Journal oflmmunological Methods, 13 (1976) 191--197 © Elsevier/North-Holland Biomedical Press
191
MEASUREMENT OF CHEMOTAXIS OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES IN FILTERS BY COUNTING THE NUMBER OF CELLS IN A SINGLE PLANE AND COMPARISON WITH LEADING FRONT METHOD *
MELVIN J. SWANSON and ELMER L. BECKER Department of Pathology, University of Connecticut, tteallh ('enter. Farmington, Connecticut 06032, U.S.A. (Received 15 April 1976, accepted 14 May 1976)
A method is described for measuring the chemotactic response in filters of human neutrophils by counting the number of cells in a single plane at a constant distance below the top of the filters. This method gives results that are similar to those obtained by counting the total number of cells that have migrated into the filters. The results obtained by this method are compared with the leading front method. Under most conditions the results are similar whether obtained by the leading front or cell number methods except that counting the numbe~ of cells is often more sensitive. In the presence of p-nitrophenyl ,l-chlorobutylphosphonate, however, the chemotaetic response appeared to be inhibited as determined by counting the number of cells stimulated to migrate but enhanced as measured by the leading front method.
INTRODUCTION N e u t r o p h i l s e x p o s e d t o a c h e m o t a c t i c f a c t o r g r a d i e n t in a f i l t e r a r e stimulated to move into the filter through the gradient (Boyden, 1962). The response can be determined either by counting the number of cells that move a certain distance or by measuring the distance that the leading front of cells moves (Zigmond and Ilirsch, 1973). Under most conditions, the s a m e r e s u l t s will b e o b t a i n e d w h e t h e r t h e cell n u m b e r o r t h e d i s t a n c e o f t h e l e a d i n g f r o n t is d e t e r m i n e d . T h e v a l i d i t y o f t h e l e a d i n g f r o n t m e t h o d , h o w ever, depends upon the assumptions that the cells respond to a chemotactic s t i m u l u s in a h o m o g e n e o u s m a n n e r a n d t h a t t h e v e l o c i t y a t w h i c h t h e y m o v e is p r o p o r t i o n a l t o t h e n u m b e r o f c e l l s s t i m u l a t e d t o m i g r a t e . T h e s e c o n d i tions do not necessarily always obtain. This report describes a method for comparing the number of cells that are s t i m u l a t e d t o m o v e w i t h t h e l e a d i n g f r o n t d i s t a n c e in a s i n g l e e x p e r i m e n t . A l s o , a n e x a m p l e is g i v e n d e m o n s t r a t i n g a d i s c r e p a n c y b e t w e e n t h e t w o methods. * This work was supported by P.H.S. grants IPO1-AI-12225 and AI-09648.
192 MATERIALS AND METHODS
Isolation of human polymorphonuclear leukocytes Human neutrophils were isolated from blood collected from normal volunteers into heparin (20 units/ml), diluted 1 : 1 with normal saline and layered o n t o a double layer of Hypaque--Ficoll similar to the m e t h o d of English and Anderson (1974). The lower layer consisted of 20 ml of 9% Ficoll (Pharmacia Fine Chemicals) and 10 ml of 50% H y p a q u e (Winthrop Laboratories). 3'he upper layer contained 4 ml of 50% H ypaque, 14 ml of 9% Ficoll and 1.9 ml of water. These were prepared in a 100-ml Rockefeller bottle and up to 50 ml of diluted blood layered on top. The gradients were centrifuged at 700 g for 20 min at r oom temperature. The p o l y m o r p h o n u c l e a r l e u c o cy te {PMN) layer (located just above the e r y t h r o c y t e layer), was removed, diluted at least 1 : 1 with saline and centrifuged for 7 min at 700 g. Red blood cells were lysed with a solution consisting of 0.15 M NH4C1, 0.01 M KHCO3 and 0.001 M ethylene diamine tetraacetic acid (Roos and Loos, 1970). The cells were washed twice in Hank's balanced salt solution buffered with 0.03 M Tris ( h y d r o x y m e t h y l ) amino methane (Tris) at pH 7.3 and suspended in Hank's solution containing 1 mg/ml bovine serum albumin.
Chemo taxis Chemotaxis was carried out in acrylic chemotaxis chambers (Alco, Inc., Southington, Conn.) using 3 micron pore size Millipore filters. A b o u t 0.75 ml o f cells (8 X l 0 s cells/ml) was applied to each chamber. The response was quantitated either by measuring the distance from the top of the filters of the leading f r o n t two cells (Zigmond and Hirsch, 1973), by counting the n u m b e r of cells in focus in a single plane measured at a constant distance from the top surface of the filters or by summing the n u m b e r of cells in focus at each ten microns from the top of the filters. The measurements of a given plane were made by focusing the microscope on the m o n o l a y e r at the top of the filters, then turning the fine focusing knob down a given distance as indicated on the micrometer. In all experiments five high power fields were averaged from each of duplicate filters.
Chemotactic factors H u man C5a was partially purified from serum prepared by collecting blood on ice w i t h o u t anticoagulant, removing the plasma, allowing it to clot and then removing the fibrin. The serum, which was either used fresh or stored at --80°C, was made 1 M in e-amino caproic acid (Sigma), left overnight at 4°C, activated with 8--10 mg/ml Z y m o z a n (Sigma) at 37°C for 1 h (Vallotta and Muller-Eberhard, ! 9 7 3 ) , centrifuged at 27,000 g for 30 min and applied to a 5.2 X 85 cm. Sephadex G-75 column (Goldstein et al.,
193 1 9 7 3 ) . T h e c o l u m n was e l u t e d with Tris b u f f e r e d H a n k ' s s o l u t i o n . A f t e r o n e liter h a d passed t h r o u g h the c o l u m n , the f r a c t i o n s c o n t a i n i n g c h e m o t a c t i c a c t i v i t y w e r e p o o l e d ( a b o u t 2 0 0 ml) and a l i q u o t s s t o r e d at - - 8 0 ° C until t h e y w e r e used. A p r o n a s e sensitive bacterial c h e m o t a c t i c f a c t o r was isolated f r o m E. coil c u l t u r e filtrates as p r e v i o u s l y d e s c r i b e d ( S c h i f f m a n et al., 1975). RESULTS Evaluation o f the n u m b e r o f cells in a single p l a n e as a measure o f chemotaxis
H u m a n P M N ' s were i n c u b a t e d in c h e m o t a x i s c h a m b e r s for 1 h at 37°C against d i f f e r e n t c o n c e n t r a t i o n s o f bacterial c h e m o t a c t i c f a c t o r . T h e n u m ber o f cells in focus at each ten m i c r o n s t h r o u g h the filters was c o u n t e d in five high p o w e r fields on each o f d u p l i c a t e filters. T h e results o f o n e such e x p e r i m e n t are s h o w n in fig. 1. F r o m these d a t a the t o t a l n u m b e r o f cells t h a t h a d m i g r a t e d into the filters was e s t i m a t e d b y s u m m i n g the n u m b e r o f cells c o u n t e d at each ten m i c r o n s t h r o u g h the filters. T h e n u m b e r o f cells in each p l a n e is p l o t t e d against the c o n c e n t r a t i o n o f c h e m o t a c t i c f a c t o r on a l o g a r i t h m i c scale in fig. 2A. In fig. 2B are s h o w n d o s e r e s p o n s e curves o f the t o t a l cell n u m b e r a n d o f the leading f r o n t d i s t a n c e s m e a s u r e d in the s a m e filters. In a t o t a l o f f o u r t e e n e x p e r i m e n t s in which the t o t a l cell n u m b e r was estim a t e d , the shapes o f the d o s e r e s p o n s e curves r e p r e s e n t i n g the t o t a l n u m b e r o f cells w e r e v e r y similar to t h o s e r e p r e s e n t i n g the n u m b e r o f cells in a single plane. T h e curves o b t a i n e d b y c o u n t i n g the n u m b e r o f cells in single planes
100.
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~ 60
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MICRONS
Fig. I. Plot of the average number of cells counted in each plane at the dilutions of bacterial chemotactie factor as indicated. The distances were measured from the top of the filters using a micrometer on the fine focusing knob of the microscope.
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TOTAL CELL NUMBER
60
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BACTERIAL CHEMOTACTIC FACTOR
Fig. 2. Chemotaxis dose response curves of dilutions of bacterial chemotactic factor as measured by (A) the average number of cells counted in the planes indicated as measured from the top of the filters and (B) the average sum of the number of cells counted at each ten microns through the filters (open circles) and the average distance from the top of the filters of the leading front two cells (closed circles).
b e t w e e n ten and f o r t y m i c r o n s were usually nearly parallel t o each o t h e r and to the curves r e p r e s e n t i n g the total cell n u m b e r . The n u m b e r o f cells in planes below f o r t y m i c r o n s resulted in dose response curves having increasingly shallower slopes. In s o m e e x p e r i m e n t s deviations f r o m parallelism occurred at either ten or f o r t y microns. E i t h e r t w e n t y or t h i r t y m i c r o n s c o n s i s t e n t l y resulted in dose response curves that paralleled those o f the total cell n u m b e r u n d e r the c o n d i t i o n s used. T h e plane at t h i r t y m i c r o n s f r o m the t o p o f the filters was usually used for measuring c h e m o t a x i s , m o s t l y because there were slightly fewer cells to c o u n t . O c c a s i o n a l l y if the response was low, h o w e v e r , or if the backg r o u n d (i.e. in the absence o f c h e m o t a c t i c factor) were zero when c o u n t e d at t h i r t y microns, then the cells were c o u n t e d at t w e n t y m i c r o n s below the t o p o f the filters. The shapes o f the dose response curves o b t a i n e d by the leading f r o n t m e t h o d were usually d i f f e r e n t f r o m those o b t a i n e d by c o u n t i n g the n u m b e r o f cells. The leading f r o n t m e t h o d resulted in dose response curves t h a t were usually linear over a wide range of c h e m o t a c t i c f a c t o r c o n c e n t r a t i o n s and that usually had a smaller total p e r c e n t d i f f e r e n c e b e t w e e n b a c k g r o u n d and maxi m u m response.
195 120
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UNDIL. DILUTIONS OF CSa
Fig. 3. Chemotaxis dose response curves with cells in the presence (closed circles) and absence (open circles) of p-nitrophenyl ethyl 4-chlorobutylphosphonate as measured by (A) the leading front method, (B) the number of ceils at thirty microns from the top of the filters and (C) the total number of cells at each ten microns through the filters.
An example o f a discrepancy between the leading front and cell number methods Cells, in the presence or absence o f 8 × 10 .4 M p - n i t r o p h e n y l ethyl 4c h l o r o b u t y l p h o s p h o n a t e , were incubated in c h e m o t a x i s chambers against different c o n c e n t r a t i o n s o f C5a. The cells were c o u n t e d at each ten microns through the filters. Leading front distances were measured in the same filters. D o s e response curves o f the total cell n u m b e r , leading front and the cell n u m b e r o f 3 0 p m are c o m p a r e d in fig. 3. The response with and w i t h o u t p h o s p h o n a t c d e t e r m i n e d by c o u n t i n g the n u m b e r o f cells (either the total n u m b e r or the n u m b e r at thirty microns)
196
was c o m p a r e d with the response d e t e r m i n e d by the leading f r o n t m e t h o d in a total o f eleven e x p e r i m e n t s . T h e r a n d o m m o t i l i t y in the absence o f c h e m o tactic f a c t o r was always inhibited by p h o s p h o n a t e w h e t h e r measured by the leading f r o n t or cell n u m b e r m e t h o d s . In the presence o f 8 × 10 -4 M phosp h o n a t e , there was c o n s i s t e n t l y e n h a n c e m e n t o f the response as measured by the leading f r o n t m e t h o d at the highest c o n c e n t r a t i o n s o f c h c m o t a c t i c f a c t o r used. T h e response as d e t e r m i n e d by c o u n t i n g the n u m b e r o f cells s t i m u l a t e d to migrate, h o w e v e r , with one e x c e p t i o n , was always inhibited by the phosphonate. DISCUSSION O f the several m e t h o d s that have bcen used to q u a n t i t a t e c h e m o t a x i s o f cells t h r o u g h gradients in filters, each has certain disadvantages or sources o f error. When the cells are allowed to migrate t h r o u g h the filters, relatively long i n c u b a t i o n times are required during which one might be c o n c e r n e d a b o u t cell viability and maintaining the c h e m o t a c t i c f a c t o r gradient. F u r t h e r m o r e , the p r o b l e m o f cells adhering to the lower surface o f the filters in variable n u m b e r s m u s t be o v e r c o m e . C o u n t i n g the n u m b e r o f cells within filters, t h e r e f o r e , offers certain advantages over some o f these o t h e r m e t h o d s . C o u n t i n g the total n u m b e r o f cells in filters, h o w e v e r , is fairly difficult and requires considerable e x p e r i e n c e in o r d e r to obtain r e p r o d u c i b l e results. Measuring the distance o f the leading f r o n t o f cells is m o r e objective and relatively easy to do. This m e t h o d , h o w e v e r , f r e q u e n t l y results in shallower dose response curves than does the d e t e r m i n a t i o n o f cell n u m b e r , therefor('. it is o f t e n less sensitive to d i f f e r e n c e s in cell response. C o u n t i n g the n u m b e r o f cells in a single plane is m u c h easier to do than c o u n t i n g t h r o u g h multiple planes and u n d e r the p r o p e r c o n d i t i o n s gives similar results. T h e r e are two possible e x p l a n a t i o n s for the c o n f l i c t i n g results o b t a i n e d by the leading f r o n t and cell n u m b e r m e t h o d s in the p h o s p h o n a t e inhibition exp e r i m e n t s . T h e r e c o u l d have been a small p o p u l a t i o n o f cells t h a t were stimulated to a m u c h greater e x t e n t than the m a j o r i t y o f cells or else, even t h o u g h fewer cells were s t i m u l a t e d to migrate, those t h a t did m o v e d at a faster average velocity. Since there s e e m e d to be a u n i m o d a l distribution o f cells t h r o u g h the filters, the s e c o n d possibility seems m o r e likely. T h e m e t h o d p r e s e n t e d in this p a p e r allows one to c o m p a r e in a single exp e r i m e n t the leading f r o n t distance with the n u m b e r o f cells stimulated to migrate. U n d e r some c o n d i t i o n s , such a c o m p a r i s o n might give m o r e inform a t i o n a b o u t the e f f e c t o f an i n h i b i t o r than does e i t h e r one o f t h e m alone. Since the same filters are used, several variables are e l i m i n a t e d as sources o f e r r o r in such a c o m p a r i s o n . F u r t h e r m o r e , if the leading f r o n t response to a c h e m o t a c t i c f a c t o r is insufficient to get useable results, f r e q u e n t l y the response is greater by the cell n u m b e r m e t h o d . This m e t h o d , however, like m o s t o t h e r filter m e t h o d s , does n o t distinguish b e t w e e n stimulated r a n d o m m o t i l i t y and true c h e m o t a x i s .
197 REFERENCES Boyden, S., 1962, J. Exp. Med. 115,453. English, D. and B.R. Anderson, 1974, J. Immunol. Methods 5,249. Goldstcin, I., S. Hoffstein, J. Gallin and G. Weissmann, 1973, Proc. Nat. Acad. Sci. 70, 2916. Roos, D. and J.A. Loos, 1970, Biochim. Biophys. Acta 222,565. Schiffman, E., H.V. Showell, B.A. Corcoran, P.A. Ward, E. Smith and E.L. Becker, 1975, J. lmrnunol. 114, 1831. Vallota, E.H. and H.J. Muller-Eberhard, 1973, J. Exp. Med. 137, 1109. Zigrnond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.
191
MEASUREMENT OF CHEMOTAXIS OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES IN FILTERS BY COUNTING THE NUMBER OF CELLS IN A SINGLE PLANE AND COMPARISON WITH LEADING FRONT METHOD *
MELVIN J. SWANSON and ELMER L. BECKER Department of Pathology, University of Connecticut, tteallh ('enter. Farmington, Connecticut 06032, U.S.A. (Received 15 April 1976, accepted 14 May 1976)
A method is described for measuring the chemotactic response in filters of human neutrophils by counting the number of cells in a single plane at a constant distance below the top of the filters. This method gives results that are similar to those obtained by counting the total number of cells that have migrated into the filters. The results obtained by this method are compared with the leading front method. Under most conditions the results are similar whether obtained by the leading front or cell number methods except that counting the numbe~ of cells is often more sensitive. In the presence of p-nitrophenyl ,l-chlorobutylphosphonate, however, the chemotaetic response appeared to be inhibited as determined by counting the number of cells stimulated to migrate but enhanced as measured by the leading front method.
INTRODUCTION N e u t r o p h i l s e x p o s e d t o a c h e m o t a c t i c f a c t o r g r a d i e n t in a f i l t e r a r e stimulated to move into the filter through the gradient (Boyden, 1962). The response can be determined either by counting the number of cells that move a certain distance or by measuring the distance that the leading front of cells moves (Zigmond and Ilirsch, 1973). Under most conditions, the s a m e r e s u l t s will b e o b t a i n e d w h e t h e r t h e cell n u m b e r o r t h e d i s t a n c e o f t h e l e a d i n g f r o n t is d e t e r m i n e d . T h e v a l i d i t y o f t h e l e a d i n g f r o n t m e t h o d , h o w ever, depends upon the assumptions that the cells respond to a chemotactic s t i m u l u s in a h o m o g e n e o u s m a n n e r a n d t h a t t h e v e l o c i t y a t w h i c h t h e y m o v e is p r o p o r t i o n a l t o t h e n u m b e r o f c e l l s s t i m u l a t e d t o m i g r a t e . T h e s e c o n d i tions do not necessarily always obtain. This report describes a method for comparing the number of cells that are s t i m u l a t e d t o m o v e w i t h t h e l e a d i n g f r o n t d i s t a n c e in a s i n g l e e x p e r i m e n t . A l s o , a n e x a m p l e is g i v e n d e m o n s t r a t i n g a d i s c r e p a n c y b e t w e e n t h e t w o methods. * This work was supported by P.H.S. grants IPO1-AI-12225 and AI-09648.
192 MATERIALS AND METHODS
Isolation of human polymorphonuclear leukocytes Human neutrophils were isolated from blood collected from normal volunteers into heparin (20 units/ml), diluted 1 : 1 with normal saline and layered o n t o a double layer of Hypaque--Ficoll similar to the m e t h o d of English and Anderson (1974). The lower layer consisted of 20 ml of 9% Ficoll (Pharmacia Fine Chemicals) and 10 ml of 50% H y p a q u e (Winthrop Laboratories). 3'he upper layer contained 4 ml of 50% H ypaque, 14 ml of 9% Ficoll and 1.9 ml of water. These were prepared in a 100-ml Rockefeller bottle and up to 50 ml of diluted blood layered on top. The gradients were centrifuged at 700 g for 20 min at r oom temperature. The p o l y m o r p h o n u c l e a r l e u c o cy te {PMN) layer (located just above the e r y t h r o c y t e layer), was removed, diluted at least 1 : 1 with saline and centrifuged for 7 min at 700 g. Red blood cells were lysed with a solution consisting of 0.15 M NH4C1, 0.01 M KHCO3 and 0.001 M ethylene diamine tetraacetic acid (Roos and Loos, 1970). The cells were washed twice in Hank's balanced salt solution buffered with 0.03 M Tris ( h y d r o x y m e t h y l ) amino methane (Tris) at pH 7.3 and suspended in Hank's solution containing 1 mg/ml bovine serum albumin.
Chemo taxis Chemotaxis was carried out in acrylic chemotaxis chambers (Alco, Inc., Southington, Conn.) using 3 micron pore size Millipore filters. A b o u t 0.75 ml o f cells (8 X l 0 s cells/ml) was applied to each chamber. The response was quantitated either by measuring the distance from the top of the filters of the leading f r o n t two cells (Zigmond and Hirsch, 1973), by counting the n u m b e r of cells in focus in a single plane measured at a constant distance from the top surface of the filters or by summing the n u m b e r of cells in focus at each ten microns from the top of the filters. The measurements of a given plane were made by focusing the microscope on the m o n o l a y e r at the top of the filters, then turning the fine focusing knob down a given distance as indicated on the micrometer. In all experiments five high power fields were averaged from each of duplicate filters.
Chemotactic factors H u man C5a was partially purified from serum prepared by collecting blood on ice w i t h o u t anticoagulant, removing the plasma, allowing it to clot and then removing the fibrin. The serum, which was either used fresh or stored at --80°C, was made 1 M in e-amino caproic acid (Sigma), left overnight at 4°C, activated with 8--10 mg/ml Z y m o z a n (Sigma) at 37°C for 1 h (Vallotta and Muller-Eberhard, ! 9 7 3 ) , centrifuged at 27,000 g for 30 min and applied to a 5.2 X 85 cm. Sephadex G-75 column (Goldstein et al.,
193 1 9 7 3 ) . T h e c o l u m n was e l u t e d with Tris b u f f e r e d H a n k ' s s o l u t i o n . A f t e r o n e liter h a d passed t h r o u g h the c o l u m n , the f r a c t i o n s c o n t a i n i n g c h e m o t a c t i c a c t i v i t y w e r e p o o l e d ( a b o u t 2 0 0 ml) and a l i q u o t s s t o r e d at - - 8 0 ° C until t h e y w e r e used. A p r o n a s e sensitive bacterial c h e m o t a c t i c f a c t o r was isolated f r o m E. coil c u l t u r e filtrates as p r e v i o u s l y d e s c r i b e d ( S c h i f f m a n et al., 1975). RESULTS Evaluation o f the n u m b e r o f cells in a single p l a n e as a measure o f chemotaxis
H u m a n P M N ' s were i n c u b a t e d in c h e m o t a x i s c h a m b e r s for 1 h at 37°C against d i f f e r e n t c o n c e n t r a t i o n s o f bacterial c h e m o t a c t i c f a c t o r . T h e n u m ber o f cells in focus at each ten m i c r o n s t h r o u g h the filters was c o u n t e d in five high p o w e r fields on each o f d u p l i c a t e filters. T h e results o f o n e such e x p e r i m e n t are s h o w n in fig. 1. F r o m these d a t a the t o t a l n u m b e r o f cells t h a t h a d m i g r a t e d into the filters was e s t i m a t e d b y s u m m i n g the n u m b e r o f cells c o u n t e d at each ten m i c r o n s t h r o u g h the filters. T h e n u m b e r o f cells in each p l a n e is p l o t t e d against the c o n c e n t r a t i o n o f c h e m o t a c t i c f a c t o r on a l o g a r i t h m i c scale in fig. 2A. In fig. 2B are s h o w n d o s e r e s p o n s e curves o f the t o t a l cell n u m b e r a n d o f the leading f r o n t d i s t a n c e s m e a s u r e d in the s a m e filters. In a t o t a l o f f o u r t e e n e x p e r i m e n t s in which the t o t a l cell n u m b e r was estim a t e d , the shapes o f the d o s e r e s p o n s e curves r e p r e s e n t i n g the t o t a l n u m b e r o f cells w e r e v e r y similar to t h o s e r e p r e s e n t i n g the n u m b e r o f cells in a single plane. T h e curves o b t a i n e d b y c o u n t i n g the n u m b e r o f cells in single planes
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1 : 0 ......... T--0 10
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0 30
0 40
0 50
~ 60
70
80
MICRONS
Fig. I. Plot of the average number of cells counted in each plane at the dilutions of bacterial chemotactie factor as indicated. The distances were measured from the top of the filters using a micrometer on the fine focusing knob of the microscope.
194 ~oo- A '~:
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Fig. 2. Chemotaxis dose response curves of dilutions of bacterial chemotactic factor as measured by (A) the average number of cells counted in the planes indicated as measured from the top of the filters and (B) the average sum of the number of cells counted at each ten microns through the filters (open circles) and the average distance from the top of the filters of the leading front two cells (closed circles).
b e t w e e n ten and f o r t y m i c r o n s were usually nearly parallel t o each o t h e r and to the curves r e p r e s e n t i n g the total cell n u m b e r . The n u m b e r o f cells in planes below f o r t y m i c r o n s resulted in dose response curves having increasingly shallower slopes. In s o m e e x p e r i m e n t s deviations f r o m parallelism occurred at either ten or f o r t y microns. E i t h e r t w e n t y or t h i r t y m i c r o n s c o n s i s t e n t l y resulted in dose response curves that paralleled those o f the total cell n u m b e r u n d e r the c o n d i t i o n s used. T h e plane at t h i r t y m i c r o n s f r o m the t o p o f the filters was usually used for measuring c h e m o t a x i s , m o s t l y because there were slightly fewer cells to c o u n t . O c c a s i o n a l l y if the response was low, h o w e v e r , or if the backg r o u n d (i.e. in the absence o f c h e m o t a c t i c factor) were zero when c o u n t e d at t h i r t y microns, then the cells were c o u n t e d at t w e n t y m i c r o n s below the t o p o f the filters. The shapes o f the dose response curves o b t a i n e d by the leading f r o n t m e t h o d were usually d i f f e r e n t f r o m those o b t a i n e d by c o u n t i n g the n u m b e r o f cells. The leading f r o n t m e t h o d resulted in dose response curves t h a t were usually linear over a wide range of c h e m o t a c t i c f a c t o r c o n c e n t r a t i o n s and that usually had a smaller total p e r c e n t d i f f e r e n c e b e t w e e n b a c k g r o u n d and maxi m u m response.
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Fig. 3. Chemotaxis dose response curves with cells in the presence (closed circles) and absence (open circles) of p-nitrophenyl ethyl 4-chlorobutylphosphonate as measured by (A) the leading front method, (B) the number of ceils at thirty microns from the top of the filters and (C) the total number of cells at each ten microns through the filters.
An example o f a discrepancy between the leading front and cell number methods Cells, in the presence or absence o f 8 × 10 .4 M p - n i t r o p h e n y l ethyl 4c h l o r o b u t y l p h o s p h o n a t e , were incubated in c h e m o t a x i s chambers against different c o n c e n t r a t i o n s o f C5a. The cells were c o u n t e d at each ten microns through the filters. Leading front distances were measured in the same filters. D o s e response curves o f the total cell n u m b e r , leading front and the cell n u m b e r o f 3 0 p m are c o m p a r e d in fig. 3. The response with and w i t h o u t p h o s p h o n a t c d e t e r m i n e d by c o u n t i n g the n u m b e r o f cells (either the total n u m b e r or the n u m b e r at thirty microns)
196
was c o m p a r e d with the response d e t e r m i n e d by the leading f r o n t m e t h o d in a total o f eleven e x p e r i m e n t s . T h e r a n d o m m o t i l i t y in the absence o f c h e m o tactic f a c t o r was always inhibited by p h o s p h o n a t e w h e t h e r measured by the leading f r o n t or cell n u m b e r m e t h o d s . In the presence o f 8 × 10 -4 M phosp h o n a t e , there was c o n s i s t e n t l y e n h a n c e m e n t o f the response as measured by the leading f r o n t m e t h o d at the highest c o n c e n t r a t i o n s o f c h c m o t a c t i c f a c t o r used. T h e response as d e t e r m i n e d by c o u n t i n g the n u m b e r o f cells s t i m u l a t e d to migrate, h o w e v e r , with one e x c e p t i o n , was always inhibited by the phosphonate. DISCUSSION O f the several m e t h o d s that have bcen used to q u a n t i t a t e c h e m o t a x i s o f cells t h r o u g h gradients in filters, each has certain disadvantages or sources o f error. When the cells are allowed to migrate t h r o u g h the filters, relatively long i n c u b a t i o n times are required during which one might be c o n c e r n e d a b o u t cell viability and maintaining the c h e m o t a c t i c f a c t o r gradient. F u r t h e r m o r e , the p r o b l e m o f cells adhering to the lower surface o f the filters in variable n u m b e r s m u s t be o v e r c o m e . C o u n t i n g the n u m b e r o f cells within filters, t h e r e f o r e , offers certain advantages over some o f these o t h e r m e t h o d s . C o u n t i n g the total n u m b e r o f cells in filters, h o w e v e r , is fairly difficult and requires considerable e x p e r i e n c e in o r d e r to obtain r e p r o d u c i b l e results. Measuring the distance o f the leading f r o n t o f cells is m o r e objective and relatively easy to do. This m e t h o d , h o w e v e r , f r e q u e n t l y results in shallower dose response curves than does the d e t e r m i n a t i o n o f cell n u m b e r , therefor('. it is o f t e n less sensitive to d i f f e r e n c e s in cell response. C o u n t i n g the n u m b e r o f cells in a single plane is m u c h easier to do than c o u n t i n g t h r o u g h multiple planes and u n d e r the p r o p e r c o n d i t i o n s gives similar results. T h e r e are two possible e x p l a n a t i o n s for the c o n f l i c t i n g results o b t a i n e d by the leading f r o n t and cell n u m b e r m e t h o d s in the p h o s p h o n a t e inhibition exp e r i m e n t s . T h e r e c o u l d have been a small p o p u l a t i o n o f cells t h a t were stimulated to a m u c h greater e x t e n t than the m a j o r i t y o f cells or else, even t h o u g h fewer cells were s t i m u l a t e d to migrate, those t h a t did m o v e d at a faster average velocity. Since there s e e m e d to be a u n i m o d a l distribution o f cells t h r o u g h the filters, the s e c o n d possibility seems m o r e likely. T h e m e t h o d p r e s e n t e d in this p a p e r allows one to c o m p a r e in a single exp e r i m e n t the leading f r o n t distance with the n u m b e r o f cells stimulated to migrate. U n d e r some c o n d i t i o n s , such a c o m p a r i s o n might give m o r e inform a t i o n a b o u t the e f f e c t o f an i n h i b i t o r than does e i t h e r one o f t h e m alone. Since the same filters are used, several variables are e l i m i n a t e d as sources o f e r r o r in such a c o m p a r i s o n . F u r t h e r m o r e , if the leading f r o n t response to a c h e m o t a c t i c f a c t o r is insufficient to get useable results, f r e q u e n t l y the response is greater by the cell n u m b e r m e t h o d . This m e t h o d , however, like m o s t o t h e r filter m e t h o d s , does n o t distinguish b e t w e e n stimulated r a n d o m m o t i l i t y and true c h e m o t a x i s .
197 REFERENCES Boyden, S., 1962, J. Exp. Med. 115,453. English, D. and B.R. Anderson, 1974, J. Immunol. Methods 5,249. Goldstcin, I., S. Hoffstein, J. Gallin and G. Weissmann, 1973, Proc. Nat. Acad. Sci. 70, 2916. Roos, D. and J.A. Loos, 1970, Biochim. Biophys. Acta 222,565. Schiffman, E., H.V. Showell, B.A. Corcoran, P.A. Ward, E. Smith and E.L. Becker, 1975, J. lmrnunol. 114, 1831. Vallota, E.H. and H.J. Muller-Eberhard, 1973, J. Exp. Med. 137, 1109. Zigrnond, S.H. and J.G. Hirsch, 1973, J. Exp. Med. 137,387.
