Immunology 1980 41 457

Effects of human IgG on locomotion of human neutrophils related to IgG binding of ahydrophobic probe*

P. C. WI LK I N SON Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary,Glasgow

Acceptedfor publication 19 May 1980

Summary. Heat-denatured (630) human IgG hadcomplex effects on locomotion of human neutrophils.At concentrations of 1 mg/ml and below, it stimu-lated chemotactic locomotion into filters judged bythe leading front assay, however, pre-treatment ofthe cells or of the filters with denatured IgG caused areduction in the number of locomoting cells, com-pared to cells locomoting in a medium containingalbumin. These effects took place in complement-freemedia. Native IgG was not chemotactic. The chemo-tactic activity of denatured IgG correlated well withincreased binding by the same IgG preparations ofthe hydrophobic probe, 1-anilinonaphthalene-8-sulphonate (ANS), and it is suggested that heatinginduces a conformational change in the IgG moleculewhich allows recognition of the altered molecule byneutrophils and activation of a chemotactic response.The integrity of the Fc fragment is required for thisactivity. As well as a direct chemoattractant activityof IgG, evidence for release of chemotactic factors bycells in contact with aggregated IgG was also ob-tained. It is suggested that the contrary effects ofdenatured IgG on neutrophil locomotion are explic-able if the protein, like other denatured proteins,activates the sensory chemotactic mechanism in the

Correspondence: Dr P. C. Wilkinson Department ofBacteriology and Immunology, University of Glasgow(Western Infirmary), Glasgow Gl 6NT, Scotland.

* Departmental Publication No. 8005.00 19-2805/80/1000/0457/$02.00c 1980 Blackwell Scientific Publications

neutrophil, while at the same time causing a modifi-cation of adhesion of cell to substratum which mayimpair the locomotor capacity of the cells.

INTRODUCTION

Human neutrophils bear receptors for the Fc frag-ment of IgG, and binding of IgG to neutrophils mayactivate various cellular functions, for example, neu-trophils bound to aggregated IgG-coated surfacesrelease hydrolases into the extracellular medium(Henson, Johnson & Spiegelberg, 1972). Severalreports suggest that IgG can modulate locomotorfunctions in neutrophils. Hayashi and colleagues re-ported that, while native IgG did not attract neu-trophils, proteolytic cleavage of a peptide from theFc fragment could result in the remainder of the IgGmolecule acting as an attractant for leucocytes infilter assays (Yamamoto, Nishiura & Hayashi, 1973;Hayashi, Yoshinaga & Yamamoto, 1974). Kemp &Brown (1980) coated filters with aggregated immu-noglobulin and found that neutrophils became im-mobilized on such filters and failed to migrate in agradient of casein diffusing from below the filter.However, though few cells migrated into immuno-globulin-coated filters, the few that did, actuallymigrated further than into serum albumin-coatedfilters. Keller, Barandun, Kistler & Ploem (1979a)had similar findings in which neutrophils adheredstrongly to IgG-coated surfaces and did not loco-

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P. C. Wilkinson

mote, yet they did migrate into filters in response togradients of IgG (Keller, Wissler & Ploem, 1979b).Thus the effects of IgG on locomotion are puzzlingsince either stimulation or inhibition of locomotionhas been observed depending on the assay conditionsused. The picture is further complicated by the obser-vation of Zigmond & Hirsch (1973) that neutrophilswhich flatten on aggregated-immunoglobulin coatedsurfaces themselves release factors that are chemo-tactic for neighbouring neutrophils.

In this paper, the hypothesis is explored that IgGmay acquire chemoattractant activity for neutrophilsif it undergoes conformational change with increasedexposure of hydrophobic sites, as has been reportedfor other proteins (Wilkinson, 1973; Wilkinson &Allan, 1978a). Conformational change was moni-tored using the fluorescent probe 1 -anilino-naphthalene-8-sulphonic acid (ANS) which fluo-resces in hydrophobic, but not in hydrophilicenvironments. This molecule binds to hydrophobicsites on proteins producing a marked increase influorescence, and can be used to monitor exposure ofsuch sites and thus to detect conformational change(Daniel & Weber, 1966; Haynes & Staerck, 1975).Cells were exposed to gradients of heat-denaturedIgG and the chemotactic response was evaluated inrelation to increases in fluorescence of ANS bound tothe same heated IgG preparations. The role of theIgG itself and of cell-released factors as attractantswas compared. The effects of IgG as an inhibitor ofneutrophil locomotion were also studied, both bypre-coating the cells with IgG, then measuring theirlocomotion into filters, and by allowing them tolocomote on IgG-coated substrata.

MATERIALS AND METHODS

IgGHuman IgG was purchased from Miles, Slough,England (Code No. 64-145, lots 28 and 31). Thismaterial (sample 1) gave a single arc on immuno-electrophoresis against polyvalent rabbit anti-humanserum and was largely in monomer form (less than5% polymer) judged by Sephadex G-200 chroma-tography. It was stored in lyophilized form. HumanIgG (sample 2) was obtained from the ScottishAntibody Production Unit, Law Hospital, Glasgow.This material gave a strong IgG arc on immuno-electrophoresis against polyvalent rabbit anti-humanserum, but also contained a small amount of albumin

and of a protein of about 40,000 mol. wt judged bySDS-polyacrylamide gel electrophoresis. Before use,it had been stored for about a year at 150 mg/ml inpreservative at 40, and about 30% of the protein waspresent in polymer form, judged by the proportion ofprotein which eluted ahead of the IgG monomerpeak from a Sephadex G-200 column.

Heat-aggregationIgG was heat-aggregated in a waterbath at 630 forvarious times detailed in the Results section.Aggregation was carried out at various protein con-centrations between 2 and 20 mg/ml followed byappropriate dilution for testing. The time at 630 wasmeasured with a thermometer immersed in the pro-tein solution. IgG sample 2, which already containedpolymers, aggregated more readily at 630 thansample 1.

Fluorescence emission off ANS bound to IgG samplesANS (BDH, Poole, Dorset) was dissolved in Gey'ssolution, acidified to approximately pH 6-0, andadded to appropriate samples of protein to give afinal ANS concentration of 10-6M. Fluorescence wasmonitored using a Perkin-Elmer 1000 fluorescencespectrophotometer with an excitation wavelength of366 nm and an emission wavelength of 475 nm. ANS(10-6 M) in the absence of protein was used as anegative control. As positive control, ANS wasadded at a final concentration of 10-6 M to humanserum albumin (HSA) at 1 mg/ml. Serum albuminhas a high-affinity binding site for ANS (Daniel &Weber, 1966) and ANS binding to albumin producedstrong fluorescence, stronger than with any of theIgG samples tested. Therefore the fluorescence ofANS-IgG complexes has been expressed in arbitraryunits where 100 units equals the fluorescence of theANS-HSA control. Before aggregation, IgG sample1 at 1 mg/ml (7 x 10-6 M) (without polymers) gave0 58-1 3 fluorescence units in different experiments,whereas sample 2 (containing polymers) at 1 mg/mlgave 2 7-4-2 fluorescence units.

Pepsin digestion of IgGF(ab')2 fragments were prepared by incubation ofthe above IgG preparations for 18 h at 370 withpepsin (Koch-Light, Colnbrook) in acetate buffer pH40 at an IgG: pepsin ratio of 20:1, followed byneutralization and prolonged dialysis against severalchanges of Gey's solution.

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Human IgG and human neutrophil locomotion

Sephadex G-200 chromatographyAggregated IgG samples were passed throughSephadex G-200 columns (Pharmacia, Uppsala,Sweden) to separate monomers from polymers.Approximately 200 mg of protein was passedthrough a column of 50 cm length, 2-5 cm internaldiameter, in phosphate buffer, pH 7-5, using bluedextran as a marker for the exclusion peak.

CellsHuman neutrophil leucocytes were prepared fromvenous blood from normal donors by dextran sedi-mentation followed by centrifugation on Ficoll-Triosil (SG 1 078). The cells were washed three timesbefore use to remove serum proteins. Cells wereprepared either in Gey's solution, pH 7-2 or inHanks's-HEPES, pH 7 2, the latter being preferredin later experiments because of its superior bufferingcapacity. Neither medium contained serum, since thestudy was concerned with direct locomotor effects ofIgG, not with complement-mediated effects. Incertain experiments, the medium was supplementedwith HSA or bovine serum albumin (BSA) usually at1 mg/ml. Whether or not albumin was added ismentioned in the various tables and figures.

Chemotaxis assaysThe micropore filter technique was used for measure-ment of neutrophil locomotion and chemotaxis asdescribed previously (Wilkinson, 1974). Filters of3 Mm pore-size (Millipore, Bedford, Mass.) wereused. In most experiments the leading-front methodwas used to measure cell locomotion, but in someexperiments, both the leading-front method was usedand the total number of cells entering the filter perfield was estimated. Chemotaxis was distinguishedfrom chemokinesis in filters using the checkerboardassay of Zigmond & Hirsch (1973).

RESULTS

Enhancement of neutrophil locomotion by IgGIgG (sample 1) was aggregated at 630 for varioustimes between 0 and 30 min. The samples were testedfor activity in chemotaxis chambers and for fluores-cence on addition of ANS. Figure 1 shows thataggregated IgG at 600 Mg/ml stimulated neutrophillocomotion to a greater extent than monomeric IgGand that the attraction was greater, the longer thetime of aggregation, up to 30 min. Furthermore,

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0 10 20 30Time (min) of heating IgG

Figure 1. The effect of time of heating at 630 on chemo-attractant activity of human IgG (sample 1) for humanneutrophils, and on fluorescence of ANS. The IgG at 2mg/ml was incubated at 63° for various times. It was thendiluted to 600 ug/ml for chemotaxis assays and placedbelow filters at this concentration. Cells in Gey's solutionwithout added protein were allowed to migrate into thefilter. Locomotion is expressed as the ratio, locomotion toaggregated IgG: locomotion to unheated IgG (± SEM).ANS fluorescence was measured on samples of IgG (1mg/ml) + ANS 10-6 M at 475 nm.

ANS fluorescence also increased with time of in-cubation at 630. Sample 2 (which contained poly-mers) had some attractant activity for neutrophilsbefore heating, which increased after heating (Table1). The relationship between enhancement of loco-motion and the increase in ANS fluorescence, for agreater number of preparations of aggregated IgGthan is shown in Table 1 and from both samples, isshown in Fig. 2. The ability of these IgG prepara-tions to attract neutrophils into filters usuallyshowed a close correlation to the ANS fluorescenceof the preparations, (evident in four out of fiveexperiments) suggesting that exposure of ANS bind-ing sites on the IgG molecule was related to theability of the IgG to bind to neutrophils and toactivate locomotion.

Separation of polymers from monomersAn aliquot of IgG (sample 2) was aggregated at 630for 20 min and passed through a column of Sephadex

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P. C. Wilkinson

Table 1. The effect of heat-aggregated IgG on neutrophil locomotion into filters with andwithout pepsin digestion

Locomotion of human neutrophils (2 x 106/ml) in HSA(1 mg/ml) in 60 min

Attractant Leading-front Total cells ANS fluorescencedistance (pm) migrating into filter of IgG sample

per field

None (cells in HSA1 mg/ml) 56 + 1-9 633

IgG (sample 2, containingpolymers but not heated) 3 0

250pg/ml 63 + 2 5 805500ug/ml 65 +4 0 716750 pg/ml 72+2-1 588

IgG (sample 2, 63°30 min) 7.3

250ug/ml 73 + 1 6 731500 yg/ml 71 +2 3, 868750 yg/ml 79 + 1-6 775

IgG (sample 2, 63'30 min, pepsin-treated) 0 3

250 pg/ml 54 + 2 2 811500 yg/ml 59 +4-2 795750 ug/ml 64 +2-0 665

G-200. Figure 3 shows the activity of the fractionsobtained. Most of the stimulatory activity for neu-trophil locomotion was found in the exclusion peak(polymers) and little activity in the monomer peak.Similar results were obtained on repeating the experi-ment. On mixing with ANS, the exclusion peakshowed much higher fluorescence than the monomerpeak. In a similar experiment, a sample of lightlyaggregated IgG with leucocyte-attracting activity wascentrifuged at 100,000 g. The supernatant from thiscentrifugation showed no activity in attracting leuco-cytes. These results suggest that the enhancement ofleucocyte locomotion by IgG is largely confined topolymerized samples which, judged by the strongcorrelation of attractant activity with ANS fluores-cence, have undergone a conformational change withincreased exposure of hydrophobic regions.

Chemotactic activity of aggregated IgGThe chemoattractant of aggregated IgG in filter

assays was analysed by the checkerboard method(Zigmond & Hirsch, 1973) to determine whether thepreparation was chemotactic or chemokinetic forhuman neutrophil leucocytes. The results of one ofthree such experiments are shown in Table 2. In allthree, cells moved further in positive gradients, andless far in negative gradients, than would be expectedon the basis of chemokinesis alone. They alsoshowed a dose-dependent increase in distance mi-grated with increasing IgG concentration. Thus theaggregated immunoglobulin had both chemotacticand chemokinetic effects. In a checkerboard assayusing unheated IgG, sample 1 (not shown), nochemotactic effect was seen, though the unaggregatedIgG did induce a slight dose-related enhancement ofchemokinesis.

Dose-response curves for chemokinesis of neutrophilstowards aggregated IgGFigure 4 shows locomotion of neutrophil leucocytes

460

Human IgG and human neutrophil locomotion

x

x

0

00

0 5 30 4 5 60 75ANS fluorescence units (475nm)

9 0

Figure 2. The relation between ANS fluorescence andchemoattractant activity of IgG samples. ANS fluorescencewas measured as in Fig. 1. Chemoattraction was also mea-

sured as in Fig. 1 except that the IgG was used at 750pg/ml. The experiment contains a variety of IgG prepara-tions from both sample 1 and sample 2 heated for a varietyof times. The first two points on the graph are for unheatedsample 1. The line through the points was derived by linearregression (r = 0 95; P = <0 0001).

into filters in various concentrations of aggregatedIgG. The IgG was present at uniform concentrationon both sides of the filter, thus the locomotion understudy was chemokinetic, not chemotactic. In theseexperiments, aggregated IgG stimulated locomotionmoderately at concentrations up to about 2 mg/ml,but at higher concentrations, the response declinedto background levels. In contrast, neutrophils mi-grating in the purely chemokinetic protein, serum

albumin, show no decline in the locomotor response

at concentrations of up to 50 mg/ml (author's.mnpublished observation).

Requirement for the Fc fragment for chemoattractantactivity of IgGIgG (sample 2) was aggregated at 63° for 30 min,then divided into two aliquots, one of which was

treated with pepsin and the other put through thesame procedure without the enzyme. Table 1 showsthe response of neutrophils to these two preparationsand to unheated (sample 2) IgG. The distance mi-grated by the leading front towards the heated IgGwas higher than towards unheated IgG, however,

ANSemission 64 28 05 057

Figure 3. Sephadex G-200 chromatography of IgG (sample

2, 630, 20 mmn) to show protein peaks and chemoattractant

activity and ANS fluorescence of the fractions. Cells in

HSA (500 pg/mi) migrated 51 pm into a filter in the absence

of lgG and the activity of each fraction (histograms) is

given as distance migrated to the IgG fraction (used at 1

mg/ml) minus distance to this control. Note that the peak

both of ANS fluorescence and of attractant activity is in the

exclusion peak (blue dextran) and that the monomer frac-

tion has little activity in either test.

after pepsin digestion of the heated preparation, the

leading front values were reduced to below those of

the unheated sample, suggesting that the activity of

the heated IgG was mediated through the Fc frag-

ment. Likewise, the increase in ANS fluorescence

following heating disappeared if the aggregated IgG

was pepsin-digested and extensively dialysed. A

second experiment gave a similar result. In Table 1

figures are also included for the total number of cells

migrating into the filter (at all levels) per field.

Measuring the total number of migrating cells does

not give information about the chemotactic response,

though attractants do frequently increase this num-

ber; however, these figures are included because at

the highest concentrations of IgG used (750 pg/mI),

fewer cells were migrating into the filters than to-

wards 500 pig IgG/ml.

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x

461

P. C. Wilkinson

Table 2. Checkerboard analysis of locomotion of human neutrophils in various con-centrations and concentration gradients of heat-treated human IgG (sample 2, 630, 15 min)

Heated IgG concentration below filter (t±g/ml)0 100 500 900

Neutrophil migrationpm in 60 mins

Heated IgG above filter 0 49 + 20 86 + 2 6(62) 97 + 2 9(65)Pg/ml) 100 65+ 16

500 67 +±23(70) 72+5 3900 68+1 7(79) 94+24

Figures in parentheses represent migration calculated as detailed by Zigmond & Hirsch(1973) on the basis that cells are responsive only to the absolute concentration of IgG andnot to concentration gradients. Figures without parentheses represent the result actuallyobtained (mean of ten readings). BSA 1 mg/ml was present in the medium on both sides ofthe filter.

Direct and indirect attractant effects of IgGIt has been shown earlier (Zigmond & Hirsch, 1973)that neutrophils which settle on an aggregated-IgGcoated surface may release chemotactic materials.Thus the chemotactic effects of IgG described abovecould be due, not to a direct effect of the proteinitself on the locomoting cells, but to its ability tocause cells to release chemotactic factors and thus toattract other cells. The possibility of a cell-derivedfactor was therefore investigated.

70-

60-

a50 0

c40-0

3000- 20

I0-

0 5 0 5 20IgG concentration (mg/ml)

Figure 4. The dose-response for chemokinetic locomotionof human neutrophils into filters in uniform concentrationsof various preparations of human IgG. , Sample 2 (630,20 min); o, sample 1 (630, 20 min); sample 1 unheated.Note that the curve for heated sample 2 has not beencarried through the point for the sample at 10 mg/ml. Thiswas because this material did not usually stimulate loco-motion at that concentration and this point was an excep-tion. Cells in Gey's solution with no added protein.

In the first group of experiments, an attempt wasmade to demonstrate the presence of a cell-derivedfactor directly using a two-filter assay. Filters weresoaked in aggregated IgG at various concentrations,then washed thoroughly. Neutrophils (2 x 106 perfilter of 6 mm diameter) were then allowed to settleon these filters, which were to be used as the lowerfilters in the two-filter assay. The upper filter wasuncoated with IgG and the population of neutrophilswhose locomotion was under study (at 2 x 105 cellsper filter in HSA 1 mg/ml) was placed on this topfilter. The lower chamber was filled withHanks's-HEPES buffer, pH 7 2. Thus the only at-tractant which could reach the cells on the top filterwould be, not the IgG itself, which was bound to thelower filter, but material, released by the cells incontact with IgG on the lower filter, which diffusedup through the upper filter. As a control, the sameassembly was used, with aggregated IgG but no cells(or cells but no aggregated IgG) on the lower filter.The results are shown in Fig. 5. Cells bound to thelower immunoglobulin-coated filters released a ma-terial which attracted cells into the upper filter, but,under the assay conditions used, this material wasdetectable only when the aggregated IgG concen-tration used to coat the lower filter was greater than5 mg/ml.

If cells locomoting into a filter in a gradient ofaggregated IgG were releasing a factor that attractedother cells, then the stimulation of locomotionshould be increased simply by increasing the number

462

Human IgG and human neutrophil locomotion

100-

_ 90--

-a 80-

E ; 70-o 0O CL

` 60-8

U' 50-

5 10 15 20Aggregated IgG (mg/ml) used to coat lower filter

Figure 5. Effect of coating a lower filter with aggregatedIgG on the locomotion of cells into an upper filter. Cells inHSA I mg/ml. & lower filter coated with IgG and 2 x 106neutrophils allowed to adhere to the upper surface of thisfilter; a, lower filter coated with IgG but no cells added toit. For a lower filter coated with cells but no IgG, thelocomotion value was 63 + 3 ,cm.

of cells used for the assay. The effects of aggregatedIgG (1 mg/ml), diffusing from below a filter, were

therefore measured using cell concentrations abovethe filter varying from 3 x 106 to 5 x 105 ml. Theattractant effect of the IgG did not vary as cellconcentration was varied. A plot of the logarithm ofcell number against the square of the distance mi-

grated by the leading front of cells gave a straightline (not shown) as would be expected for cellsresponding to a single attractant. If the cells them-selves had released an attractant, there would havebeen more of it present at high than at low cellconcentrations and a curved plot would haveresulted.

These results confirm that the presence of aggre-

gated IgG does cause neutrophils to release materialthat attracts other neutrophils. However, a directattractant effect of aggregated IgG is also likely and

Table 3. The effect of pre-treatment of human neutrophils with IgG on theirsubsequent locomotion in filters towards a chemotactic factor

Locomotion of human neutrophils (2 x 106/ml) in1FISA (1 mg/ml) in 60 min

Leading-front value Total cells migrating

Pm percentage of per percentage ofvalue for un- field value for un-treated cells treated cells(group 1) (group 1)

Group 1Untreated cellslocomoting towardsNo attractant 56 + 1-9 633Casein 350 jg/ml 92 + 3-0 741Casein 700pg/ml 110 + 2.1 816

Group 2Cells pre-treated (45 min)with aggregated IgG(63', 30 min) 750 yg/ml,locomoting towards

Noattractant 33 + 1-4 59 72 11Casein 350 pg/ml 69 + 2 2 75 225 30Casein 700 Mg/ml 69 + 3-1 63 211 26

Group 3Cells pre-treated withpepsin-digested aggregatedIgG as above locomotingtowardsNo attractant 58 + 1-7 104 318 50Casein 350 pg/ml 73 + 4-3 79 360 49Casein 700 pg/ml 103 + 2.0 94 571 63

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P. C. Wilkinson

Table 4. Locomotion of human blood neutrophils on filters coated withaggregated IgG (I mg/ml)

Locomotion of human neutrophils (5 x 105/ml)in HSA (1 mg/ml) in 60 min

Attractant below filter Distance migrated by Number of locomotingleading front (pm) cells per field

(1) Control filtersHanks's + HSA (1 mg/ml) 29 + 1-2 148Casein 250 pg/ml 39 + 1 6 111Casein 1000 pg/ml 63 + 3 5 158

(2) IgG-coated filtersHanks's + HSA (1 mg/ml) 16 + 1-9 14Casein 250 pg/ml 28 + 3 2 83Casein 1000 pg/ml 54 + 1 5 151

is supported by the findings (a) that an IgG con-centration of 5 mg/ml was required for cells to detectthe cell-released factor, whereas cells detected grad-ients of aggregated IgG optimally when the IgG wasused at concentrations below or around 1 mg/ml; (b)that increasing cell numbers did not increase theirlocomotion to aggregated IgG; (c) the checkerboardresults in Table 2 are consistent with a response to amaterial present at maximum concentration belowthe filter (the IgG) and not to a material present atmaximum concentration above or within the filter,i.e. a material released by the cells.

Inhibition of neutrophil locomotion by IgGPre-treatment of cells with IgG Jollowed by wash-

ing. Pre-treatment of neutrophils with monomericIgG followed by washing had little effect on thesubsequent locomotion of neutrophils towards a va-riety of chemotactic factors. However, in two experi-ments pre-treatment with aggregated IgG caused areduction of locomotion, not only to a variety offactors including casein, denatured albumin and for-myl methionyl peptides, but also in the absence of achemotactic agent. Table 3 shows the response tocasein, measured as both the leading-front distanceand the total number of cells entering the filter.Using the leading-front method, a reduction in loco-motion was obvious, but there was a far more ob-vious reduction in the number of cells entering thefilter. This suggests that a majority of cells wereinhibited from locomoting, but that the few that

could locomote were still able to respond to chemo-tactic factors since aggregated IgG-coated cells stillmigrated further towards casein than in the absenceof casein. The pepsin-digested IgG was less inhibi-tory than whole IgG and the leading-front valueswere near control values, however there was stillsome reduction in the numbers of cells entering thefilter.

Pre-treatment oJ filters with IgG. Filters weresoaked with aggregated IgG (sample 2; 630 for 30min; incubated with the filters at 1 mg/ml for 45min), then washed and used in a study of neutrophillocomotion towards casein, using uncoated filters ascontrols. On uncoated filters (three experiments),neutrophils showed a vigorous response to casein,while, in comparison, the response on coated filterswas inhibited in all of three experiments. Few cellsmigrated into the aggregated IgG-coated filters(Table 4). These results essentially confirm those ofKemp & Brown (1980).

DISCUSSION

The results presented above support earlier findingsthat IgG can both stimulate and inhibit leucocytelocomotion depending on the assay conditions used.In our hands, chemoattraction by IgG is correlatedwith denaturation of the IgG sample, judged both byANS fluorescence and by polymerization. The cellsshow a chemotactic response to denatured IgG

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Human IgG and human neutrophil locomotion 465

(Table 2), as they do to other denatured proteins(Wilkinson, 1973; Wilkinson & Allan, 1978b) andthis response probably represents a recognition bythe neutrophils of disordered protein structure, asdiscussed elsewhere (Wilkinson, 1978). However, thelocomotor response of the cells to denatured IgG isnot usually as pronounced as those we have observedearlier to denatured HSA, probably because at thesame time as it stimulates a chemotactic response,the IgG also depresses cell locomotion, as judged bythe decline in numbers of cells migrating into filtersas the IgG concentration is raised above 500 ig/ml(Table 1). This decline in numbers is even morestriking if the cells are pre-treated with aggregatedIgG rather than first exposed to it in the filter (Table3). It seems probable that the inhibitory effects ofIgG on neutrophil locomotion are related to its ef-fects on cell adhesion, described by Keller et al.(1979a). In their studies, pre-treatment of neutrophilswith polymerized IgG reduced their adhesion to sub-strata, and pre-treatment with monomeric IgGcaused an increase in adhesion. It is possible thateither a decrease or an increase in adhesion couldupset the balance between attachment and detach-ment which is necessary for cells to move across asubstratum, thus either could prevent cell loco-motion. Moreover, substratum-bound IgG wouldbind with fairly high affinity to Fc receptors on cellslocomoting across the substratum, and thus mighttether the cells, unless the latter possessed a mech-anism for detaching themselves, e.g. by releasingproteases. In the experiments of Keller et al. (1979b),cells in gradients of IgG showed a graded adhesionto the substratum, since cells nearest the gradientsource were strongly adherent and those furtheraway were less so. The cells tended to move towardsthe gradient source in filters, but the authors did notobserve orientation in an orientation chamber(Zigmond, 1977), thus they ascribed the accumu-lation to biased chemokinesis rather than to chemo-taxis or to haptotaxis (Carter, 1967). Our preliminaryobservations (Wilkinson & Allan, unpublishedresults) support an increase in cell-to-substratumadhesion mediated by IgG, but, in orientationassays, we observed that though few (<40%) of thecells showed any polarization in the presence ofdenatured IgG and most were flattened, between 55and 70% of those with a discernible locomotor mor-phology were oriented towards the IgG source. Wefeel that this, together with the checkerboard assayresults (Table 2), supports a tactic reaction rather

than a biased kinesis, but the point requires morestudy.

Aggregated IgG clearly has effects on leucocytelocomotion other than the direct effects discussedabove. It activates complement and also causes neu-trophils to release cell-derived factors as first shownby Zigmond & Hirsch (1973). Whether those factorsreleased by neutrophils in contact with aggregatedIgG are products of the cells themselves or representdigested fragments of the IgG aggregates has notbeen determined. It is of interest that the experimentsof Zigmond and Hirsch also showed a dual effect ofIgG aggregates, since factors released by cells boundto surfaces coated with such aggregates stimulatedchemotaxis of neighbouring neutrophils but oncethese neutrophils reached the aggregate streak, theyflattened out and became immobilized.The results presented here and those of Kemp &

Brown (1980) also pose questions about the best wayto measure leucocyte locomotion in filter assays,since cells on IgG-coated filters can show a reducedresponse if a count is made of the number of cellsmoving into the filter, at the same time as they showan enhanced response if the leading-front count isused. The two methods do not, in this case, measurethe same thing, thus it is necessary to have infor-mation about both numbers of moving cells and thedistance they move in order to make a full assess-ment of locomotion in the filter assay.

ACKNOWLEDGMENTS

The author wishes to thank Drs John Lackie andRobert Allan for helpful criticisms of thismanuscript.

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