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Chemiluminescence of asbestos-activated macrophages

Citation for published version:Donaldson, K & Cullen, RT 1984, 'Chemiluminescence of asbestos-activated macrophages', British journalof experimental pathology, vol. 65, no. 1, pp. 81-90.

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Br. J. exp. Path. (I984) 65, 8I-90

Chemiluminescence of asbestos-activated macrophages

K. Donaldson' and R.T. Cullen2'Pathology Branch, Institute of Occupational Medicine, Roxburgh Place, Edinburgh, and 2Host Defence

Laboratory, Royal Hospital for Sick Children, Sciennes Road, Edinburgh, Scotland

Received for publication I3 September I983

Summary. Chemiluminescence, a measure of reactive oxygen species release by phagocytes,was compared in peritoneal exudate macrophages elicited with chrysotile asbestos, Corynebac-terium parvum and saline. Chrysotile asbestos- and C. parvum-activated macrophages producedsignificantly more chemiluminescence than saline-elicited macrophages. In a second series ofexperiments the ability of opsonized chrysotile asbestos to act as a trigger for the release ofchemiluminescence was tested. Opsonized chrysotile asbestos produced a dose-related releaseof chemiluminescence from activated macrophages except at the highest dose wherechemiluminescence was reduced due, possibly, to a toxic effect of chrysotile during the assay.Opsonized latex also triggered a dose-related chemiluminescent response from activatedmacrophages. The potential role of toxic reactive oxygen species, released from macrophages,in the development of asbestos-related pulmonary inflammation and fibrosis are discussed.

Keywords: chemiluminescence, asbestos-activated macrophages

Pulmonary interstitial fibrosis (asbestosis) isa major disease associated with asbestosinhalation (Becklake 1976; Selikoff & LeeI 9 78) and is typified by increased depositionof interstitial collagen and fibroblast prolifer-ation. Fibrosis is a common sequel to chronicinflammation and the ability of asbestos togenerate inflammation in the lung has beendiscussed as a factor in the aetiology ofasbestos-associated pulmonary interstitialfibrosis (Hamilton I980; Miller I978). Thecentral role of the alveolar macrophage ininteracting with inhaled particles makes itlikely that alteration in the activities of thesecells in the lungs of individuals inhalingasbe.,tos could be important in the patho-genesis of asbestos-related diseases. Evidencethat the macrophage may play a central rolein asbestos inflammation has come fromstudies demonstrating macrophage-activat-ing properties of asbestos (Davies et al. I 9 74;Hamilton et al. I 9 76; Miller & Kagan I 9 76;

Sirois et al. I980; Donaldson et al. I982,I98 3a,b). Macrophages from inflammatorysites have been found to be activated andcapable of releasing an extensive array ofbiologically-active substances (Allison et al.1978; Nathan et al. I980). One group ofthese secreted molecules, the reactiveoxygen species (ROS): hydrogen peroxide(H202), superoxide (02), hydroxyl radical(OH-) and singlet oxygen (102), have beenimplicated in the tumoricidal and microbici-dal functions of leucocytes (Nathan I982)and in tissue damage at inflammatory foci(Fantone & Ward I982).

In the present study we assessed therelease ofROS from asbestos- and Corynebac-terium parvum-elicited mouse peritonealmacrophages by measuring lucigenin-amplified chemiluminescence in response toa variety of stimuli including asbestos. Luci-genin-amplified chemiluminescence in leu-cocytes is a measure of superoxide release

8i

K. Donaldson & R. T. Cullen

(Allen I 98 I; Williams & Cole I 9 8 I b). Inprevious studies we have characterized theasbestos-elicited mouse peritoneal macro-phage as a model asbestos-activated macro-phage (Donaldson et al. I982, I98 3a,b).

Materials and methods

Mice. Outbred CFi mice 6-8 weeks of age atthe time of injection were used throughout.

Peritoneal exudate cells. Peritoneal exudatecells (PEC) were harvested 5 days afterintraperitoneal injection of 2.5 mg chrysotileasbestos (Union International Contre Cancersample) in 0.5 ml Dulbecco phosphate-buf-fered saline A (DulA), 0. 5 ml DulA or 0.2 ml(0.4 mg) Corynebacterium parvum (heat-killed; Wellcome). Mice were killed by etherinhalation and the cells were harvested bythree 2-ml washes with DulA containing iounits/ml Heparin (Pularin) into ice-cold plas-tic tubes. The cells were washed, countedand maintained in Hams F1o medium con-taining io% heat-inactivated fetal calfserum(FCS) (GIBCO) on ice until assay when theywere transferred to Hanks solution withoutphenol red.

Neutrophil depletion. Neutrophil depletionwas by centrifugation through sodiummetrizoate; Io X 106 PEC in 5 ml of F1o andio% FCS were layered on top of sodiummetrizoate (Lymphoprep; Nyygaard) in aio-ml siliconized centrifuge tube and centri-fuged at 400 g for 30 min. The cells at theinterface were retrieved and washed beforeuse.

Characterization of PEC. To determine thecellular composition of the PEC, cytospinpreparations were made, stained withWright stain and differential counts carriedout.

Triggers ofROS release. The following triggersfor the stimulation of ROS release were used.(a) Opsonized zymosan: zymosan wasopsonized by incubation at 3 70C for 30 min

in Io% Human AB serum, washed andadjusted to I0 mg/ml in Hanks solutionwithout phenol red. (b) Opsonized latex:latex particles (Sigma, I.09 ,um diameter)were opsonized as above and adjusted to finalconcentrations of I, 5 or IO%. (c) Opsonizedchrysotile asbestos: chrysotile asbestos(UICC) was opsonized as above, washed andadjusted to final concentrations of I, 5, IOand 20 mg/ml.

Lucigenin-amplified chemiluminescence. Che-miluminescence (CL) was measured directlyin mV in an LKB 1250 luminometer. Cellswere prepared at 5 x Io6/ml in cold Hanksand maintained on ice. Two hundred micro-litres of cells were transferred to polystyrenecuvettes (LKB) followed by 200 P1 of luci-genin (io-3M). The cuvettes were placed inthe luminometer and the baseline CL wasallowed to develop. When this had plateaued500 ,ul of trigger were added. Followingaddition of trigger, CL was monitored until itpeaked.

Viability assay. To determine the extent ofany toxic effect of asbestos on PEC, so-,Isamples of cells were removed from a typicalCL assay (C. parvum PEC; I0-3M lucigenin;io mg/ml chrysotile) and percentage viabi-lity determined by trypan blue exclusion for200 cells.

Use ofenzymes. In order to determine the roleof different reactive oxygen species in CL,superoxide dismutase (Sigma; 50 ,ug/ml) andcatalase (Sigma; 25 Mig/ml) were included intwo separate experiments with opsonizedzymosan- and C. parvum-elicited PEC.

Statistics. The statistical significance of repli-cate experiments was analysed using Stu-dent's t-test and analysis of variance.

Results

Characteristics of CL

Baseline CL. As discussed by Williams & Cole

82

Asbestos macrophage chemiluminescence

( I 98 ia) the addition of PEC and lucigenin tothe polystyrene cuvettes resulted in a releaseof CL. Trigger was added after this reached a

plateau. In each experiment this baseline CLwas subtracted from the final peak CL toobtain the true peak CL due to the trigger.The magnitude of the baseline CL varied butwas always low for the saline-induced PEC(0.4±0.2: mean±SD) and was increasedwith C. parvum- (7.2 ± 6.5) and chrysotileasbestos- (5.4+ 6.o) elicited PECs.

Effect of enzymes on CL. Fig. i shows that CLis inhibited by superoxide dismutase (SOD)and unaffected by catalase. The same resultwas obtained in two separate experi-ments.

Role of polymorphonuclear neutrophils (PMN)in CL by peritoneal exudate cells. In order todetermine the role ofPMN in CL by activatedPEC the PEC were centrifuged through lym-

phoprep to reduce the proportion of granulo-cytes. The cells at the interface had a mean

reduction in PMN of 40% but. as shown inTable i, this had virtually no effect on peakCL when the same number of cells were

compared. It was therefore assumed thatPMN and macrophages from the activatedPEC used in these experiments releasedapproximately equal quantities of CL. Conse-

quently the proportion of CL due to PMN inany activated PEC was approximately equalto the percent PMN present (Table 2) and no

further attempt was made to separate macro-phages from neutrophils.

70 -

Catalase

No enzymew

> 50 -

E

C

E 30

E

dilruase

0 2 4 6 8 10Time after addition of zymosan (min)

Fig. i. Effet of superonde dismutase (50 pg/ml)and catalase (25 pg/il) on opsonized zymosan-triggered chemiluminescence of C. parvum-elicitedPEC (5oo pl of io mg/ml opsonized zymosan

added at time o). The same result was obtained intwo separate experiments.

Table i. Effect of separation on sodium metrizoate on proportion of polymophonuclear neutrophils andchemiluminescence of C. panrum- and chrvsotile-elicited peritoneal exudate cells: opsonized zymosan as atrigger

Polymorpho-nuclear Change

Agent eliciting neutrophils (%) Reduction in in peakperitoneal exudate Experiment polymorphonuclear chemiluminescence

cells no. Before After neutrophils %) (%)

C. panrum I 23.1 12.2 47.2 -2.72 18.4 8.3 54.9 -11.2

Chrysotile I 15.7 10.7 31.8 -4.02 31.8 23.3 26.7 + 3.1

All experiments - 40.6 - 3.1±13.9 ±6.8

83

K. Donaldson & R. T. CullenTable 2. Percent macrophages and polymorphonuclear neutrophils in various peritoneal exudate cells.Remainder of cells were lymphocytes with low (< 5%) numbers of eosinophils and basophils

Agent elicitingperitoneal exudate Polymorphonuclear Macrophages No. of

cells neutrophils (%) (%) experiments

Saline 5.8 + 2.4 70.8 i16.2 3C. parvum 15.2+5.4 69.7+6.2 5Chrysotile 21.4+ 7.3 55.8+6.3 4

Results are mean + SD.

CL responses of different PECs

Fig. 2 shows the pooled results of all experi-ments measuring the CL response, toopsonized zymosan, of PEC elicited withsaline, C. parvum or chrysotile asbestos.Initial analysis of the peak CL data from allexperiments (Table 3) suggested approxima-tely equal variance in the scale of logarithmmV and analysis of variance was thereforecarried out in this scale. Two effects wereapparent from the analysis of variance:Firstly, small but significant systematic var-iation in the absolute values of peak CLbetween experiments. The highest and low-est values differed by a factor of i.6 and itwas not possible to identify, retrospectively,the source of this variation. Secondly, takinginto account this temporal variationbetween experiments there were highly sig-nificant differences between the differentPECs with C. parvum PEC being greater thanchrysotile PEC by a factor of I .3 and chryso-tile PEC greater than saline PEC by a factor of2.0.

CL in response to asbestos and latex

Fig. 3 shows a typical dose-response of CL toopsonized latex with 5-day C. parvum-elicitedPEC. Fig. 4 shows the response of the samecells to increasing doses of opsonized chryso-tile asbestos and a clear dose-response isevident over I, 5 and io mg/ml; at 20

Tim ft.r addition10 20ww(nrmm:-*#r.atim

Fig. 2. Chemiluminescence response of peritonealexudate cells elicited with saline (0), C. parvum(-) or chrysotile asbestos (0) to opsonized zymo-san (500 pl of io mg/ml opsonized zymosanadded at time o). Mean + standard deviation ofthree (saline), four (chrysotile) and five (C. par-vum) separate experiments.

84

Asbestos macrophage chemiluminescenceTable 3. Combined results ofreplicate experimentsto measure the peak chemiluminescence responseof saline-, C. parvum- and chrysotile-elicited peri-toneal exudate cells in response to opsonizedzymosan

Agent eliciting Peak No. ofperitoneal exudate chemi- experi-

cells luminescence ments

Saline 27.4± 5.5 3C. parvum 66.i±14.2 5Chrysotile 54.5+12.7 4

Results are mean + SD.Analysis of variance showed significant

differences between peritoneal exudatecells elicited by the three different agents: C.parvum > chrysotile> saline (see results).

..J.

> >tA; ''A frs§ji / i

Fig. 3.r Ccerpse of -da C.

pavu PEC ,to ot.pson72ize latex at i,r{ an ti0% 6

separate ;exerimen*its. i4 7E-

; .Jf}' .@ tsuct')F) ,$iiz t 2J 6 tIr 6 g . w<.;§; ix

,,, ....,.,ArJ h4Xt-R;i /JO f. i 8.i.

Fi. r32.Chem|iluminscenc respns Of 5,-day C>!.}||pavm.E toi- opsonize late at 1, an i o

.,00 ..l ojd>,,flatex at the stated concentrations added0 91^'t

atFime o. Themilumiecneresulpwsobansed in twoC

separate experiments.

S 20Em

nc

.)

CDa)lo

10 mg/ml

5 mg/ml

20 mg/ml

1 mg/ml

1 2 3 4 5 6 7 8Time after addition of chrysotile (min)

Fig. 4. Chemiluminescence response of s-day C.parvum-elicited PEC to opsonized chrysotile at i,

5, iO and 20 mg/ml (5OO ,ul of opsonizedchrysotile, at the stated concentrations added attime o). The same result was obtained in twoseparate experiments.

mg/ml, however, the response is reduced toless than that found with io mg/ml.

In order to determine whether toxicitymight be a factor in the loss of dose-responseat high opsonized chrysotile concentration, a

time course of cell viability was taken duringa CL assay (C. parvum PEC; IO-3M lucigenin;io mg/ml opsonized chrysotile). As shown inFig. 5 there is 30-40% toxicity to cells withinthe timespan of a normal assay ( I0-20 min).

Table 4 shows the pooled peak CL responsefrom replicate experiments using C. parvum-elicited PEC with opsonized zymosan andopsonized chrysotile as triggers. It is evidentthat opsonized zymosan elicited a signifi-cantly greater CL response than opsonizedchrysotile on an equal-mass basis.

85

K. Donaldson & R.T. Cullen

100lr

80

6010

50

> 40E0)

30U.00)C

E 20.2

0)c5 10:

40 _

0---O

.0

.T

Ei

u20

1 5 10 20Time after addition of chrysotile (min)

Fig. _5. Change in viability of C. parvum PEC duringa chemiluminescence assay with io mg/mlopsonized chrysotile as trigger added at time o.

Table 4. Effect of different triggers on the peakchemiluminescence response of C. parvum peri-toneal exudate cells

Peak No. ofchemi- experi-

Trigger luminescence ments

Opsonized zymosan 66.i ± 14.2* 5Opsonized chrysotile 14.2 ± 7*3* 5

Results are mean± SD.* Significance of difference between zymo-

san and chrysotile: P<o.ooi.

Figure 6 shows that on reaching a plateauof CL in response to io mg/ml opsonizedchrysotile the cells are still sufficiently viableto respond to a further stimulus fromopsonized zymosan.

Discussion

In the present study we set out to determinewhether asbestos could elicit activated mac-rophages with increased ability to releasereactive oxygen species (ROS) and also

0z

2 4 6 8 10Time after addition of chrysotile (min)

12

Fig. 6. Chemiluminescence response of 5-day C.parvum PEC to opsonized chrysotile added as 500MI of io mg/ml at time o; on reaching a plateauopsonited zymosan was added (OZ); typical result.

whether asbestos could act as a trigger forthe release of ROS from activated macro-phages. Activated peritoneal macrophageselicited by asbestos injection were used sincethese have been previously characterized,while saline- and C. parvum-elicited macro-phages were used as unactivated and acti-vated controls respectively (Donaldson et al.I982; Cullen I978). As a measure of ROSrelease, lucigenin-amplified chemilumines-cence (CL) was used. Chemiluminescence iscorrelated with other indices of the respira-tory burst including 02 consumption, hexosemonophosphate shunt activity, H202 releaseand 0° release (Johnston et al. I980) whichare induced in phagocytes by phagocytic ormembrane-perturbing stimuli (Schadelin etal. I980). Lucigenin-amplified chemilu-minescence is correlated with 0° release inmacrophages (Williams & Cole I98 ib) and areaction sequence for the interaction of 02with lucigenin in the generation of CL hasbeen described (Allan I98I). In preliminaryexperiments we gained support for the in-volvement of 0° in CL by demonstrating thesuperoxide dismutase-dependent abolition ofCL while catalase had no effect on CLresponse.

86

Asbestos macrophage chemiluminescenceIn the first set ofexperiments reported here

the chrysotile asbestos-elicited PEC wereshown to release significantly more CLthan saline-elicited PEC in response to theconventional trigger of ROS release-opsonized zymosan. C. parvum-elicited PEC,however, released significantly more CL thanchrysotile-elicited PEC and phorbol myristateacetate (PMA), a non-particulate mem-brane-perturbing agent, produced the samepattern of CL response from the three differ-ent PECs (data not shown). As discussedbelow the macrophage component of thePEC contributed the major part of the CLresponse.

Asbestos is thus similar to other macro-phage-stimulating agents such as casein, C.parvum, BCG and virus infections, endotoxinand thioglycollate in causing increased che-miluminescence or ROS release in responseto membrane perturbation or phagocytosis(Nathan & Root 1977; Schleupner & Glas-gow 1978; Johnston et al. I980).The finding that the asbestos-elicited mac-

rophages used here show increased CL isconsistent with other markers of activationwhich have been reported for macrophagestreated with asbestos in vivo and in vitro(Davies et al. 1974; Hamilton et al. 1976;Miller & Kagan 1976; Sirois et al. I980;Donaldson et al. I982, I983a,b) and waspredicted in a previous study on the basis ofincreased lectin-induced capping in asbestos-activated macrophages (Donaldson et al.I983a).From the outset of these studies we were

primarily interested in the macrophage com-ponent of the PEC although substantialnumbers of PMNs could be present in somepreparations. Depletion ofPMNs from withinactivated PEC samples, by centrifugationthrough lymphoprep, resulted in an averagereduction in PMNs of 40% with no ap-preciable reduction in the CL response ofthese PEC. We therefore concluded thatCL of activated macrophages and PMNswas equal in magnitude on a cell-for-cellbasis. Consequently the CL due to PMNs inany activated PEC was equivalent to the

percentage of PMN present in the PEC. Itis important to note, however, that thestudy of Sykes et al. (I982), and ourown unpublished data, indicate that pul-monary deposition of pathogenic dust maywell result in the presence of PMNs amongstthe free alveolar cell population where theycould contribute significantly to the ac-cumulation of ROS if appropriately stimu-lated. Mixed macrophage/PMN preparationstherefore constitute a relevant population forstudy.The second set of experiments described

above confirm that asbestos can act as atrigger for the release of ROS from activatedmacrophages, as measured by CL, althoughthe response to opsonized chrysotile wasmuch less than that produced by opsonizedlatex. The dose dependency of the CL re-sponse to chrysotile asbestos which wasevident at the low and intermediate doseswas lost at the highest dose (20 mg/ml) duepossibly to a toxic effect of such a largeasbestos dose; testing for cell viability duringan assay confirmed a toxic effect of asbestosunder the conditions of the assay. Thistoxicity was evident despite the fact that theasbestos fibres were opsonized with humanserum and the acute toxic effects of asbestosare reduced by protein coating of the activefibre surface (Miller 1978). However, in-creased susceptibility of activated macro-phages to the toxic effects of asbestos in vitro,in the presence of serum, has been previouslyreported from our laboratory (Wright et al.I983).We appreciate that the asbestos dose used

to trigger CL in the present study was veryhigh and far in excess of those encountered,for example, in the lungs of animals inhalingasbestos. The relatively low sensitivity of thechemiluminescence amplification and detec-tion system, however, necessitated the use ofsuch high doses and we do not believe thatthis diminishes the potential relevance of thefindings.Two recent studies have reported the

effects of asbestos in triggering CL responsesin phagocytes; these studies revealed that

87

88 K. Donaldson & R. T. Cullenperipheral blood PMNs could be stimulatedby asbestos to release ROS (Doll et al. i 982a)while peripheral blood monocytes did notshow this response (Doll et al. i982b). Ouruse of activated macrophages as indicatorcells in the present study is supported byreports of the macrophage-activating poten-tial of asbestos (Davies et al. I 9 74; Hamiltonet al. I 9 76; Miller & Kagan I 9 76; Sirois et al.I980; Donaldson et al. I982) and the centralrole of activated macrophages in inflamma-tion (Allison et al. I 978).The in-vivo relevance of the present find-

ings lies in the possibility that activation ofmacrophages in the lungs of individualsinhaling asbestos could lead to localizedaccumulation of ROS. We do, however,recognize the metabolic and functional dif-ferences between peritoneal and alveolarmacrophages although ROS release byalveolar macrophages has been reported(Williams & Cole 198 ib). In this regard it isnotable that Miller & Kagan (I976) havereported evidence of alveolar macrophageactivation in the lungs of rats inhalingcrocidolite asbestos.

Evidence that ROS could have local toxicand tissue damaging effects is derived fromexperiments where both cell-free enzymesystem-derived, and phagocyte-derived,oxygen radicals have been found to causeendothelial cell damage (Sacks et al. I978),red cell lysis (Kellogg & Fridovich 1977),fibroblast toxicity (Simon et al. I98I) andautotoxicity to PMNs (McCord & WongI979). These toxic effects are thought to bebrought about largely by peroxidation ofmembrane lipids leading to structural disor-ganization of the cell membrane (SlaterI979). It is possible therefore that, in thelungs of individuals inhaling asbestos, afactor in the maintenance of an inflamma-tory response is localized release of increasedlevels of ROS. The asbestos-activated macro-phages could be envisaged as being primed toproduce increased amounts of ROS, therelease of which could be triggered by con-tact with any of a number of inhalableparticles reported to have this triggering

property such as bacteria, yeast (Williams etal. I 9 8o), pollen (Lindberg et al. I 98 2) and,as shown in the present study, asbestos itself.If an excess of ROS in the alveolar spacesovercame the normal scavenging systemsthis could lead to epithelial damage withsubsequent activation of inflammatory cas-cades leading ultimately to fibrosis (PickrellI98I).

Acknowledgements

We would like to acknowledge the financialassistance ofthe Asbestosis Research Council(K.D.), the Cystic Fibrosis Research Trust andE. Lilly Industries Limited (R.T.C.). We wouldlike to thank Brian Miller for statisticalanalysis and Drs J.A. Raeburn and R.E.Bolton for critical reading of the manuscript.

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