Leucocyte locomotion and chemotaxis

LEUCOCYTE

LOCOMOTION

AND CHEMOTAXIS

The Influence of Divalent Cations and Cation Ionophores P. C. WILKINSON Department

ofBacteriology

and Immunology, Glasgow, Scotland

University

of Glasgow,

SUMMARY Human blood neutrophil leucocytes and monocytes incubated in the absence of Ca*+ and MgZ’ showed reduced, but still substantial migration into micropore filters towards chemotactic agents, compared with cells migrating in a divalent cation-rich medium. This reduction in migration could he reversed by adding low doses of divalent cation ionophores (X537A or A23 187)to the CaZ+-and Mg2+-free medium which suggests that migrating leucocytes in media depleted of extracellular divalent cations can make use of intracelhrlar divalent cations and that the intracellular cation exchange necessary for locomotion is facilitated by the ionophores. At higher doses, the ionophores inhibited locomotion, as did procaine which reduces membrane permeability to cations. Little effect of K+ depletion or of ouabain on leucocyte locomotion was noted.

The isolation of actin and myosin from neutrophil leucocytes [l, 21 suggests that locomotion of these cells is mediated by contractile protein systems. However, the information is not yet available which would allow detailed comparisons to be made between the molecular mechanisms for leucocyte locomotion and those for muscle contraction. The role of divalent cations in controlling leucocyte locomotion is one thing which would require to be investigated. A recent report [3] showed that, in the absence of extracellular Ca2+, Miss+, or of both, neutrophil leucocytes were still capable of migrating towards a chemotactic source but that the migration was reduced. In this paper, which confirms this finding for both neutrophil leucocytes and for monocytes, the two major phagocytic cell types of mammalian blood, it is also shown that divalent cation ionophores [4-6], at concentrations at which they acExptl Cell Res 93 (1975)

tivate secretory processes in other cells [7-81, inhibit leucocyte locomotion. At lower concentrations they restore to normal the reduced migratory capacity of leucocytes incubated in divalent cation-free media. This suggests that the divalent cation ionophores assist the migrating leucocyte in mobilizing intracellular divalent cations which allow it to migrate efficiently in cation-depleted media. The importance of membrane permeability to cations for locomotion of leucocytes is supported by the fact that substances such as procaine which reduce this permeability [9] also inhibit leucocyte migration. MATERIALS

AND METHODS

Cells Human neutrophil leucocytes and monocytes were derived from fresh heparinized venous blood samples and used within a few hours of removal. The leucocyte preparation was separated from red cells by dextran-

Leucocyte chemotaxis and divalent cations

421

sedimentation of the latter for 1 h, followed by removal of the leucocyte-rich supematant plasma. Neutrophil leucocytes are the only blood cells which can migrate into Micropore filters of 3 pm pore size. Therefore, since these filters were used to measure neutrophil migration, no further separation of neutrophil leucocytes from other blood cells was required. Monocytes were separated from granulocytes by centrifugation over Ficoll-triosil (snec. arav. 1.078). The supematant layer containing monocytes and lymphocytes but with less than 3 % of granulocytes was removed. Depletion of cations. Gev’s solution [lo] without added protein, containing - 1.25~ 10m3 M Caz+ (as CaCl,) and 5.3 x 10V4M Ma*+ (as MaC1.J was used as the test medium. Test cellswere placed-in Gey’s solution from which either one, or both, of the divalent cations were omitted. The cells were prepared by washing three times in divalent cation-free Gev’s solution. One-half was left to stand in the divaleni cation-free 1' 1V lomedium, the other (control) half in medium containing Ca*+ and Mge+. These cells were then left for 90 min at 20°C before being placed in chemotaxis chambers. Fig. 2. Abscissa: molar cont. of procaine HCI; ordiThe control Gey’s solution contains K+ at 5x 10e3M nate: neutrophil migration to alkali-denatured HSA as (as KC1 and KH,PO,). For potassium-depletion ex- percentage of migration of untreated control cells. The effect of procaine on migration of human periments cells in potassium-free medium were prepared using a procedure similar to that described above. neutrophil leucocytes towards alkali-denatured HSA Leucocjtd migration was measured in modified in 3 pm pore size filters. Boyden [ 1l] chambers, in which the cells are placed on the top surface of the micropore falter and a chemotactic agent is placed below the filter. The cells are allowed to migrate into the filter towards the agent at 37°C. Filters of 3 urn pore size (Millinore, Bedford, (a) and (b). In the absence of a stimulant, cells did not Mass.) were used for neutrophil migration; filters of 12 migrate more than 30 pm into the filter even after 3 h urn nore size (Sartorius. Gottinnen. BRD) for mono- and the differences between the groups were small. No bytes. The test was stopped after an appropriate time effect of depletion of divalent cations on unstimulated (75 min for neutrophils; 120 min for monocytes) and random migration was observed, although the ionomigration into the filter was measured by the leading did inhibit this migration. However, since, front method of Zigmond & Hirsch r121, a method in phores which the distance reached by the leading front of cells using chemically stimulated cells, the effects of the procedures tested were similar whether or not a gradiin a fixed time is determined. The migration of these ent was present, it is likely that those effects are cells was shown by the authors to be representative of primarily on cell locomotion itself rather than on the the migration of the cell population as a whole. of locomotion in a gradient. In the absence of a chemoattractant leucocytes mi- orientation The chemoattractants used were alkali-denatured grate slowly and at random. Leucocytes placed in a human serum albumin (HSA) (Behringwerke, Marmedium containing a chemoattractant whose concen- burg, BRD) [13] or whole casein (Merck, Darmtration is uniform throughout show enhanced ran- stadf, BkD). dom migration (chemokinesis) [12]. If placed in a Different batches of cells taken on different days gradient, the movement becomes oriented towards may migrate different distances into a filter towards the source of the gmdient (chemotaxis). The results the same stimulus. Therefore, in order to make comgiven in the tables of this paper are those obtained parisons between different experiments, in tables 1 to 5 with cells migrating through filters in a gradient of and in fig. 1 the actual migration of cells in a chemoattractant. However, the effects of divalent micrometres has been converted into a nercentaae. cations and of cation ionophores have also been The migration of (control) cells towards the chemotactested under the following conditions: (a) cells stimu- tic agent in Gey’s solution containing K+, Ca2+ and lated chemically but in the absence of gradient, i.e. Mg*+ in the absence of added substances affecting cell with equal concentrations of the chemoattractant movement has been taken as 100 and the migration of on both sides of the filter; (b) cells moving against cells in the presence of added substances or in media a gradient; the chemoattractant was placed above lacking in one or other cation has been expressed as the filter, not below; (c) in the absence of a chemoattractant. Under these conditions, the effects seen Migration of test cellsx 100 were similar to those shown in the tables inasMigration of control cells much as migration was inhibited in the absence of extracellular divalent cations, but the inhibition could The figures given in the tables are the mean figures (as be reversed with low doses of ionophores. Higher a percentage) for three filters. In examining 60 doses of ionophores themselves inhibited migration. neutrophil tests, the mean for individual filters differed These effects were most clearly seen under conditions from the overall mean for the three by 5.0% Exprl Cell Res 93 (1975)

d0 “lo1’

422

P. C. Wilkinson

Table 1. Effect of extracellular divalent cations on migration of neutrophil leucocytes Migration towards alkali-denatured HSA (800 &ml) expressed as percentage of controla Cell-suspending medium

Test 1

Test 2

Test 3

Test 4

Gey’s solution containing Ca*+ and Mg2+ Gey’s solution, Ca2+-free, containing MgZ+ Gey’s solution, Mg*+-free, containing CaZ+ Gey’s solution Mg2+- and Ca*+-free Gey’s solution containing Ca2+, Mg2+ and EDTA 10V M EDTA lo+ M EDTA lo+ M EGTA lo-* M EGTA 1O-3M EGTA 1O-4M

100 (120) 82 73 69

100(84) 78 83 68

100 (145)

100(76)

67 46 81 85 56 76 85

68

74

0 Actual figures for controls in pm per 75 min in 3 pm pore size filters given in parentheses. (SD. k3.8). For 60 monocyte tests, the mean for individual filters differed from the overall mean for the three by 6.5% (S.D.+7.9). Thus the scatter in migration of cells moving in different filters using this technique is fairly small and differences of 15-20% between individual figures in the tables are of statistical significance. Reagents. Divalent cation ionophores: X537A was obtained by courtesy of Dr P. G. Philpott, HoffmannLaroche, Welwyn Garden City, UK: A23187 by courtesy of Dr W. E. Brocklehurst, Eli-Lilly, Windlesham, UK. These substances were dissolved in dimethyl formamide and 10 ~1 of this solution was added to 20 ml of the test medium. Dimethyl formamide itself at this dose had no effect on leucocyte migration. Procame HCl, ouabain, EDTA and EGTA were obtained from BDH, Poole, Dorset. The concentration of these test agents was kept uniform on both sides of the filter.

of neutrophil migration. The effect of depletion of divalent cations was reversible on addition of Ca2+ and Mg2+ to the medium. These findings are similar to those of Becker & Showell[3].

Table 2. Effect of A23187 on migration of neutrophil leucocytes in the presence and absence of divalent cations

Cell-suspending medium

Migration towards alkalidenatured HSA (800 M/ ml) expressed as percentage of controla Test 1

Test 2

RESULTS Gey’s solution containing

Effect of varying calcium and CaZ+,Mg2+ and loo (100) No A23187 100W) magnesium concentrations A23187 1O-5M 32 Table 1 shows that neutrophils in media 50 A23187 1O-BM A23187 lo-’ M 58 free of either calcium or magnesium ions A23187 1OV M 104 94 showed a slight reduction (about 20%) of A23 187 1O-gM 104 migration, Neutrophils in medium free of Ca2+-, Mg*+-free Gey’s solution containing both cations migrated still more slowly No A23187 71 77 (about 30% reduction of locomotion). 20 A23187 1O-5M A23 187 1O-6M 58 Nevertheless the locomotion of these neu64 A23187 lo-’ M A23 187 1O-8M 84 90 trophils deprived of extracellular Ca2+ and 96 A23187 lo+ M Mg2+ was still quite good. Adding EDTA or EGTA to the medium also caused a n Actual figures for controls in fl per 75 min in 3 pm diminution but not a complete suppression pore size filters given in parentheses. Exptl Cell Res 93 (1975)

Leucocyte chemotaxis and divalent cations

423

Table 3. Effect of X537A on migration of neutrophil leucocytes in the presence and absence of divalent cations Migration towards alkali-denatured HSA (800 pglml) expressed as percentage of control0 Cell-suspending medium Gev’s solution containinz CaZ+. M& and No X537A X537A IO-’ M X537A 1O-5M X537A lO-B M Ca*+-, Mg*+-free Gey’s solution containing No X537A X537A lO-4 M X537A lo+ M X537A lo-@M X537A lo-’ M

Test 1

Test 2

Test 3

loo (112)

100(56)

100 (78)

5: 100 76 0 46 96

29

79

95 95

99

LI Actual figures for controls in pm per 75 min in 3 pm pore size filters given in parentheses.

Effects of drugs affecting the permeability of membranes to ions The divalent cation ionophores X537A and A23187 are believed to increase the permeability of cell membranes to divalent cations by forming chelate compounds whose hydrophobic character allows them to transfer cations across membranes rapidly [4]. X537A transfers monovalent as well as divalent cations [5] but A23187 has been

shown to transfer Ca2+ and Mg2+ but not K+ [6]. The effects of these ionophores on neutrophil chemotaxis are shown in tables 2 and 3. Both compounds inhibited leucocyte locomotion at high doses (X537A> lop6 M; A23187>10m8 M). This inhibition was independent of the presence of Ca2+ or Mg2+ in the medium. At lower doses, i.e. A23187 at lo-* M to 1O-9M and X537A at lO-‘j M to lo-’ M, the ionophores en-

Table 4. Effects of extracellular potassium and ouabain on migration of neutrophil leucocytes Migration towards casein (500 pg/ml) expressed as percentage of control’ Cell-suspending medium Gey’s solution containing K+ No ouabain Ouabain 10m3M Ouabain 1OP M Ouabain lo+ M Gey’s solution, K+-free No ouabain Ouabain 10m3M Ouabain 10e4M Ouabain lo+ M

Test 1

Test 2

Test 3

Test 4

100 (70) 90 97 96

100 (91) E

100 (84) 96

100(68) 91

82 85 E

92 113 98 88 104

107 107

0 cell death 0

D Actual migration of control cells in pm per 75 min in 3 pm pore size filters given in parentheses. Exprl Cell Res 93 (1975)

424

P. C. Wilkinson

hanced the locomotion of neutrophils migrating in Caz+- and Mg2+-free medium. The presence of these drugs in the Ca2+and Mg2+-free medium caused a restoration of the migration of divalent cationdepleted leucocytes to values similar to those seen in divalent cation-rich media. A23187 showed these effects at lower concentrations than X537A (tables 2 and 3) which is consistent with previous reports that its ability to bind calcium is stronger than that of X537A and with previous observations of the effects of these drugs on mast cells and platelets [7, 81. The ionophores were shown not to act themselves as chemotactic agents for neutrophils. Procaine and other local anaesthetics have been reported to reduce the permeability of cell membranes to cations [9]. Fig. 1 shows that addition of procaine to the medium had a substantial inhibitory effeet on neutrophil migration.

Table 5. Migration oj’ human blood monocytes: Effects of divalent cations and cation ionophores

Cell-suspending medium Gey’s solution containing Ca*+ and Mg*+ +XS37A IO+ M + X53lA lo+ M +X537A IO-’ M +A23187 to-’ M +A23187 1O-8M Ca2+-, Mg*+-free Gey’s solution +XS37A 1O-5M +X537A 1O-6M + XS37A lo-’ M +A23187 IO-’ M +A23187 1O-8M Gey’s solution+procaine ~xIO-~ M Gey’s solution+EGTA ~xIO-~M

Migration towards alkalidenatured HSA (800 pg/ ml) expressed as percentage of controla Test I 100(62) 55 81 9.5 87 60 82 98

Test 2 -__100(89) 81 99 58 108 76 84 99 86 90

37 65

85

n Actual migration of control cells in @rnper 120min in 12 pm pore size filters given in parentheses.

Effect of varying potassium concentration medium. The results shown in table 4 fail Table 4 shows the results of four experito confirm this; ouabain had a negligible ments in which neutrophil migration was effect on neutrophil migration which was tested in media containing or lacking K+ not affected by K+ concentration. On the ions. In three of these experiments, K+ other hand, they support the observation depletion had no effect on leucocyte migraof Ramsey & Harris [16] of a lack of effect tion, in the fourth, the cells in K+-free of ouabain on the migration of leucocytes medium were grossly disorganised morphousing a slide-and-coverslip technique. logically and did not migrate. Ouabain has been reported to block the potassium pump in neutrophils at concentrations of about Studies of migration of human 10m4M [14]. Potassium exchange is believed blood monocytes Table 5 summarizes the results of experito be associated with an ouabain-inhibitable acyl phosphatase in the cell mem- ments similar to those described above but brane, not with Na+K+ATPase, an enzyme using human blood monocytes instead of which is lacking from neutrophil mem- neutrophils. Monocytes, like neutrophils, branes [14]. Ouabain has also been reported showed a slight diminution of migration in to inhibit neutrophil chemotaxis [15], an media containing no divalent cations, or effect which was reported to be reversed containing EGTA, but migration was by by adding K+ ions to the cell-suspending no means completely abolished under these Exprl Cell Res 93 (1975)

Leucocyte chemotaxis and divalent cations

425

intracellular sources of cations when migrating in a cation-depleted medium. The effects of the divalent cation ionophores, A23187 and X537A on leucocyte migration are therefore of special interest. At doses at which these drugs cause histamine release from mast cells [7] and platelet secretion [8], they inhibit leucocyte migration. Histamine release and platelet secretion may be ‘once-off events in which all that is necessary is an influx of divalent cations into DISCUSSION the cytoplasm inducing a single contracIt is probable that the reduced migration of tile event. Under such conditions a migratdivalent cation-depleted leucocytes which ing leucocyte may go into ‘rigor’ unless some mechanism is operating to allow a is restored by low doses of ionophores, and the inhibition of migration by high continued regulated influx and efflux of doses of ionophores are due to effects on cations. The inhibition of migration by the locomotion itself rather than on the way the ionophores may therefore be due to the inlocomotion is oriented. Leucocytes contain duction of rigor in leucocytes. Lower doses of ionophores, which are not themboth microfilaments and microtubules, both of which would be expected to be selves inhibitory to movement, increase the Ca2+ sensitive, and microtubules are re- migratory capacity of leucocytes depleted quired for polarization of migrating leuco- of Ca2+ and Mg2+ and, in fact, restore it cytes [18]. Effects on cell orientation can- to the levels achieved by cells migrating in not therefore be dismissed, but were not cation-rich media. Since, under such condithe primary effects seen here. It seems un- tions, the ionophores cannot act to inlikely that the ionophores could be acting crease the efficiency of cation exchange extracellularly, e.g. by binding to the from outside the cell, it seems likely that chemoattractant protein, since the latter they must be acting on cation exchange bewas present in considerable molar excess tween the cell cytoplasm and an intra(about 10V5M whereas the ionophores were cellular cation store. The presence of active at 10MgM). In any case, A23187 vesicles which take up calcium within the at 10e6M inhibits random migration of neu- leucocyte cytoplasm has been reported by trophils in the absence of a chemoat- Woodin & Wieneke [17] but evidence that intracellular cation transfer was important tractant. The migration of neutrophil leucocytes for leucocyte migration has previously and monocytes is diminished in media lack- been lacking. This paper presents prelimiing in Ca”+ and Mg2+ but the diminution is nary data suggesting that this is true but surprisingly slight, judged by the observa- leaves open many questions including the tions in this paper and that of Becker & individual roles of Ca2+ and Mg*+ ions in Showell[3]. This suggests that if, as seems this process. likely, leucocyte locomotion is dependent on contractile events regulated by cation fluxes, the cells may make efficient use of This work was supported by a grant from the M.R.C. conditions. The effects of divalent cation ionophores (inhibition of migration at high doses, restoration of the migratory capacity of divalent cation-depleted cells at lower doses) were similar with monocytes to those observed using neutrophils. Likewise, the inhibitory effect of procaine on monocyte migration was similar to the effect on neutrophils.

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P. C. Wilkinson REFERENCES

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Exptl Cell Res 93 (1975)

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