 3 p m m e m b r a n e s . T h e s e m e m b r a n e s w e r e all 140 g m thick. T h e v a r i a t i o n b e t w e e n parallel c h a m b e r s was s o m e w h a t l o w e r t h e higher emigration the membranes allowed.
Time course o f concentration gradient extinction T h e f a s t e r e x t i n c t i o n o f t h e c o n c e n t r a t i o n g r a d i e n t was seen f o r t h e l o w e r m o l e c u l a r w e i g h t t r a c e r , f o r t h e larger ' s t a r t i n g side' v o l u m e a n d f o r the 5 p m S a r t o r i u s m e m b r a n e s , as c o m p a r e d w i t h 5 p m N u c l e p o r e m e m b r a n e s (Fig. 4). T h e p r e s e n c e o f d i r e c t i o n a l l y m i g r a t i n g cells did n o t i n f l u e n c e t h e s p e e d o f g r a d i e n t e x t i n c t i o n , i n d i c a t i n g t h a t t h e d i f f u s i o n was n o t h i n d e r e d b y cells clogging t h e p o r e s ( d a t a n o t given).
Effect o f chemotactic factor gradient on neutrophil emigration N e u t r o p h i l e m i g r a t i o n was m a r k e d l y greater w h e n a t t r a c t a n t was p r e s e n t o n l y in t h e retrieval c o m p a r t m e n t (positive c h e m o t a x i s ) t h a n w h e n it was p r e s e n t at t h e s a m e c o n c e n t r a t i o n o n b o t h sides o f t h e m e m b r a n e s ( c h e m o kinesis) (Table 1). E m i g r a t i o n d u e to c h e m o k i n e s i s was m o r e p r o n o u n c e d t h a n w h e n n o c h e m o t a c t i c f a c t o r was p r e s e n t ( u n s t i m u l a t e d r a n d o m migrat i o n ) . I n t e r e s t i n g l y , u n s t i m u l a t e d , r a n d o m e m i g r a t i o n was clearly larger t h a n w h e n t h e cell i n o c u l u m o n l y c o n t a i n e d c h e m o t a c t i c f a c t o r , suggesting t h a t t h e F M L P u s e d in these e x p e r i m e n t s was really c h e m o t a c t i c to t h e n e u t r o p h i l s . R e p l a c i n g F M L P w i t h 25% E. coli CF gave q u a n t i t a t i v e l y identical results w i t h cells f r o m t h e s a m e d o n o r s ( d a t a n o t s h o w n ) .
Variations o f chamber cell inoculi Halving t h e p r o p o r t i o n o f n e u t r o p h i l s in t h e s u s p e n s i o n s ( b y enriching w i t h p u r i f i e d m o n o n u c l e a r l e u k o c y t e s ) or r e d u c i n g t h e t o t a l n u m b e r o f leukocytes to the same extent (by dilution) neither affected unstimulated r a n d o m e m i g r a t i o n n o r d i r e c t e d e m i g r a t i o n ( t o w a r d s 25% E. coli CF; d a t a not shown).
TABLE 1 Effect of chemotactic factor gradient on neutrophil migration. P < 0.02 in each of the 6 differences which can be examined for statistical significance. Results are expressed as median values and 75% confidence intervals, as described in Materials and Methods. Blood donor 1 2
Inoculum medium
1% HSA 10 nmol/1 FMLP 1% HSA 10 nmol/1 FMLP
% of neutrophils emigrated into 1% HSA
10 nmol/1 FMLP
5.9 5.3 11.1 9.4
16.2 (14.2--17.6) 7.3 (6.7-- 9.0) 17.9 (16.1--19.7) 13.9 (12.5--16.4)
(5.9-- 6.4) (5.0-- 5.3) (10.3--11.2) (9.4--10.0)
Between n chambers within 1 group
Between n identically prepared portions of 1 blood sample
Between n successive days
Between n healthy individuals
1 2
3 4 5
6 7
8--17
4.1 (--30.9, +25.9) b
4.2 (--14.3, +19.2) 4.6 (--18.1, +21.2)
6.4 (--15.3, +14.9) 3.5 (--8.7, +11.7) 1.7 (--13.0, +16.6)
4.5 (--12.2, +12.2) 2.1 (--14.3, +33.3)
1% HSA
Variability a
(10)
(4) (4)
(3) (4) (4)
(28) (30)
(n)
(--9.6, +10.2) (--7.0, +7.5)
20.8 (--13.5, +40.4) c
16.6 (--12.0, +18.8) 26.3 (--15.5, +18.1)
19.3 (--11.2, +12.8) 19.7 (--9.5, +13.1) 14.4 (--7.3, +10.0)
24.3 20.0
25% E. coil CF
(10)
(4) (4)
(3) (4) (3)
(29) (30)
(n)
a Median and --f0.25 and +f0.75 (fractiles) in per cent of the median, followed by n. The median is given as the percentage of inoculated neutrophils emigrated into the medium indicated. b Range 2.3--6.7. c Range 9.1--43.3.
Variation
Blood donor
Comparison of sources of variation in neutrophil random and directional migration.
TABLE 2
b~ bO c~
226 Using the Ficoll-Isopaque cell separation m e t h o d to obtain almost pure suspensions of granulocytes following removal of the erythrocytes with Isopaque-methylcellulose led to an average 66% increase in random cell emigration. However, under these conditions directed emigration towards 25% E. coli CF was reduced by approximately 18% (4 and 5 experiments, respectively, P < 0.10 in both cases; further data not shown). Using whole blood in the chambers as the source of the leukocytes resulted in drastically reduced directed emigration towards 20% zymosan activated serum (data not given). L e u k o c y t e isolation using the Isopaque-methylcellulose technique at 37°C resulted in optimal emigration towards 25% E. coli CF. In contrast, leukocytes obtained by letting the red cells sediment without adding anything to the blood or cells obtained by the Isopaque-methylcellulose technique at 22°C or by the dextran red cell sedimentation technique (at 22°C) were much less responsive to the chemotactic factors (2 experiments, P < 0.01 in all comparison but the one between the 2 Isopaque-methylcellulose treatments, where P = 0.4). Random emigration using 1% HSA was increased when the cells had been isolated with Isopaque-methylcellulose at 22°C as compared to the standard temperature conditions of 37°C (2 experiments, P = 0.01), but no other differences were f o u n d (P > 0.05; further data not shown). R a n d o m emigration was insignificantly increased and directional emigration into 25% E. coli CF decreased by NH4C1 lysis of red cells in the inoculum, whereas water treatment was highly detrimental to both (data not shown).
Sources o f variation in neutrophil emigration The variability in neutrophil emigration between identically treated chambers in an experiment corresponded to coefficients of variation usually below 10%. Table 2 shows that variability increased very little when variations in the preparatory procedure for the leukocyte suspension were included as well. Variability increased more when day-to-day reproducibility was added, but most on inclusion of normal interindividual variability. No evidence was f o u n d that the venipuncture t e c h n i q u e - - w h e t h e r no stasis or tight and protracted (5--10 min) stasis was used -- influenced random or directional emigration (data n o t given). Time-response and dose-response studies. Comparisons with conventional assays Neutrophil emigration was higher and variability among replicates lower with the new m e t h o d than with a classical Boyden assay (Figs. 5--8). The possibility that the difference was due to different counting methods was excluded by comparing electronic counts from a dilute cell suspension with microscopical counts obtained after cell sedimentation and adhesion to 0.22/~m porosity membranes. Cells free in suspension in the attractant
227 25 oc
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.
.
.
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.
.
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60
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120
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Fig. 5. Time course o f n e u t r o p h i l e m i g r a t i o n r e c o r d e d with t h e n e w a n d a c o n v e n t i o n a l m i c r o p o r e m e m b r a n e m e t h o d . - . . . . . , r a n d o m e m i g r a t i o n in 1% H S A ; - - , directional e m i g r a t i o n t o w a r d s 25% E . c o l i CF; o, n e w m e t h o d (n = 6); o, c o n v e n t i o n a l m e t h o d (n = 3). The e x p e r i m e n t s h o w n was r e p r e s e n t a t i v e for 3 d i f f e r e n t e x p e r i m e n t s (6 persons).
compartment of the Boyden chamber accounted for most of the difference between the 2 methods; in addition some underrating of cell numbers present on densely populated membranes may have occurred. Fig. 4 indicates that the concentration gradient is still well preserved after 2 h and Fig. 5 shows that the standard 2 h incubation time allows directional emigration to reach a plateau value without letting random emigration reach high values, although it was easily measurable. Combining 30-
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Fig. 6; E f f e c t o f c o n c e n t r a t i o n o f a t t r a c t a n t o n 2 h directional e m i g r a t i o n o f n e u t r o p h i l s in the n e w a n d c o n v e n t i o n a l assays. - . . . . . , h e a l t h y s u b j e c t 1; , h e a l t h y subject 2; ©, n e w m e t h o d (n = 6); o, c o n v e n t i o n a l m e t h o d (n = 3). The e x p e r i m e n t s h o w n was r e p r e s e n t a t i v e for 3 replicate e x p e r i m e n t s , e x c e p t for the very g o o d a g r e e m e n t b e t w e e n the results o f t h e 2 m e t h o d s c o n c e r n i n g subject 1.
228 40
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Fig. 7. Effect of varying the inoculum neutrophil load, using serial dilutions, on the counts of emigrated neutrophils obtained with the new and conventional methods. . . . . . . , unstimulated random e m i g r a t i o n ; - - - - - - , c h e m o k i n e s i s ; - - - , chemotaxis; o, new method (n = 6); e, conventional method (n = 3). The experiment shown was representative for 3 replicate experiments.
t h e d a t a o f Fig. 5 w i t h t h e k n o w n filter c h a r a c t e r i s t i c s it can be c a l c u l a t e d t h a t t h e n e t d i s p l a c e m e n t o f t h e f a s t e s t 3% o f n e u t r o p h i l s was a b o u t 10 g m / m i n . O n l y t h e fastest 1% o f n e u t r o p h i l s m o v i n g at r a n d o m h a d this high speed. Since t h e p o r e s o f t h e S a r t o r i u s m e m b r a n e s are t o r t u o u s ( i n f o r m a t i o n f r o m t h e m a n u f a c t u r e r ) , t h e real s p e e d o f t h e cells was p r o b a b l y considera b l y higher. When t h e a t t r a c t a n t was 20% z y m o s a n a c t i v a t e d s e r u m r a t h e r t h a n 25% E. coli C F , t h e cells e m i g r a t e d even f a s t e r (see b e l o w ; Fig. 8). D o s e - r e s p o n s e e x p e r i m e n t s s h o w e d t h a t t h e 25% c o n c e n t r a t i o n o f E. coli CF was t h e m o s t e f f e c t i v e o n e (Fig. 6). D o s e - r e s p o n s e e x p e r i m e n t s c o n c e r n i n g t h e n u m b e r o f i n o c u l a t e d cells i n d i c a t e d t h a t t h e curves o b t a i n e d f o r t h e n e w m e t h o d w e r e a l m o s t linear f o r all i n o c u l u m n e u t r o p h i l c o n c e n t r a t i o n s t e s t e d , ranging f r o m 0.8 t o 1 3 . 0 X 106 cells/ml (Fig. 7). F u r t h e r m o r e , variability was small t h r o u g h o u t t h e range. In c o n t r a s t , t h e c h e m o t a x i s a n d c h e m o k i n e s i s curves o f the c o n v e n t i o n a l assay d e v i a t e d m a r k e d l y f r o m linearity at t h e higher cell loads. When t h e 2 m e t h o d s were c o m p a r e d , using various c h e m o a t t r a c t a n t s o r 1% H S A , results were a l w a y s slightly s u p e r i o r w i t h t h e n e w m e t h o d (Fig. 8). This was generally t r u e , irrespective o f w h e t h e r t h e n u m b e r o f replicates in t h e n e w assay was r e d u c e d to 3, w h i c h was t h e n u m b e r a l w a y s used in t h e o t h e r assay ( d a t a n o t s h o w n ) .
229
1% (v/v/ HUMAN SERUM ALBUMIN
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AUTOLOGOUS SERUM
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2096
{v/v ) ZYMOSAN ACTIVATED SERUM
I 10
I 20
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% OF NEUIROPHILS EMIGRATED
Fig. 8. Emigration of leukocytes from 1% HSA towards various substances. In the new method n = 6, and n = 3 in the conventional method.
Others have quantified cell emigration in the Boyden assay by radiochemical means rather than by microscopy. Our experience has been that electronic or FCM counting is not inferior to radioactivity counting of SlCrlabeled cells when precision is concerned, that a procedure taking about an hour is avoided with the new m e t h o d , and that SlCr-labeled cells easily get damaged. This was shown by the observation that whereas 99.0% of both unlabeled and SlCr-labeled inoculum cells excluded trypan blue, the respective percentages were 98.0 and 95.0 among those cells that had emigrated into 25% E. coli CF. The bacterial culture filtrate per se was n o t responsible for this, as shown by the fact that cells from the same inoculum batches incubated in the filtrate for the same period of time did n o t increase their uptake of the stain. This also indicates that the cells tolerated the harvesting procedure very well. Moreover, the S~Cr-labeling decreased by 41% the percentage of cells emigrating into 25% E. coli CF (P > 0.01, further data n o t shown).
Flow cytofluorometric total and differential leukocyte counting L y m p h o c y t e s , monocytes, and granulocytes in suspensions prepared for chamber inoculation could easily be identified on the scatter plot (Fig. 9) and counted. FCM analysis confirmed the almost total granulocyte dominance in the emigrated cell populations (Fig. 9). Both total and differential
230
Fig. 9. Flow e y t o f l u o r o g r a m s of l e u k o e y t e s s t a i n e d with acridine orange. T o p left: inoculurn l e u k o e y t e s . F r o m left to right, t h e 3 m a i n clusters r e p r e s e n t l y m p h o e y t e s , m o n o eytes, a n d g r a n u l o e y t e s . T o p right: selective g r a n u l o e y t e e x h i b i t i o n of the cell preparat i o n d i s p l a y e d in t h e u p p e r left e y t o f l u o r o g r a m . B o t t o m left: all l e u k o e y t e s t h a t e m i g r a t e d in 1% HSA. B o t t o m right: all l e u k o e y t e s t h a t e m i g r a t e d t o w a r d s 2 5 ~ E. coli CF.
counts obtained with FCM analysis agreed reasonably well with those resulting f r o m com bi ned electronic particle counting and m i croscopy of smears {data n o t given). DISCUSSION
Our new m e t h o d for assaying l e u k o c y t e migration is based on the quantitative harvesting -- in suspension -- o f the cells t hat have traversed a micropore m e m b r a n e after a certain time. The suspended cells were quickly, precisely and objectively c o u n t e d with an electronic particle or flow cytof l u o r o metr ic counting apparatus. We have shown t hat the m e t h o d gives valid and reproducible results, and t hat it does n o t lead to detectable damage to the leukocytes th a t are assayed. Moreover, we have determined optimal preparative m e t h o d s for leukocyte suspensions, m e m b r a n e t y p e and poro-
231
sity, cell concentrations in the inoculi, incubation time, and concentration of chemoattractants. Sources of variability have been analysed. Finally, we have shown that in our hands the new assay is better and more simple than 2 conventional membrane assays in several respects. To our knowledge, complete quantitative data have not been presented showing that a concentration gradient of a chemotactic factor (or of a substance comparable to such a factor with regard to molecular weight etc.) does in fact persist throughout the often prolonged incubation periods. We have given such data for SlCr-EDTA with MW 362 and concentration 6.3 pmol/1, similar to those of the synthetic N-formyl-methionyl oligopeptides c o m m o n l y used to attract human granulocytes. The other marker was 12sIparathyroid hormone (MW 9500; 1.8 nmol/1), comparable to the chemotactic complement activation products C5a and C5ades A~g (Fernandez et al., 1978). Gradient decay times have been c o m p u t e d by others using diffusion equations (Zigmond, 1980a), and our results are apparently compatible with the calculations for acetic acid and bovine serum albumin diffusing through cellulose membranes. It should be noted that in contrast to chemotactins, the markers used b y us were n o t expected to bind specifically to leukocyte receptors. Degradation of chemotactic factors in the presence of cells is supposedly n o t sufficient to maintain the gradient much longer than in the absence of cells, under the conditions of filter assays (Zigmond, 1980b). However, binding of chemotactic factors to albumin might occur and contribute to maintaining the concentration gradient (Zigmond, 1980a). Even though Sartorius membranes are much thicker than Nuclepore membranes (140 vs. 10 pm), the gradients were always maintained longer when the thinner membranes were employed. Pores make up 65--85% of the Sartorius membranes (manufacturer's information), whereas the pores constitute only 6.4% of the Nuclepore membranes (calculated on the basis of information from the manufacturer). This probably explains the difference between these membranes with respect to gradient duration. We confirmed with the new method that one orientation of Sartorius membranes relative to the inoculum compartment gave markedly higher emigration and lower variability than the other (Jungi, 1978). Regardless of orientation, Millipore filters from several lots gave results comparable to those obtained with the least favorable Sartorius arrangement. We cannot explain these observations, but funnel shape of pores in Sartorius filters might play a role. It is similarly unknown w h y 8 pm membranes gave lower neutrophil emigration than 5 pm ones. While it may be necessary to use the rather laborious checkerboard assay (Zigmond and Hirsch, 1973) to formally demonstrate true chemotaxis (Keller et al., 1977), we would like to propose the 4 ~ r o u p scheme shown in Table 1 as a more practicable, routine method. However, it is not essential to include the group of unstimulated random migration. The FMLP concentration used, 10 nmol/1, was the most effective one in stimulating chemotaxis. This concentration was clearly less than corresponding to the peak of the
232 chemokinesis dose-response curve, but still evoked an undisputable chemokinetic response. The reason for using a pure chem ot act i n (FMLP) in these experiments, is that contaminants present in e.g. bacterial filtrates may disturb the effects of the c he m ot act i n unde r study. The new m e t h o d worked well with much lower cell inoculi than the standard ones (Fig. 7), which were in a range c o m m o n l y used in Boyden chamber studies of neutrophil migration. This may be i m p o r t a n t when e.g. neonates are studied. In preliminary experiments we have f o u n d the new m e t h o d suitable to d e t e c t migratory defects present in the neutrophils of patients suffering from infections or leukemia. In addition to the advantages of the new m e t h o d that have been m e n t i o n e d above, the results are obtained more rapidly than with o t h e r m e t h o d s in c o m m o n usage, thus f ur t he r indicating its clinical utility. Moreover, such cells can easily be retrieved after emigration and used for f u rt h er study. This is n o t easily done, to our knowledge, with ot her assays. ACKNOWLEDGEMENTS We are grateful to Dr. Eirik Holten for supplying the E. coli bacteria and their culture supernatants, Dr. Kaare Gautvik for providing the labeled and purified p a r a t h y r o i d h o r m o n e , Dr. Aksel Schreiner for lending us the Neuro Probe chambers, and Dr. Arne BCyum and Professor Peter A. Ward for helpful criticism o f the manuscript. We t h a n k Gro S. Lid, Maje Siebke, and Eric O. T o o g o o d for excellent technical assistance. E q u i p m e n t was obtained t h ro ug h grants f r om the Norwegian Research Council for Science and the Humanities and f r om the Norwegian Cancer Society. I.A.G. is a St udent Fellow o f the f o r m e r Council, and has received grants from Anders Jahre's F o u n d a t i o n for the p r o m o t i o n o f Science, and f r o m Johan Peter Ebbel og Hustrus Legat. Mrs. Siebke is e m p l o y e d by the Norwegian Cancer Society. All this support is gratefully acknowledged. REFERENCES Adams, L.R.and L.A. Kamentsky, 1974, Acta Cytol. 18,389. Baisero, M.H., 1973, Schweiz. Med. Wschr. 103, 1599. Benestad, H.B., 1970, Scand. J. Haematol. 7, 279. Benestad, H.B. and •. Reikvam, 1975, Exp. Hematol. 3, 249. Benestad, H.B. and I. Str~bm-Gundersen, 1982, Exp. Hematol. 10, in press. Boyden, S., 1962, J. Exp. Med. 115,453. Brade, V., G.D. Lee, A. Nicholson, H.S. Shin and M.M. Mayer, 1973, J. Immunol. 111, 1389. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97). B~yum, A., 1976, Scand. J. Immunol. 5 (Suppl. 5), 9. Clausen, J., 1971, Acta Allerg. 26, 56. Cutler, J.E., 1974, Proc. Soc. Exp. Biol. Med. 147,471. Davis, B.D. and E.S. Mingioli, 1950, J. Bacteriol. 60, 17. Fernandez, H.N., P.M. Henson, A. Otani and T.E. Hugli, 1978, J. Immunol. 120, 109.
233 Gail, M.H. and C.W. Boone, 1970, Biophys. J. 10,980. Gallin, J.I., R.A. Clark and H.R. Kimball, 1973, J. Immunol. 110,233. Gelfand, J.A., A.S. Fauci, I. Green and M.M. Frank, 1976, J. Immunol. 116, 595. Jungi, T.W., 1978, J. Immunol. Methods 21,373. Keller, H.U., P.C. Wilkinsson, M. Abercrombie, E.L. Becker, J.G. Hirsch, M.E. Miller, W. Scott Ramsey and S.H. Zigmond, 1977, Clin. Exp. Immunol. 27,377. Ketchel, M.M. and Favour, C.B., 1955, J. Exp. Med. 101,647. Kreutzer, D.L., J.T. O'Flaherty, W. Orr, H.J. Showell, P.A. Ward and B.L. Becket, 1978, Immunopharmacology 1, 39. Maderazo, E.G. and C.L. Woronick, 1978, in: Leukocyte Chemotaxis, eds. J.I. Gallin and P.G. Quie (Raven Press, New York) p. 43. Orr, W. and P.A. Ward, 1978, J. Immunol. Methods 20, 95. Van Elteren, P., 1960, Bull. Inst. Int. Statist. 37,351. Ward, P.A., 1978, in: Leukocyte Chemotaxis, eds. J.I. Gallin and P.G. Quie (Raven Press, New York) p. 405. Zigmond, S.H., 1978, in: Leukocyte Chemotaxis, eds. J.I. Gallin and P.G. Quie (Raven Press, New York) p. 57. Zigmond, S.H., 1980a, in: Mononuclear Phagocytes, Functional Aspects, ed. R. Van Furth (Martinus Nijhoff, The Hague) pp. 461 and 466. Zigmond, S.H., 1980b, in: Mononuclear Phagocytes, Functional Aspects, ed. R. Van Furth (Martinus Nijhoff, The Hague) p. 501. Zigmond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.
215
A NEW A S S A Y F O R L E U K O C Y T E C H E M O T A X I S U S I N G C E L L RETRIEVAL, ELECTRONIC PARTICLE COUNTING AND FLOW CYTOMETRY
IVAR AMUND GRIMSTAD and HAAKON B. BENESTAD Institute of Physiology, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway
(Received 4 May 1981, accepted 18 September 1981)
A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.
Key words: chemotaxis --leukocyte isolation --flow cytometry --automated cytology
INTRODUCTION Several assays have b e e n i n v e n t e d t o s t u d y crawling m o v e m e n t s o f cells. T h e results o b t a i n e d w i t h these m e t h o d s m a y e l u c i d a t e basic p r o c e s s e s o f cell m o t i l i t y . T h e y m a y also serve practical p u r p o s e s , since d e f e c t s in, f o r e x a m p l e , l e u k o c y t e m o t i l i t y dispose to disease. T h e l o c o m o t i o n o f single cells m a y b e o b s e r v e d m i c r o s c o p i c a l l y (Gail a n d B o o n e , 1 9 7 0 ; Z i g m o n d , 1 9 7 8 ) . O t h e r in vitro m e t h o d s have b e e n designed t o m e a s u r e t h e n e t d i s p l a c e m e n t o f g r e a t e r n u m b e r s o f cells: s o m e are b a s e d o n m i c r o s c o p i c a l d e t e c t i o n o f cells a d h e r e n t t o m i c r o p o r e m e m b r a n e s , a f t e r having m i g r a t e d i n t o o r t h r o u g h t h e m . T h e r e s p o n s e can b e m e a s u r e d b y assessing t h e n u m b e r o f cells t h a t have m o v e d a c e r t a i n d i s t a n c e ( B o y d e n , 1 9 6 2 ) , or t h e distance travelled b y t h e leading cells ( Z i g m o n d a n d Hirsch, 1 9 7 3 ) , or b y a c o m b i n e d a p p r o a c h e s t i m a t i n g m e a n cell d i s p l a c e m e n t ( M a d e r a z o a n d W o r o n i c k , 1 9 7 8 ) . T h e n u m b e r o f cells t h a t have m i g r a t e d a p r e d e t e r m i n e d distance can also b e c o u n t e d r a d i o c h e m i c a l l y (Gallin e t al., 1 9 7 3 ) . O t h e r m e t h o d s d e p e n d o n similar m i c r o s c o p i c m e a s u r e m e n t s o n cells crawling u n d e r agarose in dishes (Cutler, 1 9 7 4 ; Orr a n d Ward, 1 9 7 8 ) or along 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press
216
(Ketchel and Favour, 1955) or out of (Clausen, 1971) capillary tubes. Micropore membrane techniques are the most sensitive ones in assessing leukocyte chemotaxis (Kreutzer et al., 1978). However, all variants so far described render difficult further study of cells introduced to the systems, and involve either extensive handling of the cells (e.g. radiolabeling) before migration starts or tedious microscopy. The assay described here avoids these drawbacks, as well as the subjectivity of the observer. It allows extensive cell migration; it is sensitive, precise, and easy to standardize; and it gives more reproducible results than other methods. The easy retrieval of cells that have migrated may allow studies, previously n o t feasible, on motile cells other than leukocytes as well e.g., neoplastic cells capable of invasion and metastasis. M A T E R I A L S AND M E T H O D S
Cell migration chambers Chambers of the new type (Fig. 1) were made in the following way: punched-out 13 mm diam. micropore membranes of various types were heatsealed (Benestad and Reikvam, 1975) onto acrylic plastic cups designed for this purpose. The depth of a resulting cell inoculum c o m p a r t m e n t is 4 mm and its volume ~315 pl. Alternatively, membranes can be glued onto the acrylic parts (Benestad, 1970), although this was not done in the present study. Hundreds of such compartments may be assembled during one working day. Absence of leaks was verified by the 'air escape test', blowing
Fig. 1. Left: 3 cell m i g r a t i o n c h a m b e r s o f the n e w t y p e . In an a d d i t i o n a l cell i n o c u l u m c o m p a r t m e n t , the filling orifice can be seen. Right: 2 assembled cell migration c h a m b e r s . D i f f e r e n t fluid v o l u m e s can be a c c o m m o d a t e d in the cell retrieval c o m p a r t m e n t s , because the p o s i t i o n o f the cell i n o c u l u m c o m p a r t m e n t can be varied.
217 air through the 1.3 mm diam. filling orifice in the b o t t o m of the cup into the c o m p a r t m e n t under water. A stainless steel rod with a conical tip was used to plug the orifice after filling the inoculum compartment with the cell suspension to be tested. The rod also served to position this c o m p a r t m e n t in the vial constituting the cell retrieval compartment. This was the cut off lower 20 mm of a low pressure polyethylene 'polyvial' (Zinsser, F.R.G.). These had fiat bottoms, internal diam. 14 mm, and were usually filled with 540 pl (in some experiments as little as 150 pl) prewarmed (37°C) medium with or without chemotactic factor before insertion of the cell inoculum compartment. It is easy to avoid trapping of air bubbles below the membrane. The closing caps for the retrieval compartments were made of acetal plastic and had neoprene rubber o-rings to tighten both against the wail of the vial and the central rod. However, keeping the inoculum compartment just below the cap, while positioning the latter, allowed air to escape around a narrow portion of the metal rod before this was pushed down. The fluid surfaces in the two compartments were at the same level during incubation. Before use, all parts of the cell migration chambers were kept at 80°C dry heat overnight (Benestad, 1970). After use the cell inoculum compartments were kept in 2 mol/1 NaOH overnight. This treatment dissolved cellulose membranes and detached polycarbonate ones. After thorough washing, all components except the membranes could be re-used, apparently almost indefinitely, making this new assay very economical.
Micropore rnembranes We routinely used Sartorius SM 11324 cellulose nitrate membranes with average 5 pm pore diam. (Sartorius-Membranfilter GmbH, F.R.G.). Mixed cellulose acetate and nitrate membranes (Millipore Corp., U.S.A.) and polycarbonate membranes (Nuclepore Corp., U.S.A.) were also tested. For economical reasons and to reduce the notorious filter heterogeneity (Maderazo and Woronick, 1978; Ward, 1978), 13 mm diam. disks were punched o u t from large filter sheets of one fabrication lot. Both filter sides had been inspected beforehand through a stereo microscope, so that the Sartorius membrane could be sealed to the plastic cup with the smoother side turning outwards, since this arrangement gave markedly higher and more reproducible migratory responses.
Leukocyte preparation Heparinized blood (20 IU/ml) was obtained, after informed consent, by venipuncture of healthy laboratory personnel and medical students of b o t h sexes, 20--45 years old. Blood was similarly drawn from a few selected patients. We avoided prolonged venous stasis and delayed mixing with the anticoagulant, and kept the cells at ~0°C whenever the procedures did not
218 require room temperature or 37°C. Routinely, erythrocytes were removed by sedimentation, using BOyum's 2-phase Isopaque-methylcellulose technique (BOyum, 1968), at 37°C. Removal of the majority of the red cells took 15--30 min, and a supernatant containing a non-selected sample (usually >70%) of the blood leukocytes was obtained, ahnost free from contamination with separation fluid ingredients. The majority of the blood platelets remained in the leukocyte fraction, but most of them ~.~ould be removed by the following 2 centrifugations. Finally, the leukocytes were suspended, usually at - 5 × 106 neutrophils per ml, in a medium hereafter called 1% HSA. This was Medium 199 (GIBCO, U.S.A., Cat. no. 235) with 25 mmol/1 HEPES buffer, supplemented with 1% (w/v) human serum albumin (HSA), Cohn fraction V (Sigma, U.S.A.) and ampicillin 200 #l/ml (Doktacillin, Astra, Sweden). Preliminary experiments showed that the antibiotic did not affect leukocyte migration. The osmolality of the medium was adjusted to 290 mosM/kg, the pit to 7.20, and it was sterile filtered through a 0.22 #m porosity membrane. In some experiments red blood cells were sedimented by mixing 1 part of 6% (w/v) dextran in 0.9% NaCI (Macrodex, Pharmacia, Sweden) with 10 parts of blood. On other occasions almost pure suspensions of either granulocytes or mononuclear leukocytes were obtained by subjecting the leukocyte fraction of the Isopaque-methylcellulose separation to the FicollIsopaque (Lymphoprep, Nyegaard & Co. A/S, Norway) density separation technique (BCyum, 1976). Contaminating red cells were lysed in some experiments, either by exposing the cells twice to cold, pure water for 10 sec before restoration of isotonicity, or by treating them for 5 rain at room temperature with 0.75V~ NH4CI in 0.02 mol/1 Tris-HC1 buffer at pH 7.35. Chamber inoculation and incubation The chamber membranes were moistened in 1% HSA for seconds. Before filling with cell suspension, the inoculum compartments were placed in a special rack, so that the wetted membranes did n o t touch ground. The cell suspension (240 t~l) was supplied from a 10 ml Gilette disposable syringe (Gilette Ltd., U.K.) through a 25-gauge 10 mm long hypodermic needle by means of a Hamilton PB 600-10 dispenser (Hamilton Bonaduz AG, Switzerland). At least 6 replicate chambers were routinely prepared. The inoculum compartments were suspended in retrieval compartment vials, and the chamber assemblies were ordinarily incubated in air at 37°C for 120 min. The pH remained constant for much longer incubation periods. Cell harvesting procedure This was designed to disaggregate possible cell clumps and detach all cells that had migrated completely through the membrane and were adhering to its outside or to the walls of the cell retrieval c o m p a r t m e n t (i.e., the plastic vial). Addition of 60 tll of a 2.7% (w/v) Na2-EDTA solution (final conc. 0.27% or ~7.3 mmol/l) to chelate Ca 2~ and Mg 2~ and brief shaking with a
219 whirlmixer after a few minutes proved to be sufficient. Samples from the inoculum suspension had been taken for smearing and h e m o c y t o m e t r i c or electronic counting, as detailed elsewhere (Benestad, 1970). In preliminary experiments only, smears were also made of the cell suspensions in the retrieval compartments. At least 500 cells were counted in the h e m o c y t o m e t e r and 400 cells in a smear. Zaponin (Coulter Electronics Ltd., U.K.), 40 pl to 10--16 ml saline with 0.035% NAN3, were used to lyse red cells before counting with a Coulter Counter Model D Industrial (Coulter Electronics Ltd. or a Celloscope 101 (Linson Instruments, L. Ljungberg and Co., Sweden). In some control experiments, Triton X-100 (Sigma) was used as a lysing agent. The results were expressed as the percentage of inoculated neutrophils that had emigrated and were retrieved from the polyethylene vials. During a 2 h period granulocytes were virtually the only cells to cross the filter; differential counting of this population was therefore omitted after the pilot experiments. Very rarely, the cell count for one of the replicates could be much higher than for the others. If, after retesting, the cell inoculum compartment disclosed leaks, the high count was disregarded.
Conventional Boyden chambers. Preparation of membranes for microscopy Neuro Probe blind well chemotaxis chambers (Nuclepore Corp., U.S.A.) were used to compare the new m e t h o d with an established one (Boyden, 1962). The upper compartments of the conventional chambers were closed with a thick tape, so that both types of chamber could be incubated under identical conditions. The conventional chambers were prepared in triplicate. Fixation, staining, clearing and microscopical examination of cells on membranes from the conventional and in some cases also the new chambers were done according to standard techniques (Maderazo and Woronick, 1978). Chemotactic attractants Fresh serum was prepared from defibrinated blood. It was diluted and supplemented with 200 pg/ml ampicillin before use. Zymosan A (Sigma) activated serum (Brade et al., 1973) was obtained by incubating 5 mg/ml of the yeast polysaccharide in fresh serum for 30 min at 37°C in a shaking water bath, thus activating the complement system via the alternative pathway. Zymosan was removed by centrifugation, and ampicillin, 200 pg/ml, was added. Escherichia coli culture filtrate (E. coli CF) was obtained by culturing E. coli HF 4704 Thy, first for 4 h at 37°C in Davis and Mingioli (1950) medium, modified by the addition of 10 g/1 Casamino acids (Difco, U.S.A.). The suspension was then diluted 25 × with Medium 199 and agitated at 37°C for 18 h. Next, the bacteria were pelleted (5000 × g, 10 min, 4 ° C) and the supernatant modified corresponding to the recipe for 1% HSA. Small portions were frozen, only once, to --70°C and could be kept at this temperature for several months without detectable loss of chemotactic activity.
220 The synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was dissolved at 10 --2 mol/1 in d h n e t h y l sulfoxide (DMSO). These reagents were both supplied by Sigma. At the m a x i m u m effective concentration of FMLP, 10 -s mol/1, the DMSO did not affect leukocyte migration, as shown by adding it to chemotactic factors soluble in aqueous media.
Flow cytometry (FCM) In some experiments leukocytes were analysed with FCM before and after emigration in the new chambers, using a model FC 200/FC 4800A-50 Cytofluorograf e~ (Ortho Instruments, U.S.A.). Leukocytes present in a hypotonic ( - 1 6 0 mosM, pH 7.3) solution of acridine orange (~4 pg/ml, Sigma) get greenly fluorescent nuclei, redly fluorescent cytoplasmatic RNA, and -- if p h a g o c y t e s - - r e d l y fluorescent granules. Granulocytes show more intense red fluorescence than do monocytes. As a cell flows through the exciting blue laser light, the simultaneous emission of green and red light can be monitored separately by the apparatus. On this basis every cell is represented as a small dot on a scatterplot. By setting windows on this oscilloscope screen, differential counts can be obtained by FCM, which simultaneously records total cell concentrations (Adams and Kamentsky, 1974). The staining was checked with fluorescence microscopy and the FCM results controlled with the conventional techniques. Propidium iodide staining to obtain DNA histograms has been described elsewhere (Benestad and StrCm-Gundersen, 1982). Na2S~Cr04 labeling of blood cells The label (Institut for Energiteknikk, N-2007 Kjeller, Norway) was added to a suspension of Isopaque-methylcellulose separated blood cells in 1% HSA (7 X 106 cells/ml), at a concentration of 7 pCi/ml. The cell suspension was incubated for 1 h at 37 ° C, diluted with cold saline and washed twice. Fluorescein isothiocyanate (FITC) labeled E. coli phagocytosis assay FITC, isomer 1 (Sigma) and E. eoli HF 4704 Thy were used, essentially following the procedure described by Gelfand et al. (1976). Leukocytes taken before or after chamber emigration were allowed to settle and adhere onto thoroughly cleaned coverslips for 30 min, in a humified atmosphere of 5% CO2 and 95% air. Next, non-adherent cells were gently washed off. FITClabeled bacteria were deposited on the coverslips, incubation continued for 20 rain, and phagocytosis terminated by a 4°C wash. The coverslips were finally examined by alternate phase contrast and fluorescence microscopy. Membrane damage to leukocytes This was assessed by counting stained cells in a h e m o c y t o m e t e r after adding trypan blue to a concentration of 0.08%.
221 Assessing the time course o f concentration gradient extinction Two radiolabeled compounds with molecular weights close to those of various chemotactic substances were used; the extracellular space marker SlCr-EDTA (The Radiochemical Centre, Amersham, U.K.; MW 362) and 12sIparathyroid h o r m o n e (~2sI-PTH; MW 9500, positively charged at pH 7.2-7.4, and repurified by chromatography the day before use). Either 240 pl or 720 pl volumes of markers in medium were added to the retrieval compartments, omitting the HSA to avoid problems of protein binding. After incubation at 37°C, the contents of both compartments were aspirated and 100 pl samples were counted in a Packard Auto-Gamma Scintillation spectrometer, Model 5220, adjusted so that the 2 isotopes could be counted simultaneously. S ta tis tics Histogram and boxplot representation of our own data and of those given by Baisero (1973) indicated that they were not normally distributed. Nonparametric statistical methods were therefore used. Median values with 75% confidence intervals {corresponding to the range of triplicates) are given unless otherwise stated. Assays based on microscopical examination of membranes are rarely run with more than 3 parallel chambers, and we therefore chose this number for such a method. With the new assay it was easily feasible to use a greater number of parallel chambers, and this was consistently done, also in direct comparisons with the microscopy assay. The rationale was to include all relevant features of both assays in the comparisons. Twosided Wilcoxon or Wilcoxon-Van Elteren tests (Van Elteren, 1960) were used to assess the significance of differences between experiments or groups of experiments, respectively. RESULTS Retrieval of emigrated cells The main feature of the new assay is the counting of cells free in suspension rather than adherent to filters. Therefore, it must be shown i.a. that virtually all emigrated cells can be retrieved for counting. This was done in 2 different ways: by routine harvesting, the cell suspensions were removed from 18 retrieval compartments, which were then carefully rinsed with saline. Thorough flushing of these compartments with a 0.1% v/v Triton X-100/saline mixture, which under these conditions lyses the plasma membranes, did not release detectable numbers of nuclei, as assessed by electronic particle counting. Other chambers were subjected to routine harvesting and a saline rinse. Fixation, staining, and examination with light microscopy did n o t reveal any cells on the lower surface of the membranes or on split retrieval compartment vials. In separate experiments, some chambers were disassembled before the retrieval compartment vials were subjected to the routine harvesting proce-
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Fig. 2. Top curve: this was o b t a i n e d w i t h t h e f l u o r e s c e i n a t e d latex m i c r o b e a d s used for a p p a r a t u s a d j u s t m e n t s . The c o e f f i c i e n t o f variation (CV) o f the main peak was 4.5%. Middle curves: these r e p r e s e n t t w o parallel s a m p l e s o f i n o c u l u m , c o n t a i n i n g all classes o f l e u k o c y t e s . The CVs o f the main peaks were 5.5% and 5.6%. B o t t o m curves: these were o b t a i n e d b y a n a l y z i n g n e u t r o p h i l s that h a d e m i g r a t e d t o w a r d s 1% HSA (left peak) and 25c£ E. coli CF (right peak). The CVs were 3.9% and 3.4%, respectively. Fig. 3. Time course o f n e u t r o p h i l d i r e c t i o n a l e m i g r a t i o n t h r o u g h t 4 0 p m thick Sartorius c e l l u l o s e n i t r a t e m e m b r a n e s o f various average pore sizes: 3 p m (' J), 5 t/m (,)), and 8 p m ( . ) . The e x p e r i m e n t s h o w n was r e p r e s e n t a t i v e for a total n u m b e r o f 4 e x p e r i m e n t s . Results are e x p r e s s e d as m e d i a n values and 75% c o n f i d e n c e intervals, as described in Materials and M e t h o d s .
dure. When cells on the outer membrane surfaces were counted and the counts added to those obtained for the rest of the retrieval compartments, the total counts were n o t exceeded by those for parallel chamber assemblies treated in the routine way. This shows that the standard harvesting procedure did n o t liberate significant numbers of leukocytes or their nuclei from the interior of the membrane.
Characterization of emigrated cells Microscopy of cell suspensions showed that clumping was negligible, and of smears that cell m o r p h o l o g y was apparently unchanged. More than 97% of the emigrated cells were neutrophils, after both random and directional 2 h emigration. Trypan blue was excluded by 1>98% of inoculum cells, emigrated cells, and cells incubated for 2 h with the chemoattractant mostly
223
used, i.e. 25% E. coli CF. Similarly, FCM histograms of propidium iodide stained cells (Fig. 2) showed that neither cell aggregation was a problem (insignificant numbers of particles with tetraploid amounts of DNA, or more), nor 'broken nuclei' (sub-diploid amounts of DNA) were detected. Phagocytosis of FITC-labeled E. coli was assessed for neutrophils from 2 donors. For each of them 1000 cells were scored, 500 taken from the inoculum and 500 from the emigrated cells. Taken together, inoculum neutrophils contained an average of 1.90 and emigrated neutrophils 1.92 bacteria per cell. Membrane pore size
Unexpectedly, neutrophils migrated more rapidly through 5 pm than through 8 pm Sartorius membranes at all incubation times tested (Fig. 3). This was also the case with other fabrication lots. Migration was slower
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Fig. 4. Time course of gradient extinction. A mixture of the ~'-ray emitting compounds S I C r . E D T A a n d 12sI-PTH was filled into the cell retrieval c o m p a r t m e n t s ( c p m starting side) of cell-free cell m i g r a t i o n c h a m b e r s . A f t e r various periods at 37°C, samples were taken f r o m b o t h t h e cell c h a m b e r s a n d retrieval c o m p a r t m e n t s a n d t h e i r radioactivities m e a s u r e d . S o m e of the curves are labeled, since for clarity t h e e x p e r i m e n t a l points of t h e s e curves have b e e n o m i t t e d : 2 4 0 a n d 720 are t h e pl fluid volumes in the retrieval c o m p a r t m e n t s . S denotes Sartorius cellulose n i t r a t e m e m b r a n e s ; N, N u c l e p o r e polycarbonate m e m b r a n e s ; - 12sI-PTH; . . . . . . , S l C r - E D T A ; e, 2 4 0 S ; 0, 7 2 0 S ; A, 240 N; A, 7 2 0 N. Each e x p e r i m e n t a l p o i n t r e p r e s e n t s 1 c h a m b e r . In a 10 h e x p e r i m e n t , t h e results for t h a t p e r i o d were r e p r o d u c e d .
224 t h r o u g h 3 p m m e m b r a n e s . T h e s e m e m b r a n e s w e r e all 140 g m thick. T h e v a r i a t i o n b e t w e e n parallel c h a m b e r s was s o m e w h a t l o w e r t h e higher emigration the membranes allowed.
Time course o f concentration gradient extinction T h e f a s t e r e x t i n c t i o n o f t h e c o n c e n t r a t i o n g r a d i e n t was seen f o r t h e l o w e r m o l e c u l a r w e i g h t t r a c e r , f o r t h e larger ' s t a r t i n g side' v o l u m e a n d f o r the 5 p m S a r t o r i u s m e m b r a n e s , as c o m p a r e d w i t h 5 p m N u c l e p o r e m e m b r a n e s (Fig. 4). T h e p r e s e n c e o f d i r e c t i o n a l l y m i g r a t i n g cells did n o t i n f l u e n c e t h e s p e e d o f g r a d i e n t e x t i n c t i o n , i n d i c a t i n g t h a t t h e d i f f u s i o n was n o t h i n d e r e d b y cells clogging t h e p o r e s ( d a t a n o t given).
Effect o f chemotactic factor gradient on neutrophil emigration N e u t r o p h i l e m i g r a t i o n was m a r k e d l y greater w h e n a t t r a c t a n t was p r e s e n t o n l y in t h e retrieval c o m p a r t m e n t (positive c h e m o t a x i s ) t h a n w h e n it was p r e s e n t at t h e s a m e c o n c e n t r a t i o n o n b o t h sides o f t h e m e m b r a n e s ( c h e m o kinesis) (Table 1). E m i g r a t i o n d u e to c h e m o k i n e s i s was m o r e p r o n o u n c e d t h a n w h e n n o c h e m o t a c t i c f a c t o r was p r e s e n t ( u n s t i m u l a t e d r a n d o m migrat i o n ) . I n t e r e s t i n g l y , u n s t i m u l a t e d , r a n d o m e m i g r a t i o n was clearly larger t h a n w h e n t h e cell i n o c u l u m o n l y c o n t a i n e d c h e m o t a c t i c f a c t o r , suggesting t h a t t h e F M L P u s e d in these e x p e r i m e n t s was really c h e m o t a c t i c to t h e n e u t r o p h i l s . R e p l a c i n g F M L P w i t h 25% E. coli CF gave q u a n t i t a t i v e l y identical results w i t h cells f r o m t h e s a m e d o n o r s ( d a t a n o t s h o w n ) .
Variations o f chamber cell inoculi Halving t h e p r o p o r t i o n o f n e u t r o p h i l s in t h e s u s p e n s i o n s ( b y enriching w i t h p u r i f i e d m o n o n u c l e a r l e u k o c y t e s ) or r e d u c i n g t h e t o t a l n u m b e r o f leukocytes to the same extent (by dilution) neither affected unstimulated r a n d o m e m i g r a t i o n n o r d i r e c t e d e m i g r a t i o n ( t o w a r d s 25% E. coli CF; d a t a not shown).
TABLE 1 Effect of chemotactic factor gradient on neutrophil migration. P < 0.02 in each of the 6 differences which can be examined for statistical significance. Results are expressed as median values and 75% confidence intervals, as described in Materials and Methods. Blood donor 1 2
Inoculum medium
1% HSA 10 nmol/1 FMLP 1% HSA 10 nmol/1 FMLP
% of neutrophils emigrated into 1% HSA
10 nmol/1 FMLP
5.9 5.3 11.1 9.4
16.2 (14.2--17.6) 7.3 (6.7-- 9.0) 17.9 (16.1--19.7) 13.9 (12.5--16.4)
(5.9-- 6.4) (5.0-- 5.3) (10.3--11.2) (9.4--10.0)
Between n chambers within 1 group
Between n identically prepared portions of 1 blood sample
Between n successive days
Between n healthy individuals
1 2
3 4 5
6 7
8--17
4.1 (--30.9, +25.9) b
4.2 (--14.3, +19.2) 4.6 (--18.1, +21.2)
6.4 (--15.3, +14.9) 3.5 (--8.7, +11.7) 1.7 (--13.0, +16.6)
4.5 (--12.2, +12.2) 2.1 (--14.3, +33.3)
1% HSA
Variability a
(10)
(4) (4)
(3) (4) (4)
(28) (30)
(n)
(--9.6, +10.2) (--7.0, +7.5)
20.8 (--13.5, +40.4) c
16.6 (--12.0, +18.8) 26.3 (--15.5, +18.1)
19.3 (--11.2, +12.8) 19.7 (--9.5, +13.1) 14.4 (--7.3, +10.0)
24.3 20.0
25% E. coil CF
(10)
(4) (4)
(3) (4) (3)
(29) (30)
(n)
a Median and --f0.25 and +f0.75 (fractiles) in per cent of the median, followed by n. The median is given as the percentage of inoculated neutrophils emigrated into the medium indicated. b Range 2.3--6.7. c Range 9.1--43.3.
Variation
Blood donor
Comparison of sources of variation in neutrophil random and directional migration.
TABLE 2
b~ bO c~
226 Using the Ficoll-Isopaque cell separation m e t h o d to obtain almost pure suspensions of granulocytes following removal of the erythrocytes with Isopaque-methylcellulose led to an average 66% increase in random cell emigration. However, under these conditions directed emigration towards 25% E. coli CF was reduced by approximately 18% (4 and 5 experiments, respectively, P < 0.10 in both cases; further data not shown). Using whole blood in the chambers as the source of the leukocytes resulted in drastically reduced directed emigration towards 20% zymosan activated serum (data not given). L e u k o c y t e isolation using the Isopaque-methylcellulose technique at 37°C resulted in optimal emigration towards 25% E. coli CF. In contrast, leukocytes obtained by letting the red cells sediment without adding anything to the blood or cells obtained by the Isopaque-methylcellulose technique at 22°C or by the dextran red cell sedimentation technique (at 22°C) were much less responsive to the chemotactic factors (2 experiments, P < 0.01 in all comparison but the one between the 2 Isopaque-methylcellulose treatments, where P = 0.4). Random emigration using 1% HSA was increased when the cells had been isolated with Isopaque-methylcellulose at 22°C as compared to the standard temperature conditions of 37°C (2 experiments, P = 0.01), but no other differences were f o u n d (P > 0.05; further data not shown). R a n d o m emigration was insignificantly increased and directional emigration into 25% E. coli CF decreased by NH4C1 lysis of red cells in the inoculum, whereas water treatment was highly detrimental to both (data not shown).
Sources o f variation in neutrophil emigration The variability in neutrophil emigration between identically treated chambers in an experiment corresponded to coefficients of variation usually below 10%. Table 2 shows that variability increased very little when variations in the preparatory procedure for the leukocyte suspension were included as well. Variability increased more when day-to-day reproducibility was added, but most on inclusion of normal interindividual variability. No evidence was f o u n d that the venipuncture t e c h n i q u e - - w h e t h e r no stasis or tight and protracted (5--10 min) stasis was used -- influenced random or directional emigration (data n o t given). Time-response and dose-response studies. Comparisons with conventional assays Neutrophil emigration was higher and variability among replicates lower with the new m e t h o d than with a classical Boyden assay (Figs. 5--8). The possibility that the difference was due to different counting methods was excluded by comparing electronic counts from a dilute cell suspension with microscopical counts obtained after cell sedimentation and adhesion to 0.22/~m porosity membranes. Cells free in suspension in the attractant
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Fig. 5. Time course o f n e u t r o p h i l e m i g r a t i o n r e c o r d e d with t h e n e w a n d a c o n v e n t i o n a l m i c r o p o r e m e m b r a n e m e t h o d . - . . . . . , r a n d o m e m i g r a t i o n in 1% H S A ; - - , directional e m i g r a t i o n t o w a r d s 25% E . c o l i CF; o, n e w m e t h o d (n = 6); o, c o n v e n t i o n a l m e t h o d (n = 3). The e x p e r i m e n t s h o w n was r e p r e s e n t a t i v e for 3 d i f f e r e n t e x p e r i m e n t s (6 persons).
compartment of the Boyden chamber accounted for most of the difference between the 2 methods; in addition some underrating of cell numbers present on densely populated membranes may have occurred. Fig. 4 indicates that the concentration gradient is still well preserved after 2 h and Fig. 5 shows that the standard 2 h incubation time allows directional emigration to reach a plateau value without letting random emigration reach high values, although it was easily measurable. Combining 30-
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INOCULUM
Fig. 7. Effect of varying the inoculum neutrophil load, using serial dilutions, on the counts of emigrated neutrophils obtained with the new and conventional methods. . . . . . . , unstimulated random e m i g r a t i o n ; - - - - - - , c h e m o k i n e s i s ; - - - , chemotaxis; o, new method (n = 6); e, conventional method (n = 3). The experiment shown was representative for 3 replicate experiments.
t h e d a t a o f Fig. 5 w i t h t h e k n o w n filter c h a r a c t e r i s t i c s it can be c a l c u l a t e d t h a t t h e n e t d i s p l a c e m e n t o f t h e f a s t e s t 3% o f n e u t r o p h i l s was a b o u t 10 g m / m i n . O n l y t h e fastest 1% o f n e u t r o p h i l s m o v i n g at r a n d o m h a d this high speed. Since t h e p o r e s o f t h e S a r t o r i u s m e m b r a n e s are t o r t u o u s ( i n f o r m a t i o n f r o m t h e m a n u f a c t u r e r ) , t h e real s p e e d o f t h e cells was p r o b a b l y considera b l y higher. When t h e a t t r a c t a n t was 20% z y m o s a n a c t i v a t e d s e r u m r a t h e r t h a n 25% E. coli C F , t h e cells e m i g r a t e d even f a s t e r (see b e l o w ; Fig. 8). D o s e - r e s p o n s e e x p e r i m e n t s s h o w e d t h a t t h e 25% c o n c e n t r a t i o n o f E. coli CF was t h e m o s t e f f e c t i v e o n e (Fig. 6). D o s e - r e s p o n s e e x p e r i m e n t s c o n c e r n i n g t h e n u m b e r o f i n o c u l a t e d cells i n d i c a t e d t h a t t h e curves o b t a i n e d f o r t h e n e w m e t h o d w e r e a l m o s t linear f o r all i n o c u l u m n e u t r o p h i l c o n c e n t r a t i o n s t e s t e d , ranging f r o m 0.8 t o 1 3 . 0 X 106 cells/ml (Fig. 7). F u r t h e r m o r e , variability was small t h r o u g h o u t t h e range. In c o n t r a s t , t h e c h e m o t a x i s a n d c h e m o k i n e s i s curves o f the c o n v e n t i o n a l assay d e v i a t e d m a r k e d l y f r o m linearity at t h e higher cell loads. When t h e 2 m e t h o d s were c o m p a r e d , using various c h e m o a t t r a c t a n t s o r 1% H S A , results were a l w a y s slightly s u p e r i o r w i t h t h e n e w m e t h o d (Fig. 8). This was generally t r u e , irrespective o f w h e t h e r t h e n u m b e r o f replicates in t h e n e w assay was r e d u c e d to 3, w h i c h was t h e n u m b e r a l w a y s used in t h e o t h e r assay ( d a t a n o t s h o w n ) .
229
1% (v/v/ HUMAN SERUM ALBUMIN
t
~:~
C0 N VENTIONAL METHOD
[~
NEW METHOD
20%Iv/v}~/////j///////~
AUTOLOGOUS SERUM
25% (Wvl ECOL# CU
FILTRATE
,
2096
{v/v ) ZYMOSAN ACTIVATED SERUM
I 10
I 20
I 30
I 40
I 50
% OF NEUIROPHILS EMIGRATED
Fig. 8. Emigration of leukocytes from 1% HSA towards various substances. In the new method n = 6, and n = 3 in the conventional method.
Others have quantified cell emigration in the Boyden assay by radiochemical means rather than by microscopy. Our experience has been that electronic or FCM counting is not inferior to radioactivity counting of SlCrlabeled cells when precision is concerned, that a procedure taking about an hour is avoided with the new m e t h o d , and that SlCr-labeled cells easily get damaged. This was shown by the observation that whereas 99.0% of both unlabeled and SlCr-labeled inoculum cells excluded trypan blue, the respective percentages were 98.0 and 95.0 among those cells that had emigrated into 25% E. coli CF. The bacterial culture filtrate per se was n o t responsible for this, as shown by the fact that cells from the same inoculum batches incubated in the filtrate for the same period of time did n o t increase their uptake of the stain. This also indicates that the cells tolerated the harvesting procedure very well. Moreover, the S~Cr-labeling decreased by 41% the percentage of cells emigrating into 25% E. coli CF (P > 0.01, further data n o t shown).
Flow cytofluorometric total and differential leukocyte counting L y m p h o c y t e s , monocytes, and granulocytes in suspensions prepared for chamber inoculation could easily be identified on the scatter plot (Fig. 9) and counted. FCM analysis confirmed the almost total granulocyte dominance in the emigrated cell populations (Fig. 9). Both total and differential
230
Fig. 9. Flow e y t o f l u o r o g r a m s of l e u k o e y t e s s t a i n e d with acridine orange. T o p left: inoculurn l e u k o e y t e s . F r o m left to right, t h e 3 m a i n clusters r e p r e s e n t l y m p h o e y t e s , m o n o eytes, a n d g r a n u l o e y t e s . T o p right: selective g r a n u l o e y t e e x h i b i t i o n of the cell preparat i o n d i s p l a y e d in t h e u p p e r left e y t o f l u o r o g r a m . B o t t o m left: all l e u k o e y t e s t h a t e m i g r a t e d in 1% HSA. B o t t o m right: all l e u k o e y t e s t h a t e m i g r a t e d t o w a r d s 2 5 ~ E. coli CF.
counts obtained with FCM analysis agreed reasonably well with those resulting f r o m com bi ned electronic particle counting and m i croscopy of smears {data n o t given). DISCUSSION
Our new m e t h o d for assaying l e u k o c y t e migration is based on the quantitative harvesting -- in suspension -- o f the cells t hat have traversed a micropore m e m b r a n e after a certain time. The suspended cells were quickly, precisely and objectively c o u n t e d with an electronic particle or flow cytof l u o r o metr ic counting apparatus. We have shown t hat the m e t h o d gives valid and reproducible results, and t hat it does n o t lead to detectable damage to the leukocytes th a t are assayed. Moreover, we have determined optimal preparative m e t h o d s for leukocyte suspensions, m e m b r a n e t y p e and poro-
231
sity, cell concentrations in the inoculi, incubation time, and concentration of chemoattractants. Sources of variability have been analysed. Finally, we have shown that in our hands the new assay is better and more simple than 2 conventional membrane assays in several respects. To our knowledge, complete quantitative data have not been presented showing that a concentration gradient of a chemotactic factor (or of a substance comparable to such a factor with regard to molecular weight etc.) does in fact persist throughout the often prolonged incubation periods. We have given such data for SlCr-EDTA with MW 362 and concentration 6.3 pmol/1, similar to those of the synthetic N-formyl-methionyl oligopeptides c o m m o n l y used to attract human granulocytes. The other marker was 12sIparathyroid hormone (MW 9500; 1.8 nmol/1), comparable to the chemotactic complement activation products C5a and C5ades A~g (Fernandez et al., 1978). Gradient decay times have been c o m p u t e d by others using diffusion equations (Zigmond, 1980a), and our results are apparently compatible with the calculations for acetic acid and bovine serum albumin diffusing through cellulose membranes. It should be noted that in contrast to chemotactins, the markers used b y us were n o t expected to bind specifically to leukocyte receptors. Degradation of chemotactic factors in the presence of cells is supposedly n o t sufficient to maintain the gradient much longer than in the absence of cells, under the conditions of filter assays (Zigmond, 1980b). However, binding of chemotactic factors to albumin might occur and contribute to maintaining the concentration gradient (Zigmond, 1980a). Even though Sartorius membranes are much thicker than Nuclepore membranes (140 vs. 10 pm), the gradients were always maintained longer when the thinner membranes were employed. Pores make up 65--85% of the Sartorius membranes (manufacturer's information), whereas the pores constitute only 6.4% of the Nuclepore membranes (calculated on the basis of information from the manufacturer). This probably explains the difference between these membranes with respect to gradient duration. We confirmed with the new method that one orientation of Sartorius membranes relative to the inoculum compartment gave markedly higher emigration and lower variability than the other (Jungi, 1978). Regardless of orientation, Millipore filters from several lots gave results comparable to those obtained with the least favorable Sartorius arrangement. We cannot explain these observations, but funnel shape of pores in Sartorius filters might play a role. It is similarly unknown w h y 8 pm membranes gave lower neutrophil emigration than 5 pm ones. While it may be necessary to use the rather laborious checkerboard assay (Zigmond and Hirsch, 1973) to formally demonstrate true chemotaxis (Keller et al., 1977), we would like to propose the 4 ~ r o u p scheme shown in Table 1 as a more practicable, routine method. However, it is not essential to include the group of unstimulated random migration. The FMLP concentration used, 10 nmol/1, was the most effective one in stimulating chemotaxis. This concentration was clearly less than corresponding to the peak of the
232 chemokinesis dose-response curve, but still evoked an undisputable chemokinetic response. The reason for using a pure chem ot act i n (FMLP) in these experiments, is that contaminants present in e.g. bacterial filtrates may disturb the effects of the c he m ot act i n unde r study. The new m e t h o d worked well with much lower cell inoculi than the standard ones (Fig. 7), which were in a range c o m m o n l y used in Boyden chamber studies of neutrophil migration. This may be i m p o r t a n t when e.g. neonates are studied. In preliminary experiments we have f o u n d the new m e t h o d suitable to d e t e c t migratory defects present in the neutrophils of patients suffering from infections or leukemia. In addition to the advantages of the new m e t h o d that have been m e n t i o n e d above, the results are obtained more rapidly than with o t h e r m e t h o d s in c o m m o n usage, thus f ur t he r indicating its clinical utility. Moreover, such cells can easily be retrieved after emigration and used for f u rt h er study. This is n o t easily done, to our knowledge, with ot her assays. ACKNOWLEDGEMENTS We are grateful to Dr. Eirik Holten for supplying the E. coli bacteria and their culture supernatants, Dr. Kaare Gautvik for providing the labeled and purified p a r a t h y r o i d h o r m o n e , Dr. Aksel Schreiner for lending us the Neuro Probe chambers, and Dr. Arne BCyum and Professor Peter A. Ward for helpful criticism o f the manuscript. We t h a n k Gro S. Lid, Maje Siebke, and Eric O. T o o g o o d for excellent technical assistance. E q u i p m e n t was obtained t h ro ug h grants f r om the Norwegian Research Council for Science and the Humanities and f r om the Norwegian Cancer Society. I.A.G. is a St udent Fellow o f the f o r m e r Council, and has received grants from Anders Jahre's F o u n d a t i o n for the p r o m o t i o n o f Science, and f r o m Johan Peter Ebbel og Hustrus Legat. Mrs. Siebke is e m p l o y e d by the Norwegian Cancer Society. All this support is gratefully acknowledged. REFERENCES Adams, L.R.and L.A. Kamentsky, 1974, Acta Cytol. 18,389. Baisero, M.H., 1973, Schweiz. Med. Wschr. 103, 1599. Benestad, H.B., 1970, Scand. J. Haematol. 7, 279. Benestad, H.B. and •. Reikvam, 1975, Exp. Hematol. 3, 249. Benestad, H.B. and I. Str~bm-Gundersen, 1982, Exp. Hematol. 10, in press. Boyden, S., 1962, J. Exp. Med. 115,453. Brade, V., G.D. Lee, A. Nicholson, H.S. Shin and M.M. Mayer, 1973, J. Immunol. 111, 1389. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97). B~yum, A., 1976, Scand. J. Immunol. 5 (Suppl. 5), 9. Clausen, J., 1971, Acta Allerg. 26, 56. Cutler, J.E., 1974, Proc. Soc. Exp. Biol. Med. 147,471. Davis, B.D. and E.S. Mingioli, 1950, J. Bacteriol. 60, 17. Fernandez, H.N., P.M. Henson, A. Otani and T.E. Hugli, 1978, J. Immunol. 120, 109.
233 Gail, M.H. and C.W. Boone, 1970, Biophys. J. 10,980. Gallin, J.I., R.A. Clark and H.R. Kimball, 1973, J. Immunol. 110,233. Gelfand, J.A., A.S. Fauci, I. Green and M.M. Frank, 1976, J. Immunol. 116, 595. Jungi, T.W., 1978, J. Immunol. Methods 21,373. Keller, H.U., P.C. Wilkinsson, M. Abercrombie, E.L. Becker, J.G. Hirsch, M.E. Miller, W. Scott Ramsey and S.H. Zigmond, 1977, Clin. Exp. Immunol. 27,377. Ketchel, M.M. and Favour, C.B., 1955, J. Exp. Med. 101,647. Kreutzer, D.L., J.T. O'Flaherty, W. Orr, H.J. Showell, P.A. Ward and B.L. Becket, 1978, Immunopharmacology 1, 39. Maderazo, E.G. and C.L. Woronick, 1978, in: Leukocyte Chemotaxis, eds. J.I. Gallin and P.G. Quie (Raven Press, New York) p. 43. Orr, W. and P.A. Ward, 1978, J. Immunol. Methods 20, 95. Van Elteren, P., 1960, Bull. Inst. Int. Statist. 37,351. Ward, P.A., 1978, in: Leukocyte Chemotaxis, eds. J.I. Gallin and P.G. Quie (Raven Press, New York) p. 405. Zigmond, S.H., 1978, in: Leukocyte Chemotaxis, eds. J.I. Gallin and P.G. Quie (Raven Press, New York) p. 57. Zigmond, S.H., 1980a, in: Mononuclear Phagocytes, Functional Aspects, ed. R. Van Furth (Martinus Nijhoff, The Hague) pp. 461 and 466. Zigmond, S.H., 1980b, in: Mononuclear Phagocytes, Functional Aspects, ed. R. Van Furth (Martinus Nijhoff, The Hague) p. 501. Zigmond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.