Experimental Cell Research 94 (1975) 292-298
THE
EFFECT
ON THE
MOBILITY
AND
THE
ON
OF TRIISOPROPYL
OF SURFACE
LOCOMOTION
PHOSPHATE
CONCANAVALIN
A RECEPTORS
OF POLYMORPHONUCLEAR
LEUCO-
CYTES A. M. WOODIN, A. R. POOLE and G. A. DUNN Strangeways
Research Laboratory,
Cambridge
CBl4RN,
UK
SUMMARY The non-phosphorylating organophosphorus compound triisopropyl phosphate, which is known to inhibit rabbit leucocyte locomotion, can stimulate the locomotion of guinea pig leucocytes under certain conditions. Different methods of preparing guinea pig leucocyte monolayers can give preparations with different proportions of motile cells. With preparations that contain relatively slowly moving cells triisopropyl phosphate increases the number of stationary cells without significantly affecting the speed of the cells that remain motile. Most rabbit leucocytes labelled with fluorescein-labelled concanavalin A form caps within 5-10 min at 37°C. In contrast the rate of cap formation in guinea pig leucocytes is much slower and after 20 min many cells have only random patches. Triisopropyl phosphate accelerates cap formation in guinea pig leucocytes but not in rabbit leucocytes. The local acaesthetic nupercaine inhibits cap and patch formation in rabbit and guinea pig leucocytes. Inhibition of rabbit leucocyte locomotion is induced by concanavahn A at 1 pg/ml. These results are briefly related to the known effects of triisopropyl phosphate on the isolated leucocyte plasma membrane.
The non-phosphorylating organophos- that of the rabbit [3]. We show here that phorus compound triisopropyl phosphate the effect of triisopropyl phosphate on the inhibits the locomotion of rabbit poly- locomotion of guinea pig leucocytes differs morphonuclear leucocytes (called leuco- considerably from that on rabbit leucocytes in this paper) [l] and blocks conduc- cytes. The effect of triisopropyl phosphate tion in the axon [2]. The p-nitrophenyl on leucocyte locomotion could stem diphosphatase activity of the isolated plasma rectly from its action on the membrane pand membrane of the rabbit leucocyte is stimu- nitrophenyl phosphatase activity lated by triisopropyl phosphate while that sulphydryl groups but secondary effects on of the axon is inhibited and it is possible metabolism or membrane structure have that this stems from an alteration in the not been excluded. The mobility of conreactivity of the membrane sulphydryl canavalin A (ConA) receptors on the surgroups [3]. The p-nitrophenyl phosphatase face of mammalian cells is a property sensiactivity of the plasma membrane of the tive to toxic agents and we have investiguinea pig leucocyte is also stimulated by gated its modification by triisopropyl phostriisopropyl phosphate although the guinea phate. Two types of lateral movement of ConA pig enzyme differs in some respect from Expd
Cell
Res 94 (1975)
DIP on leucocyte cap formation and locomotion on ceil surfaces have been described. Small lateral movements lead to clustering of receptors into patches randomly distributed over the cell surface [4-71 while large lateral movements lead to aggregation of all the ConA receptor complexes into a cap at one part of the cell surface [S, 9, lo]. In the human leucocyte cap formation is prevented by metabolic inhibitors and local anaesthetics [ll]. We have found that in the rabbit and guinea pig leucocyte patch formation is also inhibited by the local anaesthetic nupercaine. In contrast triisopropyl phosphate does not inhibit cap formation in rabbit leucocytes and enhances the rate in guinea pig leucocytes. METHODS Materials Leucocytes were obtained from peritoneal exudates of rabbits or guinea pigs 12-14 h after injecting 0.145 M NaCl. The cells were suspended in Hanks balanced salt solution containing 1 mg/ml of bovine serum albumin (Hanks BSS-A). Triisopropyl phosphate (DIP) was synthesised by photochemical oxidation [12] of triisopropyl phosphite (Aldrich Chemical Co., Milwaukee, USA). Concanavalin A (ConA) from Miles Seravac Lab (Maidenhead, UK) was diluted as described by Woodin [13]. ConA labelled with fluorescein isothiocyanate (ConA-F) was prepared by the method of Wayne-Smith & Hollers [14]. Nupercaine was obtained from Savory & Moore (Oxford, UK).
Cinemicrography Leucocytes were filmed in the perfusion chamber described by Woodin & Harris [I]. In one experiment leucocytes were diluted into ascitic fluid obtained by centrifuging a sample of the peritoneal exudate; in other experiments the leucocytes in ascitic fluid were diluted into Hanks BSS-A to give about 2~10~ cells/ ml and perfused into the chamber. In some experiments the monolayers were allowed to form on the coverslio and the chamber was then inverted before filming. -In other experiments the chamber was not inverted. Cell movement was filmed at 37°C at 10 set intervals with a Paillard-Wild time-lapse apparatus incorporating a Bolex camera. The developed films were projected onto paper and the paths followed by individual cells traced by hand. The total number of cells and the number of stationary cells were also recorded. The distance moved by the cells was determined by measuring the length of cotton thread superimposed on each tracing with the use of an adhesive.
293
Distribution of ConA-F bound to the leucocyte surface Leucocytes in ascitic fluid were diluted in Hanks BSS-A and 5 x 106cells in 0.5 ml were allowed to settle in an area bounded by an aluminium ring attached to a microscope slide with silicone grease. After 30 min at room temperature the monolayers were gently washed with Hanks BSS-A to remove non-adherent cells and the slides placed on a pad of wet filter paper, in a closed plastic box, maintained at 37°C in an incubator, or at 0°C in an ice bath. Cells were labelled with ConA-F at 100 wg/ml in Hanks BSS-A as described in the text. To determine the time course of patch and cap formation the labelled leucocytes in Hanks BSS-A were incubated at 37°C for various times. The slides were then cooled to 0°C and treated with 8 % formaldehyde in PBS for 15 min and then for a further 15 min at room temperature. The cells were washed with PBS, mounted in 90% glycerol in 0.02 M Tris-HCl buffer, pH 8.6 and examined with dark ground fluorescence microscopy as described by Poole et al. [15] except that a 150W xenon lamp was used as light source. To permit the observation of the distribution of adsorbed ConA-F on unfixed cells the aluminium rings were replaced by rings of silicone grease, which maintained a free space between the coverslips and the microscope slides.
RESULTS Effect of DIP on locomotion of guinea pig leucocytes It was found that too vigorous perfusion in forming the monolayers of guinea pig leucocytes selectively removed the motile cells and the preparations consisted mainly of stationary cells which, while showing active surface movement and putting out pseudopodia, did not move more than 1 PM during 30 min. These stationary cells have been described by Ramsay [16] and by Woodin & Harris [ 11.With slow perfusion (2 ml/5 min) motile cells could be retained although, as seen in table 1, they were in minority. Similar results were obtained with peritoneal exudates obtained 6 or 14 h after injecting the NaCl solution. The rate of locomotion was stimulated by DIP and the effect was largely reversible. The stimulation of locomotion was mainly confined to cells that were already motile. For instance in the experiment recorded in table 1, 44 Exptl Cell Res 94 (1975)
294
Woodin, Poole and Dunn
Table 1. Effect of DIP on the locomotion
of guinea pig leucocytes
Monolayers of guinea pig leucocytes in Hanks BSS-A were formed at 37°C and filmed for 25-35 min alone, then in the presence of DIP and finally, after washing away the DIP. The medium was changed by perfusion (2 ml/5 min). The two experiments were done with the same batch of cells
Additions Expt 1 0
4 mM DIP 0 Expt 2 0
8mMDIP 0
No. cells in field
No. stationary cells 5%
Speed of cells (cLm/min) Mean (S.D.)
Range of speeds (wdmin)
52 56 54
65 59 70
2.0 (1.1) 3.2 (1.7) 2.3 (1.7)
0.6 -3.5 0.9 -6.0 0.6 6.2
80 81 75
55 56 59
1.9 (1.1) 3.1 (1.4) 2.05 (1.08)
0.3 -3.7 0.65-6.5 0.3 -3.4
stationary cells were present in the control only slowly moving cells. Table 2 shows monolayer but only two became motile in that under these conditions the stimulating the presence of 8 mM DIP. effect of DIP is not clear but the number of It is probable that, in contrast to the rab- stationary cells increases markedly in the bit leucocyte, motile guinea pig leucocytes presence of DIP. Similar results were obadhere weakly to glass. If the guinea pig tained with cells suspended in Hanks BSS-A leucocyte monolayers were inverted be- or in the ascitic fluid in which they were fore filming, the preparations contained harvested from the guinea pig. This pheTable 2. Effect of DIP on the locomotion
of guinea pig leucocytes in inverted monolayers
The experiment was carried out as described in table I except that the monolayer was inverted before filming DIP cont. (mW
No. of cells in field
Expr 1 Hanks BSS-A 0 58
2.2 0
58 47
Expt 2 Hanks BSS-A 0 59
4.0 0
64 62
Expt 3 Hanks BSS-A 0 144
8 0
134 127
Expt 4 Ascitic fluid 0 43
4 0
36 34
Exptl Cell Res 94 (1975)
Stationary cells, 5% of total
(cLm/min)
Mean (S.D.)
Range of speeds (qlmin)
27 38 49
0.90 (0.46) 0.82 (0.47) 0.81 (0.40)
0.3 -1.9 0.3 -1.3 0.3 -1.2
18 52 51
0.92 (0.34) 0.91 (0.41) 0.81 (0.33)
0.4 -1.5 0.25-l .8 0.4 -1.5
54 82 75
0.51 (0.30) 0.54 (0.37) 0.45 (0.29)
0.4 -1.4 0.4 -1.9 0.4 -1.4
34 74 67
0.64 (0.29) 0.70 (0.30) 0.60 (0.24)
0.2 -1.3 0.4 -1.1 0.2 -1.2
Speed of cells
DIP on leucocyte cap formation and locomotion
295
Effect of DIP and nupercaine on distribution of ConA receptors Monolayers of leucocytes in Hanks BSS-A were la- on surface of rabbit and belled with ConA-F (100 pg/ml) at 0°C for 10 min, washed at O”C, maintained at 0°C for 3 min alone or guinea pig leucocytes with DIP and then incubated at 37°C before fixation at 0°C. Cells without patches or caps exhibited low Leucocytes treated with ConA-F at O”C, intensity diffuse surface staining. 140-190 cells were washed free from unbound ConA-F and examined under each condition fixed at 0°C showed low intensity diffuse ConA-F distribution fluorescence over the whole cell surface. (% cells examined) IncuIf the washed cells were incubated at 37°C bation Random Nuclear Side time before fixation at O”C, the fluorescence was Additions patches patches caps (min) aggregated into patches over the surface or into a discrete cap which, in the fixed cell, 5 0 2 0 98 4mM DIP 6 0 91 appeared at one side. With all preparations 8 mMDIP 4 2 87 prolonged incubation led to a progressive 15 0 10 75 8 disappearance of the cap and the reap4mM DIP 18 78 4 8mMDIP 12 88 0 pearance of surface patches either ran25 0 0 100 0 domly distributed or concentrated over the 4mM DIP 0 100 0 nucleus. Direct nuclear capping, described 8 mM DIP 0 100 0 in immobilised human leucocytes by Ryan et al. [lo], has not been observed in rabbit nomenon may be of general significance for or guinea pig leucocytes, alone, or treated it is common practice in studying the with DIP. Complete disaggregation of the properties of leucocytes to form mono- fluorescence to the diffuse state did not oclayers in coverslips and wash them vi- cur. Preparations incubated for lo-25 min gorously [lo, 171.It is possible that this can before fixation showed some cells with both selectively remove motile cells. side caps and patches around the nucleus
Table 3. Effect of DIP on cap formation in rabbit leucocytes labelled with ConA-F
Table 4. Comparison of cap andpatch formation in two preparations of leucocytes derived from the same rabbit The experiment was carried out as described in table 3. Expt 2 was done I h after expt 1. 130-180 cells were examined for each condition ConA-F distribution (% cells examined) Incubation time (min) 5 10 15 25
Expt 1
0 8 mM 0 8 mM 0 8 mM 0 8 mM
DIP DIP DIP DIP
Expt 2
Random patches
Nuclear patches
Side caps
Random patches
Nuclear patches
Side caps
9 7 3 0 0 12 0 12
0 0 0 0 18 0 100 3
91 93 97 100 82 88 0 85
5 5 4 0 0 0 90 42
0 0 0 0 64 0 8 4
95 95 96 100 36 100 2 54 Exprl Cd Rcs 94 (1975)
296
Woodin, Poole and Dunn
Table 5. Effect of DIP on the distribution of ConA-F on the surface of guinea pig leucocytes
ment recorded in table 3 the effect of 8 mM DIP was determined on duplicate preparations of leucocytes derived from the same Monolayers of guinea pig leucocytes were treated as rabbit. Table 4 shows that no effect on cap described in table 3. The two experiments were done formation could be detected but that there with leucocytes from different animals. 1IO-130 cells were examined for each condition. Cells without was a retardation of its reversal. In conpatches or caps showed diffuse surface staining trast, 1 mM nupercaine completely inConA-F distribution hibited both patch and cap formation in four Incu(% cells examined) preparations of rabbit leucocytes. bation time Random Nuclear The possibility that DIP can affect the (mitt) Additions patches patches Caps rate of lateral movement of ConA receptors is supported by its effect on guinea pig Expr I leucocytes. With these cells the rate of cap0 4 0 90 I 0 74 21 8 mM DIP ping was much slower than with rabbit 0 84 0 8 8 leucocytes and table 5 shows that with two 80 0 8 mM DIP 20 preparations derived from different guinea 12 0 79 0 21 pigs 8 mM DIP enhanced cap formation. 58 I 8 mM DIP 41 In the presence of 1 mM nupercaine the 0 90 3 22 7 81 0 8 mM DIP 19 fluorescence remained diffuse. Woodin & Wieneke [2] found that DIP Expf 2 2 96 2 5 0 had different effects on the cation content 0 87 13 8 mM DIP of rabbit leucocytes if it was added to cells I2
26
0 8 mM DIP 0 8 mM DIP 0 8 mM DIP
80 77 97 95 100 96
II 0 0 2 0 2
9 23 3 3 0 2
and also a band of weak fluorescence connecting them. Table 3 describes the effect of DIP on cap formation and its reversal in rabbit leucocytes. Woodin & Harris [ 1] found that 8 mM DIP almost completely immobilised rabbit leucocytes. The side caps in fixed leucocytes are very distinct and duplicate counts on preparations that contained 80120 capped cells differed by less than 2%. However, the rate of cap formation and its reversal is rapid and it was uncertain if the incubation times and fixation of multiple samples were adequately synchronised. To assess the significance of the small inhibition by DIP of cap formation in the experiExprl Cell Res 94 (1975)
Table 6. Effect of DIP and nupercaine on the distribution of ConA-F on rabbit leucocytes labelled at 37°C Monolayers of leucocytes in Hanks BSS-A were maintained at 37°C for 30 min and then for a further 3 min alone or with 8 mM DIP or I mM nupercaine. ConA-F (100 pg/ml in Hanks BSS-A) was added and after incubation for various times the cells were fixed at 0°C. The two experiments were done with different batches of cells. For each condition 90-150 cells were examined and the distribution of the ConA-F recorded as diffuse (D), patched (P) or capped (C) ConA-F distribution (% of cells examined) Incubation time (min)
No additions
8 mM DIP
I mM nuperCaine
DPC
DPC
DPC
Expr I
5 IO Expr 2 10
30
5 85 5 90
IO 5
0 16 84 0 73 27
47917 2 83
I5
9028 100 0 0
0 18 82 0 85 15
98 2 0 99 1 0
DIP on leucocyte cap formation and locomotion Table 7. Effect of DIP on the distribution of ConA-F on unfured rabbit leucocytes Monolayers of leucocytes in Hanks BSS-A were labelled with ConA-F (100 pg/ml) at 0°C washed with Hanks BSS-A at 0°C and maintained at 22°C alone or with DIP. The distribution of the fluorescence was observed at different times. The experiments were done with different batches of cells. For each condition 4tL 60 cells were examined
297
bit leucocytes is slower than at 37°C and at this temperature 4 mM DIP slightly enhanced the rate cap formation, or retarded its reversal.
The effect of ConA on leucocyte locomotion It was hoped to investigate the interaction ConA-F distribution of the effects of DIP and ConA on the rabIncu(% cells examined) bit leucocyte by observing the effects of bation time Random Tail ConA on the inhibition of locomotion by Diffuse patches caps (min) Additions DIP. However, filming the cells showed that ConA is a powerful inhibitor of rabbit Expt I 12 0 0 45 55 leucocyte locomotion although the cells 14 4 mM DIP 0 47 53 preserve the characteristic shape of the 35 0 0 32 68 moving cell. With rabbit leucocytes com0 37 4 mM DIP 28 72 plete inhibition of locomotion was found Expt 2 with 1 pg/ml of ConA. A similar effect was 6 100 0 0 0 found with cells treated with 50 pg/ml of 8 100 4 mM DIP 0 0 ConA at 0°C and then washed before 12 0 91 6 14 4 mM DIP 90 7 filming at 37°C. Ryan et al. [lo] reported 17 0 67 19 4 that some human leucocytes remain motile 19 52 4 mM DIP 35 13 after treatment with 10 pg/ml of ConA. 0 52 36 13 32 This may reflect partial elution of the 12 40 4 mM DIP 33 52 ConA adsorbed on the leucocyte by the 55 0 30 14 59 58 16 4 mM DIP 12 76 serum used in their experiments. Woodin [l3] has found that relatively high and low concentrations of ConA have qualitatively maintained at 37°C rather than at 0°C. different effects in inhibiting leucocyte Table 6 compares the effect of DIP and secretion and that these stem from the ocnupercaine on cap formation on rabbit cupation of receptors of different affinity. leucocytes treated with ConA-F at 37°C. It appears that occupation of the high afThe significance of the small effects on DIP finity sites leads to inhibition of locomohas not been determined but the differtion. ence from the action of nupercaine is clear. DISCUSSION It is probable that the cap observed at the side of fixed leucocytes corresponds to In contrast to its largely irreversible inthe cap present on the tail of unfixed cells. hibitory effect on rabbit leucocyte locomoTo confirm that DIP does not inhibit the tion [l] DIP can reversibly stimulate that of formation of tail caps rabbit leucocytes the guinea pig leucocyte. The p-nitrophenyl were labelled and washed at 0°C and the phosphatase activity of the plasma memdistribution of the adsorbed ConA-F ob- brane of both cells is stimulated by DIP but served in cells maintained at 22°C without the properties of the two enzymes differ fixation. Table 6 shows that under these and while DIP enhances the inhibition of conditions the rate of cap formation in rab- the rabbit enzyme by N-ethyl maleimide it Exprl Cell Res 94 (1975)
298
Woodin, Poole and Dunn
antagonises the inhibition of the guinea pig enzyme [3]. It is possible that the effect of DIP on leucocyte locomotion stems from a modification to the membrane sulphydryl groups. At 37°C DIP increases the rate of formation of caps in guinea pig leucocytes while with rabbit leucocytes no effect on cap formation has been observed although the rate of reversal of cap formation is retarded. It should be noted that a small inhibition in the rate of cap formation could be overlooked. Thus a small reduction in the number of ConA-receptor complexes swept into the cap might not reduce the overall number of capped cells and a small decrease in the intensity of the fluorescence of the cap could pass unnoticed. It is possible that relatively rapid capping occurs in leucocytes that move relatively rapidly. The present results are consistent with this but will require confirmation by observing capping and locomotion on monolayers prepared under identical conditions. There is a clear distinction between the modification of the mobility of the ConA receptors by DIP and by other inhibitors of locomotion. DIP does not alter the position of the cap on rabbit or guinea pig leucocytes but immobilisation of human leucocytes by cytochalasin or exposure to protein-free solutions induces cap formation over the nucleus rather than at the tail. Metabolic inhibitors totally inhibit cap formation in human leucocytes [lo]. Ryan et al. [l l] found that local anaesthetics inhibited cap formation in human leucocytes but as their electron micrographs showed some aggregation of ConA receptors in cells treated with xylocaine they concluded that patch formation was unaffected. Under our conditions the distribution of the adsorbed ConA on the nupercaine-treated leucocyte is indistinguishable from that on Exprl Cell Res 94 (1975)
the cell maintained at 0°C and so some inhibition of patch formation does occur. The action of local anaesthetics is of particular interest for, like organophosphorus compounds, they act on a wide spectrum of mammalian cells. Local anaesthetics can stabilise phospholipid monolayers and protect erythrocytes against osmotic lysis [18]. Possibly by the same mechanism they could reduce the lateral diffusion of the ConA receptors in the leucocyte. The difference between the effect of DIP and local anaesthetics on capping does not establish that the former has no action on the phospholipids or other structural components of the membrane but it does suggest that the effect of DIP stems from more specific actions on the leucocyte membrane, such as those described by Woodin [3]. REFERENCES 1. Woodin, A M & Harris, A, Exp cell res 77 (1973) 41. 2. Foodin, A M & Wieneke, A A, Nature 227 (1970) 3. Woodin, A M, Exp cell res 89 (1974) 15. 4. Inbar, M, Huet, C, Oseroff, A R, Benbassat, H & Sachs, L, Biochim biophys acta 3 11 (1973) 594. 5. de Petris, S, Raff, M C & Mallucci, L, Nature new bio1244 (1973) 275. 6. Nicolson, G L, Nature new biol 243 (1973) 218. 7. Rosenbhth, J Z, Ukena, T E, Yin, H, Berlin, R D & Kamovsky, M J, Proc natl acad sci US 570 (1973) 1625. 8. Inbar. M. Benbassat, H &Sachs, L, lnt i cancer 12 (1973) 93. 9. Yahara, I & Edelman, S M, Exp cell res 81 (1973) 143. 10. Ryan, G B, Borysenko, J Z & Kamovsky, M J, J cell bio162 (1974) 351. Il. Ryan, G B, Unanue, E R & Karnovsky, M J. Nature 250 (1974) 56. 12. Cadogan, J I G, Cameron-Wood, M & Foster, W R. J them sot (1%3) 2549. 13. Woodin, A M, Exp cell res 92 (1975) 201. 14. Wavne-Smith. C & Hollers, J C, J reticuloendothel s&S (1970) 458. 15. Poole, A R, Dingle, J T & Barrett, A J, J histothem cytochem 20 (1972) 261. 16. Ramsay, W S, Exp cell res 70 (1972) 129. 17. Berlin, R D & Ukena, T E, Nature 238 (1972) 120. 18. Seeman, P, Pharmacol rev 24 (1972) 583. Received January 13, 1975 Revised version received February 24, 1975
THE
EFFECT
ON THE
MOBILITY
AND
THE
ON
OF TRIISOPROPYL
OF SURFACE
LOCOMOTION
PHOSPHATE
CONCANAVALIN
A RECEPTORS
OF POLYMORPHONUCLEAR
LEUCO-
CYTES A. M. WOODIN, A. R. POOLE and G. A. DUNN Strangeways
Research Laboratory,
Cambridge
CBl4RN,
UK
SUMMARY The non-phosphorylating organophosphorus compound triisopropyl phosphate, which is known to inhibit rabbit leucocyte locomotion, can stimulate the locomotion of guinea pig leucocytes under certain conditions. Different methods of preparing guinea pig leucocyte monolayers can give preparations with different proportions of motile cells. With preparations that contain relatively slowly moving cells triisopropyl phosphate increases the number of stationary cells without significantly affecting the speed of the cells that remain motile. Most rabbit leucocytes labelled with fluorescein-labelled concanavalin A form caps within 5-10 min at 37°C. In contrast the rate of cap formation in guinea pig leucocytes is much slower and after 20 min many cells have only random patches. Triisopropyl phosphate accelerates cap formation in guinea pig leucocytes but not in rabbit leucocytes. The local acaesthetic nupercaine inhibits cap and patch formation in rabbit and guinea pig leucocytes. Inhibition of rabbit leucocyte locomotion is induced by concanavahn A at 1 pg/ml. These results are briefly related to the known effects of triisopropyl phosphate on the isolated leucocyte plasma membrane.
The non-phosphorylating organophos- that of the rabbit [3]. We show here that phorus compound triisopropyl phosphate the effect of triisopropyl phosphate on the inhibits the locomotion of rabbit poly- locomotion of guinea pig leucocytes differs morphonuclear leucocytes (called leuco- considerably from that on rabbit leucocytes in this paper) [l] and blocks conduc- cytes. The effect of triisopropyl phosphate tion in the axon [2]. The p-nitrophenyl on leucocyte locomotion could stem diphosphatase activity of the isolated plasma rectly from its action on the membrane pand membrane of the rabbit leucocyte is stimu- nitrophenyl phosphatase activity lated by triisopropyl phosphate while that sulphydryl groups but secondary effects on of the axon is inhibited and it is possible metabolism or membrane structure have that this stems from an alteration in the not been excluded. The mobility of conreactivity of the membrane sulphydryl canavalin A (ConA) receptors on the surgroups [3]. The p-nitrophenyl phosphatase face of mammalian cells is a property sensiactivity of the plasma membrane of the tive to toxic agents and we have investiguinea pig leucocyte is also stimulated by gated its modification by triisopropyl phostriisopropyl phosphate although the guinea phate. Two types of lateral movement of ConA pig enzyme differs in some respect from Expd
Cell
Res 94 (1975)
DIP on leucocyte cap formation and locomotion on ceil surfaces have been described. Small lateral movements lead to clustering of receptors into patches randomly distributed over the cell surface [4-71 while large lateral movements lead to aggregation of all the ConA receptor complexes into a cap at one part of the cell surface [S, 9, lo]. In the human leucocyte cap formation is prevented by metabolic inhibitors and local anaesthetics [ll]. We have found that in the rabbit and guinea pig leucocyte patch formation is also inhibited by the local anaesthetic nupercaine. In contrast triisopropyl phosphate does not inhibit cap formation in rabbit leucocytes and enhances the rate in guinea pig leucocytes. METHODS Materials Leucocytes were obtained from peritoneal exudates of rabbits or guinea pigs 12-14 h after injecting 0.145 M NaCl. The cells were suspended in Hanks balanced salt solution containing 1 mg/ml of bovine serum albumin (Hanks BSS-A). Triisopropyl phosphate (DIP) was synthesised by photochemical oxidation [12] of triisopropyl phosphite (Aldrich Chemical Co., Milwaukee, USA). Concanavalin A (ConA) from Miles Seravac Lab (Maidenhead, UK) was diluted as described by Woodin [13]. ConA labelled with fluorescein isothiocyanate (ConA-F) was prepared by the method of Wayne-Smith & Hollers [14]. Nupercaine was obtained from Savory & Moore (Oxford, UK).
Cinemicrography Leucocytes were filmed in the perfusion chamber described by Woodin & Harris [I]. In one experiment leucocytes were diluted into ascitic fluid obtained by centrifuging a sample of the peritoneal exudate; in other experiments the leucocytes in ascitic fluid were diluted into Hanks BSS-A to give about 2~10~ cells/ ml and perfused into the chamber. In some experiments the monolayers were allowed to form on the coverslio and the chamber was then inverted before filming. -In other experiments the chamber was not inverted. Cell movement was filmed at 37°C at 10 set intervals with a Paillard-Wild time-lapse apparatus incorporating a Bolex camera. The developed films were projected onto paper and the paths followed by individual cells traced by hand. The total number of cells and the number of stationary cells were also recorded. The distance moved by the cells was determined by measuring the length of cotton thread superimposed on each tracing with the use of an adhesive.
293
Distribution of ConA-F bound to the leucocyte surface Leucocytes in ascitic fluid were diluted in Hanks BSS-A and 5 x 106cells in 0.5 ml were allowed to settle in an area bounded by an aluminium ring attached to a microscope slide with silicone grease. After 30 min at room temperature the monolayers were gently washed with Hanks BSS-A to remove non-adherent cells and the slides placed on a pad of wet filter paper, in a closed plastic box, maintained at 37°C in an incubator, or at 0°C in an ice bath. Cells were labelled with ConA-F at 100 wg/ml in Hanks BSS-A as described in the text. To determine the time course of patch and cap formation the labelled leucocytes in Hanks BSS-A were incubated at 37°C for various times. The slides were then cooled to 0°C and treated with 8 % formaldehyde in PBS for 15 min and then for a further 15 min at room temperature. The cells were washed with PBS, mounted in 90% glycerol in 0.02 M Tris-HCl buffer, pH 8.6 and examined with dark ground fluorescence microscopy as described by Poole et al. [15] except that a 150W xenon lamp was used as light source. To permit the observation of the distribution of adsorbed ConA-F on unfixed cells the aluminium rings were replaced by rings of silicone grease, which maintained a free space between the coverslips and the microscope slides.
RESULTS Effect of DIP on locomotion of guinea pig leucocytes It was found that too vigorous perfusion in forming the monolayers of guinea pig leucocytes selectively removed the motile cells and the preparations consisted mainly of stationary cells which, while showing active surface movement and putting out pseudopodia, did not move more than 1 PM during 30 min. These stationary cells have been described by Ramsay [16] and by Woodin & Harris [ 11.With slow perfusion (2 ml/5 min) motile cells could be retained although, as seen in table 1, they were in minority. Similar results were obtained with peritoneal exudates obtained 6 or 14 h after injecting the NaCl solution. The rate of locomotion was stimulated by DIP and the effect was largely reversible. The stimulation of locomotion was mainly confined to cells that were already motile. For instance in the experiment recorded in table 1, 44 Exptl Cell Res 94 (1975)
294
Woodin, Poole and Dunn
Table 1. Effect of DIP on the locomotion
of guinea pig leucocytes
Monolayers of guinea pig leucocytes in Hanks BSS-A were formed at 37°C and filmed for 25-35 min alone, then in the presence of DIP and finally, after washing away the DIP. The medium was changed by perfusion (2 ml/5 min). The two experiments were done with the same batch of cells
Additions Expt 1 0
4 mM DIP 0 Expt 2 0
8mMDIP 0
No. cells in field
No. stationary cells 5%
Speed of cells (cLm/min) Mean (S.D.)
Range of speeds (wdmin)
52 56 54
65 59 70
2.0 (1.1) 3.2 (1.7) 2.3 (1.7)
0.6 -3.5 0.9 -6.0 0.6 6.2
80 81 75
55 56 59
1.9 (1.1) 3.1 (1.4) 2.05 (1.08)
0.3 -3.7 0.65-6.5 0.3 -3.4
stationary cells were present in the control only slowly moving cells. Table 2 shows monolayer but only two became motile in that under these conditions the stimulating the presence of 8 mM DIP. effect of DIP is not clear but the number of It is probable that, in contrast to the rab- stationary cells increases markedly in the bit leucocyte, motile guinea pig leucocytes presence of DIP. Similar results were obadhere weakly to glass. If the guinea pig tained with cells suspended in Hanks BSS-A leucocyte monolayers were inverted be- or in the ascitic fluid in which they were fore filming, the preparations contained harvested from the guinea pig. This pheTable 2. Effect of DIP on the locomotion
of guinea pig leucocytes in inverted monolayers
The experiment was carried out as described in table I except that the monolayer was inverted before filming DIP cont. (mW
No. of cells in field
Expr 1 Hanks BSS-A 0 58
2.2 0
58 47
Expt 2 Hanks BSS-A 0 59
4.0 0
64 62
Expt 3 Hanks BSS-A 0 144
8 0
134 127
Expt 4 Ascitic fluid 0 43
4 0
36 34
Exptl Cell Res 94 (1975)
Stationary cells, 5% of total
(cLm/min)
Mean (S.D.)
Range of speeds (qlmin)
27 38 49
0.90 (0.46) 0.82 (0.47) 0.81 (0.40)
0.3 -1.9 0.3 -1.3 0.3 -1.2
18 52 51
0.92 (0.34) 0.91 (0.41) 0.81 (0.33)
0.4 -1.5 0.25-l .8 0.4 -1.5
54 82 75
0.51 (0.30) 0.54 (0.37) 0.45 (0.29)
0.4 -1.4 0.4 -1.9 0.4 -1.4
34 74 67
0.64 (0.29) 0.70 (0.30) 0.60 (0.24)
0.2 -1.3 0.4 -1.1 0.2 -1.2
Speed of cells
DIP on leucocyte cap formation and locomotion
295
Effect of DIP and nupercaine on distribution of ConA receptors Monolayers of leucocytes in Hanks BSS-A were la- on surface of rabbit and belled with ConA-F (100 pg/ml) at 0°C for 10 min, washed at O”C, maintained at 0°C for 3 min alone or guinea pig leucocytes with DIP and then incubated at 37°C before fixation at 0°C. Cells without patches or caps exhibited low Leucocytes treated with ConA-F at O”C, intensity diffuse surface staining. 140-190 cells were washed free from unbound ConA-F and examined under each condition fixed at 0°C showed low intensity diffuse ConA-F distribution fluorescence over the whole cell surface. (% cells examined) IncuIf the washed cells were incubated at 37°C bation Random Nuclear Side time before fixation at O”C, the fluorescence was Additions patches patches caps (min) aggregated into patches over the surface or into a discrete cap which, in the fixed cell, 5 0 2 0 98 4mM DIP 6 0 91 appeared at one side. With all preparations 8 mMDIP 4 2 87 prolonged incubation led to a progressive 15 0 10 75 8 disappearance of the cap and the reap4mM DIP 18 78 4 8mMDIP 12 88 0 pearance of surface patches either ran25 0 0 100 0 domly distributed or concentrated over the 4mM DIP 0 100 0 nucleus. Direct nuclear capping, described 8 mM DIP 0 100 0 in immobilised human leucocytes by Ryan et al. [lo], has not been observed in rabbit nomenon may be of general significance for or guinea pig leucocytes, alone, or treated it is common practice in studying the with DIP. Complete disaggregation of the properties of leucocytes to form mono- fluorescence to the diffuse state did not oclayers in coverslips and wash them vi- cur. Preparations incubated for lo-25 min gorously [lo, 171.It is possible that this can before fixation showed some cells with both selectively remove motile cells. side caps and patches around the nucleus
Table 3. Effect of DIP on cap formation in rabbit leucocytes labelled with ConA-F
Table 4. Comparison of cap andpatch formation in two preparations of leucocytes derived from the same rabbit The experiment was carried out as described in table 3. Expt 2 was done I h after expt 1. 130-180 cells were examined for each condition ConA-F distribution (% cells examined) Incubation time (min) 5 10 15 25
Expt 1
0 8 mM 0 8 mM 0 8 mM 0 8 mM
DIP DIP DIP DIP
Expt 2
Random patches
Nuclear patches
Side caps
Random patches
Nuclear patches
Side caps
9 7 3 0 0 12 0 12
0 0 0 0 18 0 100 3
91 93 97 100 82 88 0 85
5 5 4 0 0 0 90 42
0 0 0 0 64 0 8 4
95 95 96 100 36 100 2 54 Exprl Cd Rcs 94 (1975)
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Woodin, Poole and Dunn
Table 5. Effect of DIP on the distribution of ConA-F on the surface of guinea pig leucocytes
ment recorded in table 3 the effect of 8 mM DIP was determined on duplicate preparations of leucocytes derived from the same Monolayers of guinea pig leucocytes were treated as rabbit. Table 4 shows that no effect on cap described in table 3. The two experiments were done formation could be detected but that there with leucocytes from different animals. 1IO-130 cells were examined for each condition. Cells without was a retardation of its reversal. In conpatches or caps showed diffuse surface staining trast, 1 mM nupercaine completely inConA-F distribution hibited both patch and cap formation in four Incu(% cells examined) preparations of rabbit leucocytes. bation time Random Nuclear The possibility that DIP can affect the (mitt) Additions patches patches Caps rate of lateral movement of ConA receptors is supported by its effect on guinea pig Expr I leucocytes. With these cells the rate of cap0 4 0 90 I 0 74 21 8 mM DIP ping was much slower than with rabbit 0 84 0 8 8 leucocytes and table 5 shows that with two 80 0 8 mM DIP 20 preparations derived from different guinea 12 0 79 0 21 pigs 8 mM DIP enhanced cap formation. 58 I 8 mM DIP 41 In the presence of 1 mM nupercaine the 0 90 3 22 7 81 0 8 mM DIP 19 fluorescence remained diffuse. Woodin & Wieneke [2] found that DIP Expf 2 2 96 2 5 0 had different effects on the cation content 0 87 13 8 mM DIP of rabbit leucocytes if it was added to cells I2
26
0 8 mM DIP 0 8 mM DIP 0 8 mM DIP
80 77 97 95 100 96
II 0 0 2 0 2
9 23 3 3 0 2
and also a band of weak fluorescence connecting them. Table 3 describes the effect of DIP on cap formation and its reversal in rabbit leucocytes. Woodin & Harris [ 1] found that 8 mM DIP almost completely immobilised rabbit leucocytes. The side caps in fixed leucocytes are very distinct and duplicate counts on preparations that contained 80120 capped cells differed by less than 2%. However, the rate of cap formation and its reversal is rapid and it was uncertain if the incubation times and fixation of multiple samples were adequately synchronised. To assess the significance of the small inhibition by DIP of cap formation in the experiExprl Cell Res 94 (1975)
Table 6. Effect of DIP and nupercaine on the distribution of ConA-F on rabbit leucocytes labelled at 37°C Monolayers of leucocytes in Hanks BSS-A were maintained at 37°C for 30 min and then for a further 3 min alone or with 8 mM DIP or I mM nupercaine. ConA-F (100 pg/ml in Hanks BSS-A) was added and after incubation for various times the cells were fixed at 0°C. The two experiments were done with different batches of cells. For each condition 90-150 cells were examined and the distribution of the ConA-F recorded as diffuse (D), patched (P) or capped (C) ConA-F distribution (% of cells examined) Incubation time (min)
No additions
8 mM DIP
I mM nuperCaine
DPC
DPC
DPC
Expr I
5 IO Expr 2 10
30
5 85 5 90
IO 5
0 16 84 0 73 27
47917 2 83
I5
9028 100 0 0
0 18 82 0 85 15
98 2 0 99 1 0
DIP on leucocyte cap formation and locomotion Table 7. Effect of DIP on the distribution of ConA-F on unfured rabbit leucocytes Monolayers of leucocytes in Hanks BSS-A were labelled with ConA-F (100 pg/ml) at 0°C washed with Hanks BSS-A at 0°C and maintained at 22°C alone or with DIP. The distribution of the fluorescence was observed at different times. The experiments were done with different batches of cells. For each condition 4tL 60 cells were examined
297
bit leucocytes is slower than at 37°C and at this temperature 4 mM DIP slightly enhanced the rate cap formation, or retarded its reversal.
The effect of ConA on leucocyte locomotion It was hoped to investigate the interaction ConA-F distribution of the effects of DIP and ConA on the rabIncu(% cells examined) bit leucocyte by observing the effects of bation time Random Tail ConA on the inhibition of locomotion by Diffuse patches caps (min) Additions DIP. However, filming the cells showed that ConA is a powerful inhibitor of rabbit Expt I 12 0 0 45 55 leucocyte locomotion although the cells 14 4 mM DIP 0 47 53 preserve the characteristic shape of the 35 0 0 32 68 moving cell. With rabbit leucocytes com0 37 4 mM DIP 28 72 plete inhibition of locomotion was found Expt 2 with 1 pg/ml of ConA. A similar effect was 6 100 0 0 0 found with cells treated with 50 pg/ml of 8 100 4 mM DIP 0 0 ConA at 0°C and then washed before 12 0 91 6 14 4 mM DIP 90 7 filming at 37°C. Ryan et al. [lo] reported 17 0 67 19 4 that some human leucocytes remain motile 19 52 4 mM DIP 35 13 after treatment with 10 pg/ml of ConA. 0 52 36 13 32 This may reflect partial elution of the 12 40 4 mM DIP 33 52 ConA adsorbed on the leucocyte by the 55 0 30 14 59 58 16 4 mM DIP 12 76 serum used in their experiments. Woodin [l3] has found that relatively high and low concentrations of ConA have qualitatively maintained at 37°C rather than at 0°C. different effects in inhibiting leucocyte Table 6 compares the effect of DIP and secretion and that these stem from the ocnupercaine on cap formation on rabbit cupation of receptors of different affinity. leucocytes treated with ConA-F at 37°C. It appears that occupation of the high afThe significance of the small effects on DIP finity sites leads to inhibition of locomohas not been determined but the differtion. ence from the action of nupercaine is clear. DISCUSSION It is probable that the cap observed at the side of fixed leucocytes corresponds to In contrast to its largely irreversible inthe cap present on the tail of unfixed cells. hibitory effect on rabbit leucocyte locomoTo confirm that DIP does not inhibit the tion [l] DIP can reversibly stimulate that of formation of tail caps rabbit leucocytes the guinea pig leucocyte. The p-nitrophenyl were labelled and washed at 0°C and the phosphatase activity of the plasma memdistribution of the adsorbed ConA-F ob- brane of both cells is stimulated by DIP but served in cells maintained at 22°C without the properties of the two enzymes differ fixation. Table 6 shows that under these and while DIP enhances the inhibition of conditions the rate of cap formation in rab- the rabbit enzyme by N-ethyl maleimide it Exprl Cell Res 94 (1975)
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Woodin, Poole and Dunn
antagonises the inhibition of the guinea pig enzyme [3]. It is possible that the effect of DIP on leucocyte locomotion stems from a modification to the membrane sulphydryl groups. At 37°C DIP increases the rate of formation of caps in guinea pig leucocytes while with rabbit leucocytes no effect on cap formation has been observed although the rate of reversal of cap formation is retarded. It should be noted that a small inhibition in the rate of cap formation could be overlooked. Thus a small reduction in the number of ConA-receptor complexes swept into the cap might not reduce the overall number of capped cells and a small decrease in the intensity of the fluorescence of the cap could pass unnoticed. It is possible that relatively rapid capping occurs in leucocytes that move relatively rapidly. The present results are consistent with this but will require confirmation by observing capping and locomotion on monolayers prepared under identical conditions. There is a clear distinction between the modification of the mobility of the ConA receptors by DIP and by other inhibitors of locomotion. DIP does not alter the position of the cap on rabbit or guinea pig leucocytes but immobilisation of human leucocytes by cytochalasin or exposure to protein-free solutions induces cap formation over the nucleus rather than at the tail. Metabolic inhibitors totally inhibit cap formation in human leucocytes [lo]. Ryan et al. [l l] found that local anaesthetics inhibited cap formation in human leucocytes but as their electron micrographs showed some aggregation of ConA receptors in cells treated with xylocaine they concluded that patch formation was unaffected. Under our conditions the distribution of the adsorbed ConA on the nupercaine-treated leucocyte is indistinguishable from that on Exprl Cell Res 94 (1975)
the cell maintained at 0°C and so some inhibition of patch formation does occur. The action of local anaesthetics is of particular interest for, like organophosphorus compounds, they act on a wide spectrum of mammalian cells. Local anaesthetics can stabilise phospholipid monolayers and protect erythrocytes against osmotic lysis [18]. Possibly by the same mechanism they could reduce the lateral diffusion of the ConA receptors in the leucocyte. The difference between the effect of DIP and local anaesthetics on capping does not establish that the former has no action on the phospholipids or other structural components of the membrane but it does suggest that the effect of DIP stems from more specific actions on the leucocyte membrane, such as those described by Woodin [3]. REFERENCES 1. Woodin, A M & Harris, A, Exp cell res 77 (1973) 41. 2. Foodin, A M & Wieneke, A A, Nature 227 (1970) 3. Woodin, A M, Exp cell res 89 (1974) 15. 4. Inbar, M, Huet, C, Oseroff, A R, Benbassat, H & Sachs, L, Biochim biophys acta 3 11 (1973) 594. 5. de Petris, S, Raff, M C & Mallucci, L, Nature new bio1244 (1973) 275. 6. Nicolson, G L, Nature new biol 243 (1973) 218. 7. Rosenbhth, J Z, Ukena, T E, Yin, H, Berlin, R D & Kamovsky, M J, Proc natl acad sci US 570 (1973) 1625. 8. Inbar. M. Benbassat, H &Sachs, L, lnt i cancer 12 (1973) 93. 9. Yahara, I & Edelman, S M, Exp cell res 81 (1973) 143. 10. Ryan, G B, Borysenko, J Z & Kamovsky, M J, J cell bio162 (1974) 351. Il. Ryan, G B, Unanue, E R & Karnovsky, M J. Nature 250 (1974) 56. 12. Cadogan, J I G, Cameron-Wood, M & Foster, W R. J them sot (1%3) 2549. 13. Woodin, A M, Exp cell res 92 (1975) 201. 14. Wavne-Smith. C & Hollers, J C, J reticuloendothel s&S (1970) 458. 15. Poole, A R, Dingle, J T & Barrett, A J, J histothem cytochem 20 (1972) 261. 16. Ramsay, W S, Exp cell res 70 (1972) 129. 17. Berlin, R D & Ukena, T E, Nature 238 (1972) 120. 18. Seeman, P, Pharmacol rev 24 (1972) 583. Received January 13, 1975 Revised version received February 24, 1975
