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J. Cell Sci. 16, 167-180 (1974)
Printed in Great Britain
THE AGGREGATION OF RABBIT
POLYMORPHONUCLEAR LEUCOCYTES:
EFFECTS OF ANTIMITOTIC AGENTS,
CYCLIC NUCLEOTIDES AND
METHYL XANTHINES
J. M. LACKIEStrangeways Research Laboratory, Wort's Causeway, Cambridge, England
SUMMARY
The aggregation of rabbit peritoneal exudate polymorphonuclear leucocytes (PMNs) wasinvestigated by following total particle number with time, in a shaken suspension. Aggregationwas rapid and essentially complete by 30 min, but could be inhibited by microtubule-blockingagents, adenosine and methyl xanthines. The addition of cyclic adenosine 3':5'-mono-phosphate (cAMP), or its dibutyryl derivative, also inhibited aggregation slightly, butguanosine nucleotides had no significant effect. By consideration of the maximum inhibitionbrought about by colchicine or theophylline alone, or the two in combination, it appears thatthey act at different sites to inhibit aggregation, though the effects of either were partiallyreversed by the addition of adenine. Adenine, lumicolchicine and high concentrations ofvinblastine sulphate (io~* M), enhanced aggregation.
INTRODUCTION
Polymorphonuclear leucocytes (PMNs) are a convenient cell type for a generalstudy of cell-cell adhesion, they can be obtained in large numbers, and much is knownabout their general cell biology. An additional point of interest is their role in in-flammation, involving as it does the transition from the circulating non-adhesivePMN, to the adhesive PMN migrating up a chemotactic gradient. A variety ofmethods have been employed to investigate the adhesion of PMNs to substrates invitro (Garvin, 1968; Bryant & Sutcliffe, 1972), and Berlin & Ukena (1972) investigatedthe agglutination of PMNs by Concanavalin A, which is inhibited by vinblastine andcolchicine. An estimate of the strength of cell-cell adhesion is provided by observingthe rate and extent of aggregation of cells in a shaken suspension. Aggregation canbe measured by following the decline in total particle number with time, an operationmost conveniently done using an electronic particle counter (Edwards & Campbell,1971).
Microtubule-blocking agents such as colchicine and vinblastine are known to affectcell locomotion (Vasiliev et al. 1970; Ramsey & Harris, 1973), the rate of certaintransport processes in PMNs (Berlin, 1973), the state of certain membrane components(Yahara &Edelman, 1973; Berlin, Oliver, Ukena & Yin, 1974), but not the adhesionof cells to glass (Weiss, 1972). It has, however, been reported that the aggregation of
168 J. M. Lackie
BHK-21 Clone 13 cells is inhibited by these agents (Waddell, Robson & Edwards,J974)-
In some systems it has been proposed that cyclic 3':5'-adenosine monophosphatewill reverse or inhibit the effects of microtubule-blocking agents (Hsie & Puck, 1971;Roisen, Murphy & Braden, 1972; Kirkland & Burton, 1972), and that the converseis also true (Kram & Tomkins, 1973). The effects of antimitotic drugs, methyl xan-thines, cyclic nucleotides, adenine and adenosine, both alone and in combination, onthe aggregation of rabbit PMNs, are described here.
MATERIALS AND METHODS
Cells
New Zealand White rabbits were injected intraperitoneally with 350-40x21111 of 154 mMNaCl containing 01 % oyster glycogen (Sigma Ltd), and the fluid was drained off after 4 h.At this time the exudate consists mainly of PMNs (up to 95 %). The exudate was collectedinto plastic beakers with heparin (Evans Medical Ltd) present to give a final concentration ofapproximately 10 units/ml, and then filtered through surgical gauze to remove fibrin clots.
Cells were either used immediately or stored at 4 CC in the exudate fluid, under whichconditions viability remains high for up to 3 days. Prior to use, the cells were washed in calcium-and magnesium-free Hanks' HEPES solution with 1 mM EDTA (ethylenediaminetetra-acetate) at pH 8-o, and then washed twice in normal Hanks' HEPES. If necessary, red bloodcells in the exudate were lysed by suspending the cell in 30 % Hanks' for 1 min. Cell viabilitywas tested by trypan blue dye exclusion.
Solutions
Hanks' salts without bicarbonate (obtained in powder form from Flow Laboratories, Scot-land) were made up in glass-distilled water buffered to pH 7-6 with 5 mM HEPES (iV-2hydroxyethylpiperazine N'-z ethanesulphonic acid) and this solution (Hanks' HEPES) usedboth for washing cells and as the aggregation medium.
Aggregation
Cell suspensions were placed either in 25-ml siliconed conical flasks or in plastic scintillationvials, the former with 4 ml of initial solution, the latter with 2 ml. Flasks were shaken at 90 rev/min and vials at n o rev/min, in a reciprocating shaker with a 4 m"1 stroke and at a temperatureof 37 °C. Total particle number at various times was estimated by removing an aliquot ofo-2 ml with an 'Oxford' micropipette, the aperture of which had been enlarged to approxi-mately 2 mm diameter. This sample was diluted in 10 ml of cold filtered 154 mM NaCl andcounted on a model 401 Celloscope (Ljungberg & Co., Stockholm). The counter was fittedwith a ioo-/»m aperture tube, the amplifier setting was 10 units, and the discriminator setting40 units. Only particles with a diameter greater than 6 fim were registered, and coincidencecorrections were made when appropriate.
Chemicals
Adenosine 3':5'-cyclic monophosphoric acid (cAMP), A^.C-dibutyryl adenosine 3': 5'-cyclic monophosphate (dibutyryl cAMP), guanosine 3':5'cyclic monophosphate (cGMP),N1^1' dibutyryl cGMP, colchicine, theophylline (1,3-dimethyl xanthine), caffeine (1,3,7-trimethyl xanthine), theobromine (3,7-dimethyl xanthine), adenine and adenosine, were allobtained from Sigma Ltd (London). Vinblastine sulphate was obtained from Eli Lilly Co. Ltd.Lumicolchicine was prepared according to the method of Wilson & Friedkin (1966).
Aggregation of rabbit PMNs 169
Presentation of data
The data have, in general, been normalized with respect to the internal control, and theparticle ratio (JR() defined as
Nmt treatmentN^t control '
where Nml is the total number of particles at time t. A particle ratio greater than one indicatesan inhibition of aggregation in the experimental flasks. Comparisons have been made usingpairs of results, each pair from a separate set of flasks. Using paired comparisons, the mostappropriate statistic was considered to be z, the mean difference between the particle ratios of2 treatments, even when one of the particle ratios was by definition unity, when comparinga treatment with the control. Comparing treatment 1 and treatment 2:
N
st = i/n£ (Rti-Rn) for «'1
pairs of flasks at time t. A positive mean difference indicates that the amount of inhibition dueto the first treatment is less than the inhibition due to the second treatment, or when the com-parison is between the control and a treatment, a positive mean difference indicates that thetreatment inhibits aggregation. When calculating z only pairs of flasks are used and the valuesfor z (control vs colchicine) in Tables 1, 3 and 7 differ since the pairs have been drawn fromdifferent sets of flasks according to the other comparisons being made.
Confidence interval estimates (c.i.) were calculated according to the formula
S.
where *S, is the standard deviation of z, n the number of observations, and t is obtained fromthe appropriate statistical table ( 5 % probability level, n— 1 degrees of freedom).
RESULTS
In general the aggregation of these cells was extremely rapid, and was almost com-plete by 30 min. Aggregation rate depends, in this system, upon the number ofcollisions, and the rate is therefore affected both by the initial cell concentration, andby the concentration at any particular instant. The kinetics are complex, but for manypurposes it is sufficient to realize that comparison between aggregations carried outon different days is unlikely to be valid, and to restrict comparisons accordingly.These limitations were additionally appropriate since the variation observed betweenbatches of cells was very marked. The precise cause of this variation is obscure, andthere appeared to be no correlation with the age of the experimental animal or thenumber of times that the animal had been used. Use has therefore been made of pairedcomparisons, though wherever possible muliple replicates within an experiment werecompared. For purposes of illustration, averaging over a number of experimentssmoothes the curve, but the average has little predictive value, and it is inappropriateto affix a standard deviation. The normal pattern of aggregation is shown in Fig. 1.
Colchicine
The addition of colchicine, even at 5 XIO~7M, inhibited aggregation and theresponse to different levels of colchicine is shown in Table 1 and Fig. 2. Concentra-tions greater than io~6 M appeared to have no further effect. The effect of colchicinewas rapid, a degree of inhibition being obvious within 1 min of adding colchicine
J. M. Locke
150
125 -
100
Si 75
« SO
10 15 20 25Time, mln
30 60
Fig. i. Decline of total number of particles per ml (Nmi) with time for untreatedPMNs in Hanks' HEPES. The figure was based on 14 sets of data selected, fromthe much larger set of control aggregations, on the basis of the similarity of their originalcell concentrations. As explained in the text, it is unjustifiable to put s.D. bars on thepoints.
Table 1. The effects of various concentrations of colchicine onthe aggregation of PMNs
Concentrationof colchicine,
M
Meandifference
5 % C.I.No. of
observations
2-5 x io~7
5 0 x io~7
125 x io~25 x 10"'5-0 x io~8
125 x 10-2-5 x io~5
S o x 10-5
125 x 10"2-5 x io"4
5 x 10-4
0085 ±01500-374 ±0209O-457 ±0172°455 ±0055O'6oi ±O'2IO°'599± 0-2280-598 ±0-1930648 ±0143o-644±o-22io-66o ±0-2290-654 ± 0-308
12
12
11
911
12
11
11
11
10
10
The mean differences between controls and colchicine-treated cells at 30 and 60 min havebeen combined, and are given ± 5 % confidence interval (c.i.). All concentrations, with theexception of 2-5 x io~7 M, cause a significant inhibition of aggregation (i.e. give a positivemean difference greater than zero).
Aggregation of rabbit PMNs 171
Table 2. The inhibitory effect of colchicine following addition at time zero
Time,z±S% c.i.
No. ofobservations
I
2
35
0-094 ±0-0570-078 ±0061008710-0530-07910-063
o-oi0-05o-oi0 0 5
6666
P is the probability that the null hypothesis (of no mean difference) is correct. Colchicinewas added to give a concentration of 2-5 x io~* M.
10 r
0 9
0 8
0 7
u 0-6o
o(J
2 0-5"o
8 0-4
0-3
0-2
01 -
10-7 10"' 10"' 10-4 10-'
Colchicine cone, M
Fig. 2. Inhibition of aggregation by increasing concentrations of colchicine; notethat the abscissa is plotted on a logarithmic scale. The figure is based on the data inTable 1, and the points are shown with 5 % interval estimates.
(Table 2). This represents the lower limit of resolution of the assay, since aggregationdepends upon collisions and requires some time to occur. The addition of colchicineto cells which have been allowed to aggregate for 30 min leads to an increase in totalparticle number, implying that the aggregation, at this stage at least, was readilyreversible (see Fig. 3). There was no indication of cell mortality with this or any ofthe other treatments.
172 J. M. Lackie
Colchicine will undergo a photochemical rearrangement, and the products, /? and ylumicolchicines, do not bind to the microtubule monomer (tubulin) (Wilson & Fried-kin, 1966), though they will bind non-specifically to membranes (Berlin, 1973). Ascan be seen from Table 3, lumicolchicine enhances rather than inhibits aggregation.Vinblastine sulphate, which binds to a different site on tubulin, will also inhibit theaggregation of PMNs, though the effect is rather more complex than with colchicine.High levels (io^1 M) of vinblastine sulphate markedly enhanced aggregation, whilstlow concentrations (icr7 to I O ^ M ) inhibited.
175r
15 20 25 30 35 40
Time, min45 50 55 60
Fig. 3. The effect of adding colchicine (2-5 x io~' M) to PMNs which have beenallowed to aggregate for 30 min. # , control; O> with colchicine, A, the meandifference (in units of total particle number) between colchicine-treated andcontrol flasks. Bars indicate S.E. for the total particle number, and the 5 % confidenceinterval for mean difference.
Cyclic nucleotides
Cyclic AMP is involved in the mediation of many cellular processes (Kram, Mamont& Tomkins, 1973), and of particular interest is the probable involvement of cAMPin platelet aggregation (Mills & Smith, 1971; Shio & Ramwell, 1972). When used atconcentrations comparable to those producing marked effects on other systems, bothcAMP and the dibutyryl derivative inhibit aggregation slightly (Table 4). Theeffects of cGMP and dibutyryl cGMP, which may be antagonistic to cAMP (Hadden,Hadden, Haddox & Goldberg, 1972; Kram & Tomkins, 1973) were negligible, and
Aggregation of rabbit PMNs 173
probably not significant (Table 4). The addition of exogenous cyclic nucleotides, inthe absence of any inhibition of the phosphodiesterases or stimulation of synthesis,may make little difference to the intracellular concentration, a factor which shouldbe borne in mind.
Table 3. The effects on aggregation of PMNs of colchicine at 5 XIO~6M, vinblastinesulphate at high ( I O ^ M ) and low (icr7
M) concentrations, and the photo-inactivatedform of colchicine, lumicolchicine, at 5 XIO~BM
Comparison
Control vs CControl vs L-CColchicine vs L-CControl vshigh VLB
Control vslow VLB
Low VLB vshigh VLB
z « ± 5 % C i .
+ 0123 ±0-137— 0203 ±0082+ 0-312 ±0-081— 0267 ±0050
+ 0-155 ±0-085
+ 0-422 ±0065
No. ofobserva-
tions
888
1 0
1 0
1 0
2« ± 5 % C.I.
+ 0169 ±0-083-0-305 ±0-075+ 0-475 ±0036-0-553 ±0-050
+ 0267 ±0119
+ 0819 ±0-150
No. ofobserva-
tions
888
15
15
IS
The mean difference at 30 min {z^) and 60 min (zM) is shown with a 5 % interval estimate.A negative value of z indicates that aggregation was enhanced.
C, colchicine; L-C, lumi-colchicine; VLB, vinblastine sulphate.
Table 4. The effects of the addition of cyclic nucleotides on theaggregation of PMNs
Treatment
Control1 mM cAMP0-5 mM dibutyryl
cAMP1 mM cGMP05 mM dibutyrylcGMP
N M 0 ± S . E .( x 10-1)
T23'2± I Oii7-8± 1-4ii9-8± 15
ii5'2± 1-4118-5 ±2-0
Nto30±S.E.(xio-1)
368 ±2-24S'S±3-3t50-8 ±3-2*
43'7±2-2f40-2 ± i-4f
NtoM±S.E.( x 10-1)
277 ±3-°37-2 ±4-649'3 ±3'4**
347 ±2-832-3 ±3'4
Since the comparison was within an experiment paired comparisons have not been used,rather the mean total particle number per ml at time t min (Nroi)±s.E.; 6 replicates of eachtreatment were run. P is the probability that the null hypothesis, of no difference from thecontrol, is correct.
f o-i > P > 005; * P < o-oi; • • P < 0001.
Theophylline
Theophylline is commonly used in conjunction with cAMP or dibutyryl cAMPand the major effect is normally assumed to be on the cAMP phosphodiesterase. Theeffect of theophylline alone on the aggregation of PMNs was drastic, though relatively
174 J- M. Lackie
high concentrations have been used. The inhibition brought about by i mM theo-phvUine, the concentration commonly used in conjunction with cAMP, was of thesame order as the maximum inhibition produced by colchicine, but the maximumlevel of inhibition approached 90 % as the concentration of theophylline was increased.Caffeine was similarly effective; the effect of theobromine was probably limited by its
Table 5. The inhibitory effect of methyl xanthines on aggregation of PMNs
No. of No. ofobserva- observa-
Treatment Z&, 1 5 % C.I. tions 2K ± 5 % c.l. tions
1 mM Theophylline 0-5010-14 7 0-7710-23 81 mM Caffeine 0-5910-07 8 1-0710-25 81 mM Theobromine 0-2310-07 8 0-4210-19 85 mM Theophylline 1-6910-35 6 2-5810-45 65 mM Caffeine 2-0910-23 6 3-3110-30 6
Abbreviations as in Table i, p. 170.
Table 6a. Reversibility of the theophylline-induced inhibition of aggregation
Time,min s i 5% c.i.
o 3-6718-7530 15-8317-506 0 21-67 1 II-O2
Groups A and B were both aggregated in 15 mM theophylline for 30 min, washed andthen resuspended: Group A in normal Hanks' HEPES, Group B in 15 mM theophylline.Group A, in Hanks'-HEPES, aggregate more than Group B. The mean difference is in unitsof (Nmtx io~*). There were 6 replicates in both Group A and Group B.
Table 6 b. The effect of adding 2 mM theophylline after cells havebeen allowed to aggregate in normal Hanks' HEPES for 30 min
The mean difference between the particle number (N^tX io"4), immediately after additionand after 30 min in theophylline.
• s i 5 % c.i. = 6-6 1 286:71 = 9
low solubility. Table 5 summarizes these results. Methyl xanthines are known toinhibit platelet aggregation (Bygdeman & Johnsen, 1971), and the effect is supposedto be mediated through intracellular cAMP.
Theophylline, like colchicine, reversed preformed aggregations (Table 6) and itsinhibitory action was reversible.
Adenine and adenosine
Because of the structural similarity between methyl xanthines and purines, bothadenine and adenosine were tested. The results were somewhat surprising in that
Aggregation of rabbit PMNs 175
adenine will enhance aggregation of PMNs, whereas adenosine inhibits, as it doeswith platelets (Mills & Smith, 1971). At 30 min, the effect of adenine was less markedthan at 60 min, suggesting that the time course was different from that of theophylline.Adenosine was effective in inhibiting at 30 min, but at 60 min no inhibition wasobserved, probably because the adenosine had been taken up by the cells (Table 7).
Addition of adenine partially reversed the effects of both theophylline and colchicine(Table 7), suggesting that there might be some common intermediate.
Table 7. A comparison of the effects of colchicine (1-25 XIO~*M) , theophylline (1 miw),adenine (1 WJM) and adenosine (1 m ) , both alone and in combination
Comparison
Control vs colchicineControl vs theophyllineControl vs adenineControl vs adenosineControl vs colchicine +adenine
Control vs theophylline+ adenine
Colchicine vs colchicine+ adenine
Theophylline vs theophylline+adenine
*so±5%C.I.
0-409 ±00840-361 ±0-071
— 0-083 ±0-1080-094 ±00490102 ±0-130
0-083 ±0126
-o-3O7±o-i33
— 0-246 ±0-106
"so
11
16
171511
17
1 1
16
*»±5%C.I.
0381 ±03180269 ±0067
— 0217 ±0088o-ooi ±0-0790-046 ±0-258
— O-O4S ±O-I22
— O45O ± 0 2 6 2
-o-339±oi3i
" 6 0
71 2
17
IS7
13
6
1 1
The mean difference between particle ratios at 30 min («M) and 60 min (sM) is given ± 5 %confidence interval (c.i.) A positive value of z indicates that the second treatment (of the com-parison) inhibited aggregation, a negative value indicates enhancement. Only adenine at t^ andadenosine at tM failed to produce a significant difference from the control value (i.e. 2 is notsignificantly different from zero). The number of pairs used in the comparison (n) is indicated.
Theophylline and colchicine
By analysis of the effect of saturating levels of both theophylline and colchicine, itshould be possible to determine whether there is a common site of action. Theobservation that the maximum level of inhibition obtained with colchicine differsfrom that of theophylline, might be thought to make this improbable from the outset;it could, however, be explained on the basis of 2 different sites, one for theophyllinealone, one for either theophylline or colchicine.
Because of the difficulty of getting complete saturation with theophylline, theanalysis has been based on curves fitted to data on different concentrations of theo-phylline, and the same concentrations with the addition of a saturating amount ofcolchicine (Fig. 4). On the assumption that the form of the curve was best fitted byan equation of the form: y = a + b{rY then the asymptotic value a (the concentrationrequired to give maximum inhibition) can be determined. Asymptotic values fromseveral experiments, in each of which a complete range of both treatments (theophyl-line with and without colchicine) was carried out, have been compared using a paired ttest (Table 8 and Fig. 5). The results indicate that there is probably no interaction,
176 J. M. Lackie
2-2
20
1-8oCv 1-6
1-4
1-2
1010
Theophylline cone, mM
15 20
Fig. 4. Particle ratio (see text) at 30 min for flasks with different concentrations oftheophylline, with and without (A and A, respectively) the addition of a saturatingamount of colchicine (125 x io~* M). The data for one set of theophylline concentra-tions only is plotted together with the computer-fitted curve.
Table 8. A comparison of the effects of different concentrations of theophylline'jvith andwithout the addition of a saturating amount of colchicine (1-25 x io"4 M), and a comparisonof the asymptotic values (see text)
Concentrationof theophylline,
mM
1
51 0
15170 0 *
*30±5%C.I.
0409 ±o-mO'22O±Oo8l0-196 ±0-0660090 ±0-1530038 ±0-175o-533 ±0-511
15
15151368
*«o±5%c.i.
o-593 ±0-385O-3II ±O-I22O-I9O ± 0 1 4 3O-I23 ±O-I9O
0-259 ±0-487
"60
I I
I I
I I
9—
7
The asymptotic values for the 'theophylline' and 'theophylline + colchicine' curves, indicatethe maximum inhibition that would have been obtained, had the concentration of theophyllinebeen increased sufficiently. If the values of zm and zm for the asymptotes are combined, thenz = 0-405 ±0316, indicating that the null hypothesis, of colchicine having no further effectat maximal theophylline concentrations, has approximately 2 % probability of being correct.Abbreviations as for Table 7, p. 175.
Comparisons: theophylline vs theophylline + colchicine.• i.e. asymptotic value.
Aggregation of rabbit PMNs 177
and that colchicine continues to inhibit, even at maximum concentrations oftheophylline.
Two observations point to the independence of the colchicine and theophyllineeffects, the marked difference between the maximum inhibition which can be pro-duced by saturating doses of either drug, and the continued inhibitory effect ofcolchicine at high theophylline concentrations.
07 r
« 0-6
8 0-5
T. 0-4.ca.2* 0-3
£
I- 0-2a.I& 0-1
10
Theophylline cone, mM
15 20
Fig. 5. The mean difference (z) between flasks with theophylline alone and with boththeophylline and colchicine (as in Fig. 4), at different concentrations of theophylline.The figure is based upon the data in Table 8, but the values of zM and zK have beencombined.
Table 9. Total particle number (Nxt) ± S.D. at 3 different times (30, 60 and 75 min)in flasks with theophylline, adenine. or both
Treatment 10-4)
N006O ± S.D.
( X IO"«)<c76
Control17 mM theophylline17 mM theophylline+adenine
1 mM adenine
3991046 ± 56io6'4± 2'8
18-0 ±4'i
38-911501063 ±2-5108-4 ±2-7
12-4 ±4-6
39-4 ± 168107-4 ±4-3no -o l 5-6
i3-o±3-8
These results were from a single experiment with 6 replicates of each treatment, and themean difference method is therefore unnecessary. The results for theophylline with andwithout adenine show a mean difference of 2-i6±2'70, not significantly different from zeroat the S % level.
C EL 16
178 J. M. Lackie
Theophylline and adenine
A simpler method was used to study the interaction of theophylline and adenine.Theophylline, at approximately the saturating level, was used to inhibit aggregationwith or without adenine present, and the results compared within a single experiment(Table 9). It appears that adenine competitively inhibits the theophylline effect,since it was ineffective when the concentration of theophylline was high. Adenine israther insoluble, which restricts the design of these experiments.
DISCUSSION
From the above results, it appears that aggregation can be inhibited by micro-tubule-blocking agents such as colchicine and vinblastine, and by increasing the intra-cellular concentration of cAMP, though the mechanisms are independent. Whethermethyl xanthines act solely by inhibiting the cAMP phosphodiesterase, or by a com-bination of this and some other mechanism, is not clear. If decreasing the intracellularconcentration of cAMP enhances aggregation, then this would explain the effect ofadenine in competing with, and reversing the effect of theophylline, though there isno evidence to support this hypothesis.
Internal cAMP levels can be increased in 3T3 cells by serum starvation or treat-ment with dibutyryl cAMP and theophylline; however achieved, this leads to areduced rate of transport of uridine, leucine and 2-deoxyglucose (Kram et al. 1973).Adenosine also inhibits transport in 3T3 cells, probably through the same mechanism.Colcemid and vinblastine, though they do not affect the cAMP concentration, willrelieve the inhibition of transport.
Thus if metabolic energy were required for aggregation, the inhibition of transportmight cause a subsequent inhibition of aggregation. This explanation is, however,inadequate since colchicine should release the inhibition of transport and therebyantagonize the effects of theophylline.
Methyl xanthines, cAMP and microtubule blocking agents all inhibit the plateletrelease reaction (Mustard & Packham, 1970), and also affect the release of variouslysosomal enzymes from PMNs (Weissmann, Dukor & Zurier, 1971; Goldstein,Hoffstein, Gallin & Weissmann, 1973.) The observation that theophylline andcolchicine appear to operate at different sites to effect the inhibition of aggregation,suggests that the search for a common mechanism is probably futile.
It is by no means obvious how colchicine could affect adhesiveness though Waddellet al. (1974) propose 3 schemes. Changes in gross surface topography, involving theloss of microvilli, might affect the ease of close approach, and hence adhesiveness, ifthe arguments of Bangham & Pethica (i960) are accepted. Alternatively, microtubulesmay be involved in maintaining clusters of adhesive sites on the cell surface, the dis-persal of these local patches, following colchicine treatment, leading to a weakeningof cell-cell adhesions. The scheme which Waddell et al. favour, involves the loss ofpolarity in the insertion of new membrane, putatively the only adhesive surface, byanalogy with the loss of locomotory polarity in colchicine-treated cells (Vasiliev et al.
Aggregation of rabbit PMNs 179
1970). That microtubules are indeed involved is supported by the observation thatboth colchicine and vinblastine are effective, whereas lumicolchicine is not, but therole of microtubules in adhesion remains uncertain.
The other effects may then be ascribed to some change consequent upon anincrease in the intracellular concentration of cAMP, and the analogy with plateletaggregation is obvious. Since both platelets and PMNs respond to inflammatorystimuli, which may be mediated through prostaglandins and cAMP (Williams &Morley, 1973), this would not be too surprising, though extrapolation from in vitrostudies to the situation in vivo is dangerous.
I would like to thank the Eastwood Memorial Foundation and Trinity Hall, Cambridge, forfinancial support, Mr E. M. Walters, A.R.C. Statistics Unit for help with the regressionanalysis, Mr M. Abercrombie for reading the manuscript, and many of my colleagues forhelpful discussions.
REFERENCESBANGHAM, A. D. &PETHICA, B. A. (i960). The adhesiveness of cells and the nature of the chemi-
cal groups at their surfaces. Proc. R. phys. Soc. Edinb. 28, 43-52.BERLIN, R. D. (1973). Temperature dependence of nucleoside membrane transport in rabbit
alveolar macrophages and polymorphonuclear leucocytes. J. biol. Chem. 248, 4724-4730.BERLIN, R. D., OLIVER, J. M., UKENA, T. E. & YIN, H. H. (1974). Control of cell surface
topography. Nature, Lond. 247, 45-46.BERLIN, R. D. & UKENA, T. E. (1972). Effect of colchicine and vinblastine on the agglutination
of polymorphonuclear leucocytes by Concanavalin A. Nature, Netv Biol. 238, 120—122.BRYANT, R. E. & SUTCLIFFE, M. C. (1972). A method for quantitation of human leucocyte
adhesion to glass. Proc. Soc. exp. Biol. Med. 141, 196-202.BYGDEMAN, S. & JOHNSEN, O. (1971). Methylxanthines in the inhibition of platelet aggregation.
Ada med. scand., Suppl. 525, 179-180.EDWARDS, J. G. & CAMPBELL, J. A. (1971). The aggregation of trypsinized BHK-21 cells.
J. Cell Sd. 8, 53-71-GARVIN, J. E. (1968). Effects of divalent cations on adhesiveness of rat polymorphonuclear
neutrophils in vitro, jf. cell. Physiol. 72, 197-212.GOLDSTEIN, I., HOFFSTEIN, S., GALLIN, J. & WEISSMANN, G. (1973). Mechanisms of lysosomal
enzyme release from human leucocytes: microtubule assembly and membrane fusioninduced by a component of complement. Proc. natn. Acad. Sd. U.S.A. 70, 2916-2970.
HADDEN, J. W., HADDEN, E. M., HADDOX, M. K. & GOLDBERG, N. D. (1972). Guanosine3:5"Cyclic monophosphate: a possible intracellular mediator of mitogenic influences inlymphocytes. Prcc. natn. Acad. Sd. U.S.A. 69, 3024-3027.
HSIE, A. W. & PUCK, T. T. (1971). Morphological transformation of Chinese hamster cellsby dibutyryl adenosine 3': s'-monophosphate and testosterone. Proc. natn. Acad. Sci. U.S.A.68, 358-361.
KIRKLAND, W. L. & BURTON, P. R. (1972). Cyclic adenosine monophosphate-mediated stabili-zation of mouse neuroblastoma cell neurite microtubules exposed to low temperature. Nature,New Biol. 240, 205-207.
KRAM, R., MAMONT, P. & TOMKINS, G. M. (1973). Pleiotypic control by adenosine 3':5'-cyclicmonophosphate: a model for growth control in animal cells. Proc. natn. Acad. Sci. U.S.A.7*>, 1432-1436.
KRAM, R. & TOMKINS, G. M. (1973). Pleiotypic control by cyclic AMP: interaction withcyclic GMP and possible role of microtubules. Proc. natn. Acad. Sci. U.S.A. 70, 1659-1663.
MILLS, D. C. B. & SMITH, J. B. (1971). The influence on platelet aggregation of drugs thataffect the accumulation of adenosine 3':s'-cyclic monophosphate in platelets. Biochem. J.121, 185-196.
180 J. M. Lackie
MUSTARD, J. F. & PACKHAM, M. A. (1970). Factors influencing platelet function; adhesion,release, and aggregation. Pharmac. Rev. 22, 97-187.
RAMSEY, W. S. & HARRIS, A. (1973). Leucocyte locomotion and its inhibition by anti-mitoticdrugs. Expl Cell Res. 82, 262-270.
ROISEN, F. J., MURPHY, R. A. & BRADEN, W. G. (1972). Dibutyryl cyclic adenosine mono-phosphate stimulation of colcemid-inhibited axonal elongation. Science, N.Y. 177, 809-811.
SHIO, H. & RAMWELI., P. (1972). Effect of prostaglandin E2 and aspirin on the secondaryaggregation of human platelets. Nature, Netv Biol. 236, 45-46.
VASILIEV, J. M., GELFAND, I. M., DOMNINA, L. V., IVANOVA, O. Y., KOMM, S. G. &OLSHEVSKAJA, L. V. (1970). The effect of colcemid on the locomotory behaviour of fibroblasts.J. Evibryol. exp. Morph. 24, 625-640.
WADDELL, A. W., ROBSON, R. T. & EDWARDS, J. G. (1974). Colchicine and vinblastine inhibitfibroblast aggregation. Nature, Lond. 248, 239-241.
WEISS, L. (1972). Studies on cellular adhesion in tissue culture. XII. Some effects of cyto-chalasins and colchicine. Expl Cell Res. 74, 21-26.
WEISSMANN, G., DUKOR, P. & ZURIER, R. B. (1971). The effect of cyclic AMP on release oflysosomal enzymes from phagocytes. Nature, New Biol. 231, 131-135.
WILLIAMS, T. J. & MORLEY, J. (1973). Prostaglandins as potentiators of increased vascularpermeability in inflammation. Nature, Lond. 246, 215-217.
WILSON, L. & FRIEDKIN, M. (1966). The biochemical events of mitosis. I. Synthesis and pro-perties of colchicine labelled with tritium in its acetyl moiety. Biochemistry, N. Y. 5, 2463-2468.
YAHARA, I. & EDELMAN, G. M. (1973). Modulation of lymphocyte receptor redistribution byConcanavalin A, anti-mitotic agents and alterations of pH. Nature, Lond. 246, 152-155.
{Received 7 February 1974)
