Experimental Cell Research 99 ( 1976) 207-220 THE ROLE OF CYTOPLASMIC POLYMORPHONUCLEAR
Evidenw for the Releuse Hypothesis by Means of Time-lapse Anc1lysi.y of‘PMN Movement Relative to Dot-like Attrnctrtrzts I>. RYDGREN,
and B. NORBERG
SUMMARY The present experiments were designed to elucidate the role of cytoplasmic microtubules in the chemotaxis of human polymorphonuclear leukocytes (PMNs) by means of the Boyden chamber technique and by means of analysis of PMN locomotion around a dot-like attractant. Casein induced positive chemotaxis in a small and variable fraction of the PMNs in the Boyden chamber. The movements of individual PMNs in coverslip preparations of clotted autoplasma were analysed as regards velocity of locomotion, locomotive index and net radial dislocation relative to the cell centre, with or without a yeast-phagocytosing leukocyte as a dot-like attractant. PMNs without obvious attractants tended to leave the visual field, i.e. they had a negative net radial dislocation relative to the centre of the visual field. Their locomotive indices suggested that their disappearance from the visual field was due to random movement. In contrast, the locomotive indices of PMNs influenced by attractants suggested the presence of both positive and negative chemotaxis in the population of moving PMNs. Yeast-phagocytosing leukocytes attracted wandering PMNs isolated by the Isopaque-Ficoll method (IF-PMNs) with a force which approximately balanced the basic tendency of the IF-PMNs to leave the visual field. Selective pretreatment of the moving IF-PMNs with podophyllic acid ethylhydrazide (SPI), 0.5 pg/ml (I .05 x IOm6 M), did not inhibit their attraction towards the central yeast phagocyte. The attraction of wandering IF-PMNs towards the central yeast phagocyte was inhibited by selective pretreatment of the phagocytes with SPI, 0.5 pg/ml. These observations indicate that cytoplasmic microtubules have an essential role in the release of chemotactic substances from phagocytosing leukocytes but not in the direction-finding of attractant-approaching PMNs. From the present observations by means of SPI, it is suggested that antitubulin inhibition of the release of chemotactic substances from phagocytosing leukocytes is the mechanism of inhibition of PMN chemotaxis by sub-antimitotic antitubulin concentrations in vitro. The latter phenomenon is thought to reflect the cellular basis of the anti-inflammatory action of the antitubulins.
Sub-antimitotic concentrations of antitubulins-colchicine and its derivatives, Vincu alkaloids, griseofulvin, podophyllic acid derivatives-have been reported to inhibit the chemotaxis of polymorphonuclear leukocytes (PMNs) in vitro but not PMN random movement [ 1, 2, 31. At least two hypotheses have been advanced which could explain the effect of antitubulins on PMN chemotaxis. According to the release hypothesis , the antitubulins may interfere
with the release of chemotactic substances from PMNs which have already arrived to the attractant by means of random movement. According to the conduction hypothesis [3, 5, 61, the antitubulins may inhibit the direction-finding of wandering PMNs by interference with the conduction of the chemotactic message from the cell surface to the contractile machinery of the cell. a mechanism which thus is supposed to be microtubule dependent.
Rydgren et trl.
Table I. Experimentrrl design Control IF-PMNs Control Donor GYP
SPI-treated IF-PMNs Control GYP
Control IF-PMNs SPI-treated GYP
I 7 3 4 5 6 7 8 9 IO II 12 z
14 4 IO 0 2 5 0 7
0 2 x IO
14 IX 5 6 IX x 7 IO IO 2 0 2 100
2 3 55
I2 8 II 17 s 90
2: 28 24 23 16 27 16 19 25 28 20 12 245
IF-PMNs, polymorphonuclear leucocytes isolated by the Isopaque-Ficoll method. These were the moving PMNs. GYP, glass-adherent yeast-containing leukocyte (phagocite), supposed togttract the IF-FMNs. SPI-treated, metreatment for 30 min with SPI, 0.5
Control, no pretreatment with SPI. The figures refer to the observed number of moving IF-PMNs.
It has been reported by previous authors [4, 7, 81 that PMNs release chemotactic substances during phagocytosis. Laserkilled erythrocytes, granulocytes and lymphocytes attract PMNs in the vicinity, as reported by Bessis 1973 . Accidental chemotaxis of PMNs towards two nonidentified cells has been observed in our laboratory during experiments designed for the study of PMN random movement . The non-identified cells provided dot-like attractants around which the chemotactic movements of wandering PMNs were available for analysis . It thus seemed desirable to reproduce the chemotaxis towards dot-like attractants in further experiments. The aim of the present study was to test the conduction hypothesis and the release hypothesis by means of analysis of PMN chemotaxis towards dot-like attractants. Exp Cell Res 99 (1976)
With the reported release of chemotactic substances from phagocytosing leukocytes in mind [4, 7, 81, we allowed leukocytes from peripheral blood to phagocytose yeast cells, adhere to a slide and then we recorded the movements of added PMNs around the yeast-containing leukocytes by means of time-lapse microcinematography. The conduction hypothesis was tested by selective treatment of the wandering PMNs with the antitubulin podophyllic acid ethylhydrazide (SPI). The release hypothesis was tested by selective SPI treatment of the glassadherent yeast-containing leukocytes. For comparison, we also re-examined the chemotactic response of PMNs towards casein in the Boyden chamber, the random movement of PMNs in coverslip preparations of clotted autoplasma and the accidental chemotaxis of PMNs towards dotlike attractants . MATERIAL
The Boyden chamber technique The present modification of the Boyden chamber technique has been described in a previous study . The modified Bovden chamber consisted of two compartments, separated from each other by a 3 gm pore biter (MilhDore Filter CorDoration, Bedford, Mass.). A 0.45 irn Millipore tilt& was placed at the bottom of the lower compartment. PMNs, approx. 1x 106,were injected into the upper compartment and the attractant, casein, to a final concentration of 10 mglml, into the lower compartment at the beginning of the experiment. The Boyden chambers were then incubated for 43 h at +3PC, the bottom filter was stained, and the PMNs were counted along a diameter of the filter at a magnification x320. The observed PMNs were multiplied by a factor 26 in order to assess the total number of PMNs on the bottom filter (total area of the filter/ examined area of=67.3 mm2/2.6 mmz=26). The obtained number of PMNs was then expressed in pro mille of the PMNs in the upper compartment (tip. 2).
Random movement and accidental chemotaxis Random movement and accidental chemotaxis were studied in coverslip preparations of thrombin-clotted leukocyte-rich autoplasma, obtained by means of
non-purified PMNs in coverslip preparations of clotted autoplasma at +37”C Direction of moving PMNs Donot
+ 6 3 2 8 5 3 I 4 4
I:, 8, IO
0 I 0 0 I 0 0 0 0
8 8 I5 II I2 9 7 13 8
Donors l-9, PMNs without obvious attractant. supposed to move at random. Donor IO, accidental chemotaxis towards two nonidentified cells. NRD. sum of net radial dislocation in the observed PMNs which ate specified in the next column: the radial dislocation towards (+) or away from (-) the centre of the visual field. dextran sedimentation. as described in a previous study . PMN locomotio,I wound glass-udherent yeast-containing leukocytes. Pretreatment of blood samples. Venous blood was obtained from the antecubital fossa of I2 healthy volunteers with [email protected]
The blood was collected in four IO ml glass tubes loaded with heparin. 160 IU as dry substance. To each tube was added I ml of a solution containing 0. I5 M L-histidine hydrochloride and 5 % w/v Dextran TSOO, producing a final histidine concentration of 7.5 ~mollml. The blood in each tube was allowed to sedimknt at an angle of 45 degrees at +37”C for 30-40 min. The leukocvte-rich supernatants were then pipetted off. Preparation of wandering PMNs. The supernatant of tube 1 was centrifuged twice at 150 g for IO min to provide plasma free of erythrocytes and leucocytes. The PMNs from the leukocyte-rich plasma of tubes 11 and II1 were separated from the mononuclear leukocytes by means of the Isopaque-Ficoll method described by Bbyum [IO], except that the washings were performed at +4”C. The isolated PMNs were suspended in 0.5 ml isotone saline from the last washing and 0.5 ml autologous plasma from tube I. Control smears showed that approx. 98% of the cells were PMNs. The PMN suspension was divided in two portions, one without antitubulin treatment and one treated with SPI in a final concentration of 0.5 pglml. Both control suspension and SPI-treated suspension were incubated at +37”C for 30 min in order to allow the SPI to penetrate into the cells and bind to the tubulin in the test specimen.
Prepurution of ,gloss-crdhrrent yeust phagoc’,vie\. The cell concentration of the leukocyte-rich supernatant of tube IV was adiusted to 1000 PMNsltil with cell-free plasma. Then 2i)x IO” yeast cells werd added in 50 ~1 of isotone saline to I &I of PMN suspension. orovidina a final concentration of 20000 yeast cells! ;I. The resulting suspension of leukocytes and yeast cells was divided into two equal portions. One portion provided the non-treated control. The other portion was treated with SPI. 0.5 pg/ml. A drop of non-treated cell suspension was placed on 6-7 slide\. A drop 01 SPI-treated cell suspension was placed on 3-1 slide\. The slides were incubated in a moist chamber at +37”C for 30 min. During this period the yeast cell\ were phagocytosed by the leukocytes and these were sedimented down onto the slide and adhered to the glass surface. Control smears showed that approx 50% of the PMNs had ingested yeast cells during this incubation. Then excess suspension with non-adherent leukocytes and non-phagocytosed yeast cells wa\ removed by means of a filter paper. Wandering PMN\. isolated by means of the Isopaque-Ficoll method and resuspended in autologous plasma as described above. were added. The droplet was spread between slide and coverslip. clotted with thrombine and sealed with Vaseline. Experimrntul de.siRn. The coverslip preparatlom were filmed at +37”C in a Wild time-lapse microscope with phase contrast equipment, basic magnification x60. one exposure per 2 sec. as previously described . The visual field covered 120x I80 pm of the covet.slip preparation. Glass-adherent phagocytes, easily detectable because of their ingested yeast cells. with wandering PMNs in the same visual field were selected and filmed for 7-X min (fig. 8). Three combinations were filmed (tables I. 4): (I) Wandering PMNs not treated with SPI (control IF-PMNs) and glass-adherent yeast phagocytes nc>t treated with SPl (control GYP). (2) Wandering PMNs treated with SPI (SPI-treated IF-PMNs) and glass-adherent yeast phanocvte\ not treated with SPI?control GYP). . . - (3) Wanderine PMNs not treated with SPI (control IF-PMNs) and glass-adherent yeast phagocytex treated with SPI (SPI-treated GYP). The coverslip preparations from one donor were filmed the same day. The time sequence between the above-mentioned combinations was varied systematically. The film was analysed frame by frame by prqjection on a screen. The traiectories of individual PMNs were depicted by IO set intervals with the edge of the advancing lamellipodium as the reference point. Only PMNs in active locomotion were analysed. The IO yet marks were connected by straight lines. The total length of an individual trajectory was determined by means of a map-measurer. the median value of three independent measurements. These measurements differed by approx. 2 pm. The velocity of the individual PMNs was then calculated (cf figs 5. 7). Individual PMNs were observed for 120 set (median value. 0,-O, -. _- 80-160 sec. extreme values 40-420 set). Net radiul dislocation. The difference in distance between the individual wandering PMN and the central phagocyte, at the beginning and at the end of the observation period. was calculated. This difference i? detined as the “net radial dislocation” with a minus
Rydgren et al.
50 * L
0.1 0.5 1.0 Fig. I. Abscissu: SPI cont. (&ml); ordinute: oxalateinduced RS with control values as 100%.
sign if the movement was directed away from the phagocyte and with a plus sign, if the movement was directed towards the phagocyte (fig. 8). In the control experiment, locomotion of non-purified PMNs without obvious attractants (table 2), the net radial dislocation was calculated relative to the centre of the visual field. Locomotive index. The locomotive index is the quotient between the distance start point-end point of the cell and the length of the path actually travelled by the cell (fig. 8). Assay of antitubulin activ&v. The antitubulin activity of the SPI-loaded test tubes was measured as inhibition of the oxalate-induced RS of the nuclei of lymphocytes and monocytes from peripheral blood, according to Norbera & Uddman 1973 rlll, extent that onl; 100 cells/&de were counted.‘The doseresponse curve indicated that the test tubes contained the expected concentration and activity of SPI (fig. I). Stafistics. The final populations to be analysed consisted of 129 non-purified PMNs without obvious attractant from 9 donors (table 2, fig. 3) and 245 IFPMNs from 12 donors (table 4, fig. 4). The 245 IFPMNs consisted of subpopulations, n =24-100 (tables I, 4,5). The Mann-Whitney U test [ 121or the Wilcoxon matched-pairs signed-ranks test [I21 were applied to these pouulations, as described in “Results” bv means of the- parameters and symbols used by Siegel 1956 . Levels of significance: pbO.05, not significant; 0.05>p>0.01, almost significant; O.Ol>p>O.OOl, significant; O.OOl>p, highly significant. Chemicals and solutions. Dextran T500 was ob-
Fig. 2. Abscissa: no. of PMNs which moved towards the casein through the intercompartmental filter, in %o of PMNs at the starting side; ordinate: no. of donors, total 28. For comments, see “Results” (casein responders). Exp Cell Res 99 (1976)
Fin. 3. Abscissa velocitv of locomotion (umlmin); ordinate: no. of PMNs. . The velocity of locomotion of 129 non-purified PMNs without obvious attractant (cf table 3). tained from Pharmacia Fine Chemicals, Uppsala, Sweden, casein according to Hammarsten from Merck, Darmstadt, BRD, chromatographically homogeneous L-histidine hydrochloride from British Drug House Chemicals Ltd, London, and bovine thrombin from Parke-Davies, Detroit. MI. Podoohvllic acid ethvlhydrazide (SPI) was provided by Sandoz AG, Basel. The other chemicals used were of analytical grade. The yeast cell solution was prepared according to Brandt 1967 [l3]. SPI was added to the test tubes in IO ~1 sterile water to I ml cell suspension. The glassware was washed with bichromate-H,SO, and rinsed in demineralized water. The plastic material was soaked in ethanol for 30 min and rinsed in demineralized water. Double distilled water was used for the preparation of solutions.
RESULTS Casein responders
The results of experiments on chemotaxis with a modified Boyden chamber and ca-
0 60 20 40 Fig. 4. Abscissa: velocity of locomotion (pmlmin); ordinate: no. of PMNs. The velocity of locomotion of 245 IF-PMNs (PMNs isolated bv means of the Isooaaue-Ficoll method). The SPI treatment of 55 wandering IF-PMNs or of the central glass-adherent yeast phagocyte of 90 IF-PMNs was not expected to influence the velocity of IF-PMN locomotion (cf table 3).
Cytoplasmic microtubrrlt~s rrnd PMN chcmottr.\-i.5 211 Table 3. The ,trlocity of‘ locomotion (pmlmin) of’ PMNs lrndrlr I,trriou.r r.rpr~rimotltrrl conditions All observations
of clotted PMN
Non-purified accidental Control Control
PMNs during chemotaxis
Donors Non-purified PMNs obvious attractant
SPI-treated IF-PMNs Control GYP
Control IF-PMNs SPI-treated GYP
Non-purified PMNs, coverslip preparations of leukocyte-rich plasma. IF-PMNs, coverslip preparations of PMNs, isolated by means of the suspended in autologous plasma. GYP, glass-adherent yeast phagocyte. SPI-treated cell. pretreatment with SPI, 0.5 pg/ml, for 30 min. Control cell. no pretreatment with SPI.
sein, 10 mg/ml, as attractant are shown in fig. 2. The figures indicate PMNs which have crossed the 3 pm pore filter between compartment A and B, a distance of I50 pm, and settled down on the bottom filter. It is evident from fig. 2 that the distribution of the numbers of PMNs recovered at the bottom filter is skew and has a wide and heterogeneous dispersion. These properties are further evidenced by the arithmetic mean (0.64%0) and standard deviation +0.97%0. The latter parameter suggests the presence of negative chemotaxis, which is impossible in the present modification of the Boyden chamber. The distribution shown in fig. 2 is better characterized by its median (13.1 %c), interquartile range Q1-Q3 (I J-18.8 %c)and extreme values (O-130%0). These parameters and the distribution shown in fig. 2 indicate that only a small and variable fraction of the PMNs show positive chemotaxis towards casein in most
probands, as studied by the Boyden chamber technique. Random movement The median velocity of the non-purified PMNs was approximately twice the velocity of IF-PMNs under comparable experimental conditions (table 3). When IO-15 wandering PMNs without obvious attractant were observed in a visual field of a coverslip preparation of clotted autoplasma, there was a tendency for the PMNs to move away from the visual field which was usually empty within 10 min. This tendency was quantified as negative net radial dislocation (table 2). The median locomotive index in these 129 PMNs from 9 donors was 0.63 with an interquartile range Q1-Q3 0.50-0.75, i.e. these parameters were significantly lower than the corresponding parameters of non-purified PMNs in accidental chemotaxis (table 5) and also signifi-
Rydgren et al.
Table 4. Net rmdiul dislocation (pm) C$ Isopuque-Ficoll-separated polymorphonuclear leukocytes (IF-PMNs) reluti\>e to a glass-adherent yeast phugocyte (GYP) in the centre of the visual field
Direction of cells Donor
I 2 3 4 5 6 7 8 9 IO II 12 Cl-l.2
- 69.5” +107.5 + 41.5 - 52” + 68” - 47.5” + 29 - 31.5 - 0.5 - 4 + 18 + 59
Control IF-PMNs SPI-treated GYP
SPI-treated IF-PMNs Control GYP
Control IF-PMNs Control GYP
Direction of cells
Direction of cells
7 IO 3 2 13 I 5 5 4 I
0 0 0 0 0 I 0 0 0 0
7 8 2 4 5 6 2 5 6 I
-134 + 63.5 +154.5= + 41 + 0.5
- 14.5 - 8 - 15 - 2.5 + 37 +122.5
5 4 7
0 0 0
9 0 3
4 3 0 I 3 31
6 0 0 0 0
5 4 I I 0 24
+ 17” + 18.5 - 620 - 4 +1.5 - 79 - 95 - 53.5 -272 + 31.5 - 81.5 -579
2 4 4 5 2 5 3 4 4 2 3 38
0 I 0 0 0 0 0 0 0 :,
0 3 6 2 I 7 5 7 I3 2 4 50
SPI-treated cell, pretreatment with podophyllic acid ethylhydrazide for 30 min. Control cell, no pretreatment with SPI. NRD, the sum of net radial dislocation in the IF-PMNs observed. D Presence of small aggregates of PMNs around a GYP at the beginning of the observation period.
cantly lower than the corresponding parameters of moving IF-PMNs with a yeast phagocyte not treated with SPI in the centre of the visual field (table 5). There was no difference Cs,=0.07) in locomotive indices between the 36 PMNs which moved towards the centre of the visual field (median 0.66, Ql-Q3 0.53-0.77) and the 91 PMNs which moved away from the centre of the visual field (median 0.63, Q1-Q3 0.46-0.74). Accidental chemotaxis The velocity of non-purified PMNs during accidental chemotaxis was high (table 3), the locomotive index was high (table 5), two thirds of the wandering PMNs moved towards the attractants (table 5, fig. 7), and the net radial dislocation was positive (table 2), due to the formation of an aggregate of PMNs around the two non-identified cells (fig. 5). Exp Cell Res 99 (1976 j
PMN movement relative to a central glass-adherent phagocyte The basic material is described in table 1 and figs 6,8. It consisted of 245 PMNs from 12 donors. In 3 of these PMNs, the locomotion did not produce any net radial dislocation of the cell relative to the central phagocyte (table 4). In 7 out of the 48 coverslip preparations studied, we noted small aggregates of PMNs around the yeast-containing phagocytes (table 4). These aggregates were present at the beginning of the observations and did not grow in size during the observation period. They may have been produced by chemotactic aggregation during the preparation or by random clumping during the preparation. It is evident from table 3 that IF-PMNs moved with approximately half the velocity of non-purified PMNs in autoplasma clots. The paths travelled by the IF-PMNs were
Cytoplasmic microtubules trnd PMN chemotrrsis Table 5. The locomotive indices of polymorphonuclear experimental conditions
Non-purified PMNs obvious attractant
leukocytes (PMNs) under various ---
Direction of PMNs
Comparison between indices of approach. and leaving PMNs
Comparison of indices with those of non-purrf. PMNs without obv. attr. -.-
SPI-treated IF-PMNs Control GYP
Not significant, p=o. 19
Control IF-PMNs SPI-treated GYP
Not significant. p=O.IO
p =0.052 p 5 0.00003
Not significant. p =0.23
,I < 0.00003 p=O. 16X.5
Non-purified PMNs during accidental chemotaxis
not sigp ~0.46
Locomotive index. distance start point-end point/length of travelled path (fig. 7). Non-purified PMNs. obtained directly from leukocyte-rich plasma. IF-PMNs, isolated by means of the Isopaque-Ficoll method. GYP, glass-adherent yeast phagocyte. Control PMN, no pretreatment with podophyllic acid ethylhydrazide (SPI). All experiments were performed at +37”C, in coverslip preparations of clotted autoplasma. +. PMNs approaching the central GYP or the centre of the visual field; -, leaving PMNs. Statistical method: the Mann-Whitney U test. Q,-Q:$. interquartile range.
tortuous (figs 6, 8) but the locomotive indices were higher than the locomotive indices of non-purified PMNs without obvious attractants (table 5), although lower than the locomotive indices of non-purified PMNs during accidental chemotaxis (table 5). Then the net radial dislocation of the IFPMNs was analysed in relation to the central glass-adherent yeast phagocyte, first without SPI treatment of movers and phagocytes (table 4). It is evident from table 4 that the net radial dislocation of 100 control IF-PMNs relative to their control phagocytes was approximately zero. This net radial dislocation was more positive than the net radial dislocation of non-purified PMNs without obvious attractant (0.001 >p, calculated on the sum of dis-
location in each donor, n,=9 from table 2, n2= II from table 4, R,=47), i.e. the IFPMNs were attracted towards the central phagocyte by a force which approximately balanced the basic tendency of the PMNs to leave the visual field. The conduction hypothesis was tested by means of SPI treatment of the wandering IF-PMNs. It is evident from table 4 that the sum of the net radial dislocation of SPItreated IF-PMNs relative to the central glass-adherent phagocyte was approximately zero for the 55 IF-PMNs analysed, i.e. the basic tendency of the PMNs to leave the visual tield was balanced by an attraction towards the central phagocyte. This attraction could hardly be mediated by redistribution of microtubules in the wandering IF-PMNs, since they were pre-treated
Rydgren et al.
Fig. 5. Aggregate of PMNs around a non-identified cell (NIC). The aggregate was produced by accidental chemotaxis of non-purified PMNs towards the nonidentified cell in the centre (cf fig. 7). x400. Exp Cell Res 99 (1976)
Fig. 6. Two central GYPS with moving IF-PMNs in the same visual field (arrows in the direction of locomotion). Y, Yeast cells. From the time-lapse film (donor 7, table 1). x60.
Fig. 7. Trajectory of 13 PMNs during accidental chemotaxis towards a non-identified cell in the centre of the visual field (not shown here, cf fig. 5). Ten out of 13 PMNs moved towards the non-identified cell in the centre (cf fig. 5). Bars indicate 10 set intervals.
with 0.5 g/ml of SPI. This finding provides evidence against the conduction hypothesis. The release hypothesis was tested by means of SPI pretreatment of the glassadherent yeast phagocytes. The net radial dislocation of 90 non-treated IF-PMNs relative to the central SPI-treated yeast phagocyte was then analysed (table 4) and found to be negative relative to the net radial dislocation of 100 control IF-PMNs towards control phagocytes (O.Ol>p>O.O01, nl= n,=lO, T=3). It was, however, less negative than the net radial dislocation of nonpurified PMNs without obvious attractant relative to the centre of the visual field (O.Ol>p>O.OOl, n,=9 from table 2, n,=ll from table 4, R,=60). The attraction of IFPMNs towards the central yeast phagocyte was thus partly inhibited by pretreatment of the phagocytes with 0.5 pg/ml of SPI. These findings provide strong evidence in favour of the release hypothesis, i.e. the release of chemotactic substances from phagocytosing leukocytes is at least partially microtubule-dependent. The data of table 5 suggest that the
median locomotive index of a population of wandering PMNs may reflect the presence or absence of chemotaxis. During chemotaxis, it was notable that both approaching PMNs and disappearing PMNs had their distribution of indices on the same absolute level and without statistical significance. as assessed by the Mann-Whitney U test. The locomotive indices of PMNs influenced by chemotaxis were significantly higher than the locomotive indices of PMNs without obvious attractant (table 5). It seems reasonable to conclude from the data of table 5 that a median locomotive index above 0.70 in a population of wandering PMNs suggests the presence of defined attractants. DISCUSSION The present study provides evidence that PMNs without obvious attractant disappear from the visual field when observed by means of phase contrast microscopy, that PMN chemotaxis is induced towards leukocytes which have ingested yeast cells, and
Fig. 8. Trajectory of the locomotion of IF-PMNs relative to a central glass-adherent yeast phagocyte. Locomotive index (PMN no. 2): the quotient between straight distance (dotted line) and actual distance (barred line) travelled by the PMN. Net radial dislocation (PMN no. 3): the distance of the IF-PMN to the central glass-adherent yeast phagocyte at the beginning of observation minus the same distance at the end of observation. Bars indicate IO set intervals.
Rydgren rt 01
that the release of chemotactic substances from yeast phagocytes is at least partially microtubule-dependent, i.e. the PMN chemotaxis towards yeast phagocytes was partially inhibited by pretreatment of the phagocytes with low concentrations of SPI. SPI pretreatment of the wandering PMNs did not inhibit chemotaxis. These observations imply that the antitubulin inhibition of PMN chemotaxis is due to an effect on PMNs which have arrived at the attractant by means of random movement. Chemotaxis appears to be a highly complex process, which includes initiation of movement, amoeboid movement, directionfinding, aggregation at the site of the attractant and termination of movement [ 1, 3, 14, 151. It is evident that inhibition of every factor mentioned will inhibit PMN chemotaxis, provided that the PMNs can escape from the site of the attractant. Premature aggregation and premature termination of movement will also inhibit chemotaxis in most test systems. In contrast, a locomotory paralysis of the cell during contact with an agent restricted to a limited area of the visual field would mimic the final stage of chemotaxis in a test system where only the last stage of chemotaxis is studieddirectional movement, aggregation at the site of the attractant and termination of movement [3, 91. In the Boyden chamber technique [cf 16191, chemotaxis is assessed by determination of the advancing border of wandering PMNs or, as in the present study, by collecting and counting the PMNs which have crossed the intercompartmental filter during a defined period of time. The latter method appears to be less sensitive to fluctuations in the time course of the chemotactic process. The PMNs cannot escape from the bottom filter, except by cytolysis. The effect of random movement is asExp Cell Res 99 (1976)
sessed by means of control chambers without attractant. In previous studies we have by definition considered PMN chemotaxis to be present when at least 10 PMNs were found in the casein control, if the PMNs in the non-casein control were 5 or less [3, 5, 61. It is obvious from fig. 2 that the modified Boyden chamber technique provided a sensitive recording of chemotaxis when only a small fraction of the PMNs move towards the attractant. The cells collected at the bottom filter are, however, a net result of many interacting and counteracting processes, e.g. initiation of movement, amoeboid movement, direction-finding and premature aggregation. Inhibition of chemotaxis in Boyden chambers may be effectuated by influence on any one of the mentioned processes. Our figures on caseinresponding PMNs in the modified Boyden chamber are not directly comparable to other reports known by us, since PMN chemotaxis can be assessed in many ways and the basic chemotactic response of the PMNs is by tradition often veiled as an “index”. Since such indices may distort the statistical analysis, we have avoided them entirely. In contrast to lymphocytes and monocytes, the PMNs show a vigorous motility in fresh coverslip preparations of clotted autoplasma. The basic mechanism of this immediate motility in the absence of defined attractants is not known. Since attractants appear to induce directional locomotion in resting PMNs, as reported by Zigmond 1974, it is reasonable to assume that the “random movement” of PMNs could simply be a hunt for indistinct and competing attractants in the preparation. The basic tendency of the PMNs to leave the visual field warrants the introduction of a control preparation without attractant in
future studies by means of the present experimental design (tables 1, 4). The disappearance of the PMNs without obvious attractant from the visual field raises the question if their locomotion was random or non-random, e.g. due to “photophobia” of the PMNs or negative chemotaxis between non-releasing PMNs (cf ). This problem will be subject to further analysis by means of other methods. The low locomotive indices of these PMNs suggest, however, that their movement was random at least in some respects (table 5). It is thus assumed in the following discussion that the nonpurified PMNs without obvious attractants (table 2) moved at random. The negative net movement of these PMNs relative to the centre of the visual field provides an essential background material in the analysis of PMN locomotion relative to a central yeast phagocyte. The velocity difference between nonpurified PMNs and IF-PMNs (table 3) could be due to cell damage during isolation on the Isopaque-Ficoll column, to the elongated isolation process (3-4 h) or to varying cell concentrations in the preparations. The latter factor can, however, be assumed to vary at random in all preparations of nonpurified PMNs and IF-PMNs. The experimental conditions were comparable apart from the Isopaque-Ficoll separation, which thus was the likely reason for the slower movement of the IF-PMNs. Moreover, the low temperature during the isolation process, desirable in order to prevent bacterial growth in the cell suspension, was expected to induce a reversible disassembly of the cytoplasmic microtubules of the IF-PMNs (cf ). A weak positive chemotaxis was demonstrated in both control IF-PMNs and SPI-treated IF-PMNs (table 4) by comparison with non-purified PMNs without obvious attractant (table 2). The cold isola-
und PMN chrmottr.ris
tion could, however, have reduced the response of the IF-PMNs to the attraction towards the yeast phagocytes. It is desirable to reinvestigate PMN chemotaxis without preincubation of the wandering PMN\ at +4”C for 3-4 h. Chemotaxis in individual cells is readily inferred when a clear majority of the PMNs in a visual field move towards the attractant and aggregate around the attractant [3, 9, 141.Problems arise, however. when only a fraction of the moving PMNs respond to the attractant. It is evident that a moving PMN has to approach or leave a defined point in a visual field, attractant or nonattractant. The chance that PMN locomotion would not produce a net radial dislocation in relation to the centre of the visual field, with or without yeast phagocyte, was in the present study 0.013 (5 out of 374 PMNs, tables 2. 4), which from a statistical point of view appears to correspond to the case when the tossed coin lands on its edge, neither heads nor tails. A consequence of the above-mentioned considerations is that chemotaxis cannot be assumed in one individual cell which moves towards a suspected attractant but rather in a population of cells, the movements of which significantly deviate from a random pattern in the absence of other systematic factors. Since a moving cell may hypothetically display positive chemotaxis. random movement or negative chemotaxis. we prefer to term the quotient between straight distance and actually travelled distance of a PMN “locomotive index”. which does not a priori imply the presence of chemotaxis. This index reflects the straightness or the tortuosity of the path travelled by the cell. In the present study it was expected that (1) a population of wandering PMNs is composed of one fraction (cz) of cells which
Rydgren et ul.
move by chemotaxis to the attractant, and another fraction (1-o) of cells which approach or leave the attractant by random movement; (2) PMNs in chemotaxis move straighter than PMNs in random movement, i.e. the chemotactic index is higher for chemotactic PMNs than for randommoving PMNs. Actual observations of PMNs influenced by an attractant showed, however, that the locomotive indices, although higher than those of non-purified PMNs without obvious attractants, were approximately equal for attractant-approaching PMNs and attractant-leaving PMNs (table 5). These observations suggest that the attractants influenced the movements of at least a fraction of both approaching and leaving PMNs, i.e. positive chemotaxis was induced in some PMNs and negative chemotaxis in some PMNs. Random-moving PMNs were presumably present but could not be identified under the prevailing conditions. Negative chemotaxis has previously been reported in bacteria  but not with conclusive evidence in PMNs (cf ). There was an occasional formation of small PMN aggregates around the yeast phagocytes, already present at the beginning of the observation and without further growth during the observation period (table 4). The moving -IF-PMNs were thus not arrested at the site of the yeast phagocytes during the observation period. These observations support the idea that the attractive force of the yeast phagocytes was weak during the observation period, and that this force had been stronger in a previous stage of the experiment. These conclusions are in agreement with the finding of Ward & Hill 1970  that the production of chemotactic activity from PMN granules is timedependent with a rapid decrease after 30 min , Exp Cell Res 99 (1976)
In previous studies, chemotactic activity has been obtained from leukocytes by means of cell destruction [4, 9, 241 from cultures of living leukocytes  and especially from preparations of living leukocytes fed with immune complexes [7, 141. The present study indicates that living leukocytes release chemotactic substances during phagocytosis of yeast cells in vitro. This release was blocked by low concentrations of an antitubulin, SPI, i.e. the release was microtubule dependent. This finding has interesting bearings on our understanding of PMN chemotaxis and the cellular mechanism of the anti-inflammatory action of the antitubulins in, e.g., acute gout (cf ). The present study favours the release hypothesis by positive evidence, i.e. inhibition of PMN chemotaxis by selective and controlled antitubulin treatment of the central phagocyte. The present study also provides evidence against the conduction hypothesis, i.e. no inhibition of PMN chemotaxis by selective and controlled antitubulin treatment of the wandering IFPMNs. The conduction hypothesis is, nevertheless, still attractive and may be worth a few more tests under varied experimental conditions. The cold isolation of the IF-PMNs could, e.g., interfere with their subsequent microtubule function. Direct observations by previous authors show, in any case, that wandering PMNs are able to recognize an attractant and move towards this attractant [3, 8, 9, 151. This observed behaviour of the PMNs obviously must have an intracellular correlate on the ultrastructural and molecular level. It is assumed that membrane receptors sense the concentration gradient of an attractant and initiate movement and direct the moving PMNs towards the attractant [14, 261. The distribution of surface receptors is influenced by microtubules, as
Cytoplasmic microtubules rend PMN chemottr.ui.\ inferred from antitubulin experiments by previous authors . Studies on the role of divalent cations in PMN chemotaxis also appear to support the microtubule conduction hypothesis, as reported by Gallin & Rosenthal in 1974. The above-mentioned observations of previous authors and the present findings may be applied to a tentative model of PMN chemotaxis, according to which a randommoving PMN ingests a bacterium, a ureate crystal or an immune complex. This PMN releases chemotactic substances during phagocytosis, and these substances attract more PMNs to the site of the phagocytosable material. The mechanism of direction-finding within the approaching PMNs is at present entirely unknown, except that a role of surface receptors is postulated from direct observations on cell behaviour, theoretical considerations [ 14, 261 and membrane blocking with complement factors . PMN chemotaxis is inhibited in vitro by sub-antimitotic concentrations of the antitubulins studied hitherto, i.e. colchicine , demecolchine , SPI , and griseofulvin . The antitubulin inhibition of PMN chemotaxis is the likely cellular basis of the long-known anti-inflammatory action of antitubulins, e.g. in acute gout (cf [5, 6, 25, 301). It is mediated by antitubulin interference with the release of chemotactic substances from phagocytosing leukocytes, as evidenced in the present study, i.e. microtubule redistribution is an essential link in the release phenomenon. The present tentative model of PMN chemotaxis does not exclude complementary and alternative mechanisms. It implies that antitubulins can be expected to inhibit the inflammatory reaction mediated by the release of chemotactic ,substances from living PMNs but not a priori the release of
chemotactic substances from dying cells and tissues. The wide and skew dispersion of individual values in experiments on PMN chemotaxis by means of the Boyden chamber technique may at least in part be cxplained by the dependence of the whole process on the random-moving PMNs which first reach the attractant, and by the dependence on microtubule redistribution. which is sensitive to many physical and chemical factors (cf [ 11, 2 I]). It seems reasonable to conclude from the present study that PMN locomotion relative to a dot-like attractant in the centre of the visual field has proved useful in experiments designed to elucidate the role ofcytoplasmic microtubules in PMN chemotaxts. The presence of chemotaxis is suggested by a non-random pattern of movement in a population of PMNs or by high locomotive indices (by conjecture a median value above 0.70) in a population of moving PMNs. The presence of chemotaxis cannot be detected in single cells by this method, except when the fraction of PMNs in positive chemotaxis approaches 1 (a- I). We thank Mr HBkan Brodin, Mrs Astrid Norberg. Dr Inge Olsson. Professors Nils SGderstriim and Tor Zelander for advice and constructive criticism. Mrs Gunilla Naumann and Mrs Sylvie Persson for technical assistance and Mrs Patricia Wetterberg. for revising the English. This study was supported by grant no. B75-19X2294-09A from the Swedish State Medical Board for Medical Research and grants from the Medical Faculty of Lund.
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