Supernatants from in vivo asbestos-activated macrophages failed to show any cytostatic activity against a syngeneic fibrosarcoma cell line in vitro. UICC chrysotile-induced peritoneal exudate cells also failed to demonstrate any growth inhibitory effect on the same cells in Winn assays of tumor growth. Mixing UICC crocidolite with inoculated tumor cells resulted in a dose-dependent inhibition of tumor growth; this could, however, be explained by a direct cytostatic effect on the tumor cells of high doses of crocidolite, which was observed in vitro

A Ewing

Ames B. N.

Cohn Z. A.

Davies P.

Gabizon A.

J M Davis

K Donaldson

K James

Keller R

Lowry 0.

Pylev L. N.

Raz A.

Topping D. C.

Environmental Health Perspectives

English

PubMed

Donaldson, Ken

Davis, J. M.

Ewing, A

James, K

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Interactions of asbestos-activated macrophages with anexperimental fibrosarcomaCitation for published version:Donaldson, K, Davis, JM, Ewing, A & James, K 1983, 'Interactions of asbestos-activated macrophages withan experimental fibrosarcoma' Environmental Health Perspectives , vol 51, pp. 97-101.Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher final version (usually the publisher pdf)Published In:Environmental Health PerspectivesGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015Environmental Health PerspectivesVoL 51, pp. 97-101, 1983Interactions of Asbestos*ActivatedMacrophages with an ExperimentalFibrosarcomaby K. Donaldson,* J. M. G. Davis,* A. Ewingtand K. JamestSupernatants from in vivo asbestos-activated macrophages failed to show any cytostatic activityagainst a syngeneic fibrosarcoma cell line in vitro. UICC chrysotile-induced peritoneal exudatecells also failed to demonstrate any growth inhibitory effect on the same cells in Winn assays of tu-mor growth. Mixing UICC crocidolite with inoculated tumor cells resulted in a dose-dependent inhi-bition of tumor growth; this could, however, be explained by a direct cytostatic effect on the tumorcells of high doses of crocidolite, which was observed in vitro.IntroductionRecently there has been an accumulation of ex-perimental evidence suggesting that asbestos canhave a stimulatory or activating effect on macro-phages in vivo and in vitro. Davies et al. (1) reportedthat peritoneal macrophages treated with asbestosin vitro released lysosomal hydrolases without evi-dence of cell death, and Hamilton and co-workers (2,3) demonstrated that asbestos in vivo and in vitroinduces selective release of the neutral proteaseplasminogen activator from peritoneal macro-phages. Humes et al. (4) and Sirois (5) have reportedthe induction of prostaglandin release by peritonealand alveolar macrophages treated with asbestos invitro. Selective release of lysosomal hydrolases andneutral proteases from macrophages can be inducedby a range of macrophage activating agents, bothimmunological and nonimmunological (6).Miller and Kagan (7) have reported that the alve-olar macrophages of rats inhaling crocidolite showevidence of activation by morphology, Fc receptoravidity and the ability to stimulate T-lymphocytesto take up thymidine. We have studied the macro-phages induced in the peritoneal cavity by intra-peritoneal asbestos injection and have found maco-phages to be activated by several criteria (8, 9) but*Pathology Branch, Institute of Occupational Medicine,Roxburgh Place, Edinburgh, Scotland.tUniversity of Edinburgh, Department of Surgery, TeviotPlace, Edinburgh, Scotland.full activation to the tumor cell cytotoxic state wasnot found (8). Small significant reductions in the sizeof experimental subcutaneous fibrosarcomas were,however, found in mice which had received UICCchrysotile intraperitoneally compared with micewhich had received saline intraperitoneally.In this paper we present further studies into theinteractions of asbestos, macrophages and an ex-perimental fibrosarcoma.Materials and MethodsMacrophage Plasma Membrane5'-Nucleotidase and Lysosomal AcidPhosphataseThese assays were carried out according to themethod of Raz et al. (10) using glycerophosphate(Sigma) and 5'-adenosine monophosphate (Sigma) assubstrates, and assaying for phosphate release bythe method of Ames and Dubin (11). Protein was as-sayed by the method of Lowry (12).Experimental FibrosarcomaThe experimental tumor line used was the CCH,fibrosarcoma derived from a subcutaneous methyl-cholanthrene-induced fibrosarcoma in CBA/Ca mice.It has been maintained in long-term culture and pas-saged through mice at intervals (13).DONALDSONETAL.Macrophage Supernatants, Leachate andAsbestosPeritoneal exudate cells (PEC) were harvested 3days after intraperitoneal injection of UICC crocido-lite, chrysotile, latex spheres (0.81 pm diameter, Dif-co) or saline. The macrophages were maintained inculture over 24 hr in complete RPMI (RPMI 1640,Gibco) containing 10% fetal calf serum (Gibco) andantibiotics (cRPMI). The supernatant was centri-fuged, aliquoted and stored frozen at - 200C untiluse. Leachate was prepared by incubating crocido-lite or chrysotile (5 mg/mL) in cRPMI for 3 days at370C then spinning out the fiber by centrifuging at3500g for 1 hr. Asbestos was serum-coated to re-duce direct cytotoxic effects by preincubating for 1hr at 370C in cRPMI.CCH1 Tumor Cell Proliferation AssayThe same assay system was used to measure theeffect of macrophage supernatants, asbestos or as-bestos leachate on CCHI cell proliferation in vitro.5 x 104 CCH, cells were inoculated into microtiterplates (Sterilin) in 100 ML of cRPMI. Various vol-umes of supernatant, leachate or asbestos (preincu-bated in cRPMI for 1 hr at 370C) were then added,and the final volume was adjusted to 200,L withcRPMI. After 24 hr, 0.25 ,ACi of 3H-thymidine wereadded to each well and after a further 24 hr thecells were harvested in a cell harvester (Skatron);cell-bound 3H-thymidine counts were measured byliquid scintillometry.Winn AssayThe assay used was essentially the same as thatdescribed by Gabizon and Trainin (14). PECs ob-tained 3 days after IP injection of saline or chryso-tile (5 mg) were harvested, counted and mixed withCCH, tumor cells in the following ratios: (1) 10:1, i.e.,105 CCH, cells plus 106 PEC in 0.1 mL Dulbecco'sphosphate buffered saline (Dul. A) and (2) 100:1, i.e.,5 x 101 CCH, cells plus 5 x 104 PEC in 0.1 mL DulA. Groups of 10 mice received 0.1 mL subcutane-ously in the right hind limb. Tumor growth wasmonitored by taking the mean of the two major di-ameters of the tumor.Table 1. Plasma membrane 5'-nucleotidase andlysosomal acid phosphatase activity in 3-day saline-and 3-day chrysotile-induced macrophages.5'-Nucleotidase, Acid phosphatase*mole phosphate! *mole phosphate/Macrophage source mg protein/hr mg protein/hrSaline-induced(3-day) 3.7 0.4Chrysotile-induced(3-day) 0.5 4.7Chrysotile was arbitrarily chosen for this assaysince previous studies (8) had shown crocidolite andchrysotile to be similar in their ability to activatemacrophages.Inclusion of Crocidolite in Tumor CellInoculumUICC crocidolite was incubated in normal mouseserum for 1 hr at 370C and then mixed with CCH,cells so that 0.1 mL of inoculum contained 10 ,L ofnormal mouse serum; 0, 5, 50, or 500 lAg of crocido-lite; and 5 x 102 CCH, cells. Groups of 10 mice re-ceived 0.1 mL subcutaneously in the right hind limband tumor growth was monitored.Statistical AnalysisAll differences were examined for statistical sig-nificance using Student's t-test.ResultsTable 1 shows that chrysotile-induced macro-phageshave decreased plasma membrane 5'-nucleo-tidase and increased acid phosphatase content, com-pared to saline-induced macrophages.The effect of 24-hr supernatants of macrophagesinduced by various agents on tumor cell prolifera-tion is shown in Figure 1. Statistical analysis re-w4( 1000-WZzo>Wc10~SAL. LATEX CRO. CHRYS.SUPERNATANT (%')FIGURE 1. Effect of supernatants from 3-day saline-, latex-,crocidolite- or chrysotile-induced peritoneal exudate macro-phages on proliferation of tumor cells in vitro as measured by3H-thymidine uptake.i l Lro V . n98EXPERIMENTAL FIBROSARCOMA INTERACTIONvealed no significant effects of macrophage super-natants from any source, on CCH1 cell proliferation,compared to CCH, cells alone. There was consider-able variation in the response of CCH1 cells to thesesupernatants between experiments, and this is re-flected in the large amount of variation evident inFigure 1, which represents the mean and standarderrors of five separate experiments.Although there were differences in the mean tu-mor diameter produced by tumor cells alone, tumorcells mixed with saline-induced PEC and tumor cellsmixed with chrysotile-induced PEC, at both 10:1and 100:1 (Figs. 2 and 3), these differences were notstatistically significant.Figure 4 shows a dose-dependent inhibition of tu-OTUMOUR CELLS ONLYo +SALINE PE.C* +CHRYSOTILE PECi 5 *-i,E5LULUj+1+Z10 1 3 72Qxw0DAYSFIGURE 2. Effect of 3-day saline- and 3-day chrysotile-inducedperitoneal exudate cells on subcutaneous tumor growth in aWinn assay; peritoneal exudate cell: tumor cell ratio of 10:1 atsubcutaneous inoculation. Although no positively cytostaticWinn assay control was included in these experiments, in-traperitoneal C. parvum is known to inhibit this tumor modelby inducing tumor cell cytotoxic macrophages (8).* TUMOUR CELLS ONLYO +SALINE PE.C.1 4 + CHRYSOTILE PE.C.,E_ 12.H10.<+I 8-CZDWu 6-:D 4-2-DAYSFIGURE 3. Effect of 3-day saline- and 3-day chrysotile-inducedperitoneal exudate cells on tumor growth in a Winn assay;peritoneal exudate cell:tumor cell ratio of 100:1 at subcutane-ous injection.mor growth produced by mixing crocidolite thathad been preincubated with normal mouse serumwith the CCH1 tumor cell inoculum.The effects of crocidolite and chrysotile, at doseswhich extend over four orders of magnitude, on pro-liferation of CCH, tumor cells in vitro are shown inFigure 5; remarkably little effect is seen except atthe very highest doses of asbestos, where crocido-lite caused marked inhibition of 3H-thymidine up-take by tumor cells. Figure 5 also shows that croci-dolite leachate, at the highest concentration, pro-duced inhibition of 3H-thymidine uptake.0Iff ~~~~~~~~~~~~~5E10IUW500z 01 |Ul5DM:0I-~~~~~~~~~~~~,10 20 24 28 30DAYSFIGURE 4. Effect of tumor growth of 0, 5, 50 or 500 Mg of cro-cidolite percent along with tumor cells at subcutaneous inocu-lation.%LEACHATEFIGURE 5. Effect of various doses of asbestos and asbestosleachate on tumor cell proliferation in vitro.99DONALDSONETAL.DiscussionIn this paper we describe experiments whichwere aimed at further elucidating the degree of ac-tivation of macrophages induced in the murine peri-toneal cavity by injection of asbestos.Macrophage activation apparently involves thesequential adoption of properties which can culmi-nate in the fully tumoricidal state (15). In keepingwith other reports, we have found that asbestos,can lead to activation of macrophages in vivo but,using an in vitro assay, we failed to detect any evi-dence of tumor cell cytotoxicity by asbestos-acti-vated macrophages although Corynebacterium par-vum-activated macrophages were tumoricidal inthis system (8). However, in the same study, therewas the contradictory finding in vivo that the ad-ministration of asbestos by intraperitoneal injectionto mice caused a small but significant reduction inthe growth of an experimental subcutaneous tumor.The present study follows on from this work andexamines whether the tumor-retarding effect thatwas noted in vivo could be associated with macro-phage activation.Macrophage cytostatic activity, as well as cyto-toxic activity, can play a role in retarding tumorgrowth, and this can be mediated by soluble factors(16). The supernatants from asbestos-activated mac-rophages were therefore tested in an assay of CCH,tumor cell proliferation in vitro. No significant in-hibitory activity against tumor cell proliferationwas detected in crocidolite, chrysotile, latex or sa-line-induced macrophage supernatants using Stu-dent's t-test.In a further attempt to detect cytostatic activityin asbestos-activated macrophages, and in an effortto circumvent the problem produced by differencebetween the site of the tumor (leg) and the site ofprimary macrophage activation (the peritoneal cav-ity), two approaches were tried. In the first ap-proach, Winn assays were used to bring 3-day as-bestos- or saline-induced PEC into close contactwith the target tumor cells by mixing them to-gether at various effector: target ratios; this mix-ture was then inoculated and tumor growth com-pared to that of tumors produced with the appropri-ate number of tumor cells alone and with similar ef-fector: target ratios of saline-induced PEC andtumor cells. No significant differences in tumor sizewere obtained with chrysotile-activated macro-phages at ratios of either 10:1 or 100:1 compared tocontrols, and therefore no evidence of tumor cellcytostatic activity in asbestos-activated macro-phages was detected.In a second approach various doses of crocidoliteasbestos that had been preincubated in serum-con-taining medium for 1 hr were mixed with inocu-lating tumor cells and tumor growth monitored. Thedose-dependent decrease in tumor size which wasobtained could have been due to local macrophageactivation to the cytostatic state or could have beendue to a direct toxic effect of crocidolite on the tu-mor cells. It was specifically to avoid such a directtoxic effect that crocidolite was chosen rather thanchrysotile, since the amphiboles have a less activesurface than chrysotile, and this is also why the cro-cidolite was precoated by incubation in serum-con-taining medium. However, in order to test for sucha direct toxic effect of crocidolite on tumor cells,crocidolite was used in the in vitro CCH1 cell prolif-eration assay. It was clear that both chrysotile andcrocidolite and a fiber-free leachate of both asbestostypes produced very little effect at all doses, exceptfor inhibition at the highest doses with crocidolite.Since the doses of crocidolite present in inoculacompared closely with, or exceeded, the doses usedin the in vitro assay, it is evident that the reductionof tumor size produced could be accounted for by di-rect toxic effects of crocidolite on CCH, tumor cells.The induction of local tumor cell cytostatic macro-phages does not therefore have to be evoked as amechanism.It seems likely that the small but significant re-duction in the size of experimental tumors in miceinjected with chrysotile which we reported pre-viously (8) was not macrophage-mediated and thatthe overall adjuvant effect of asbestos could resultin a nonspecific mobilization of some of the otherwell-documented antitumor immune responses.The tumoricidal potential of macrophages hasbeen used in these studies in the context of assess-ing the degree of macrophage activation and the re-sults suggest that asbestos-activated macrophageshave not attained the fully activated tumoricidalstate. Tumor cell cytotoxic macrophages have beenconsidered to be part of the surveillance systemwhich acts to eliminate transformed cells before tu-mors can develop, and so the potential of asbestos-activated macrophages to attain full tumoricidal sta-tus has relevance for asbestos carcinogenesis. Pre-vious studies have produced evidence that asbestoscan act as a tumor promoter in classical two-stagecarcinogenesis after initiation by polycyclic hydro-carbons (17, 18). A recent report has shown that atumor promoter can block lymphokine-mediated ac-tivation of macrophages to the tumoricidal state(19). If this is shown to be a general property of tu-mor promoters, then it could be of particular rele-vance to asbestos carcinogenesis.The authors would like to acknowledge the financial sup-port of the Asbestosis Research Council (KD, JMGD) and theCancer Research Campaign (AE, JF).100EXPERIMENTAL FIBROSARCOMA INTERACTION 101REFERENCES1. Davies, P., Allison, A. C., Ackerman, J., Butterfield, A.,and Williams, S. Asbestos induces selective release of ly-sosomal enzymes from mononuclear phagocytes. Nature251: 423-424 (1974).2. Hamilton, J., Vassalli J.-D., and Reich, E. Macrophageplasminogen activator: induction by asbestos is blockedby anti-inflammatory steroids. J. Exp. Med. 144:1648- 1694 (1976).3. Hamilton, J. Macrophage stimulation and the inflam-matory response to asbestos. Environ. Health Perspect.34: 69-74 (1980).4. Humes, J. L., Bonney, R. J., Pelus, L., Dahlgren, M. C.,Sadowski, J. J., Kuehl, F. A., and Davies, P. Macrophagessynthesize and release prostaglandins in response to in-flammatory stimuli. Nature 269: 149- 151 (1977).5. Sirois, P., Rola-Pleszczynski, M., and Begin, R. Phospholi-pase A activity and prostaglandin release from alveolarmacrophages exposed to asbestos. Prostagl. Med. 5: 31-37(1980).6. Davies, P., and Bonney, R. J. The secretion of hydrolyticenzymes by mononuclear phagocytes. In: Handbook of In-flammation, Vol 2. The Cell Biology of Inflammation. (G.Weissman, Ed.), Plenum Press, New York-London, 1980,pp. 497-542.7. Miller, K., and Kagan, E. Manifestations of cellular immu-nity in the rat after prolonged asbestos inhalation. II.Alveolar macrophages induce splenic lymphocyte prolifer-ation. Environ. Res. 26: 182- 194 (1981).8. Donaldson, K., Davis, J. M. G., and James, K. Character-istics of peritoneal macrophages induced by asbestosinjection. Environ. Res., Environ. Res. 29: 414-424 (1982).9. Donaldson, K., Davis, J. M. G., and James, K. Concanava-lin A receptors and capping in control and activatedmacrophages. Histochem. J. 15: 59-69 (1982).10. Raz, A., Shahar, A., and Goldman, R. Characterization ofan in vivo induced peritoneal macrophage population fol-lowing intraperitoneal injection of Concanavalin A. J. Re-ticuloendothel. Soc. 22: 445-460 (1977).11. Ames, B. N., and Dubin, D. Y. The role of polyamines in theneutralization of bacteriophage deoxyribonucleic acid. J.Biol. Chem. 235: 769-775 (1960).12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall,R. J. Protein measurements with the Folin phenol re-agent. J. Biol. Chem. 193: 265-275 (1951).13. Woodruff, M. F. A., Inchley, M. P., and Dunbar, N. Fur-ther observations on the effect of C. parvum and anti-tumour globulin on syngeneically transplanted tumours.Brit. J. Cancer 26: 67-76 (1972).14. Gabizon, A., and Trainin, N. Enhancement of tumourgrowth by peritoneal macrophages of normal and tumourbearing mice. In: Macrophage and Lymphocytes: Nature,Function and Interactions (M. R. Escobar and H. Fried-man, Eds.), Plenum Press, New York-London, 1980, pp.485- 491.15. Cohn, Z. A. Macrophage activation: fact fancy and future.J. Immunol. 121: 813-816 (1978).16. Keller, R. The cytostatic and cytocidal effects of macro-phages: are they really specific for tumour cells? In:Lymphokines, Vol. III. Lymphokines and Macrophage Ac-tivation. (E. Pick, Ed.), Academic Press, New York-Lon-don, 1981, pp. 283-292.17. Pylev, L. N., and Shabad, L. B. Some results of experi-mental studies on asbestos carcinogenesis. In: BiologicalEffects of Asbestos (P. Bogovski, V. Timbrell, J. C. Gilsonand J. C. Wagner, Eds.), IARC Pub. No. 8, InternationalAgency for Research on Cancer, Lyon, 1973, pp. 99-105.18. Topping, D. C., and Nettesheim, P. Two stage carcinogen-esis studies with asbestos in Fischer 344 rats. J. Natl.Cancer Inst. 65: 627-630 (1980).19. Keller, R., Keist, R., and Hecker, E. Tumour promotingditerpene esters prevent macrophage activation. Brit. J.Cancer 45: 144-146 (1982).

Asbestos induces selective release of lysosomal enzymes from mononuclear phagocytes.

Characteristics of peritoneal macrophages induced by asbestos injection.

Characterization of an in vivo induced peritoneal macrophage population following intraperitoneal injection of Concanavalin

Concanavalin A receptors and capping in control and activated macrophages.

Enhancement of tumour growth by peritoneal macrophages of normal and tumour bearing mice. In: Macrophage and Lymphocytes: Nature, Function and Interactions

Further observations on the effect of C. parvum and antitumour globulin on syngeneically transplanted tumours.

Macrophage activation: fact fancy and future.

Macrophage plasminogen activator: induction by asbestos is blocked by anti-inflammatory steroids.

Macrophage stimulation and the inflammatory response to asbestos.

Macrophages synthesize and release prostaglandins in response to inflammatory stimuli.

Manifestations of cellular immunity in the rat after prolonged asbestos inhalation. II. Alveolar macrophages induce splenic lymphocyte proliferation.

Phospholipase A activity and prostaglandin release from alveolar macrophages exposed to asbestos.

Protein measurements with the Folin phenol reagent.

Some results of experimental studies on asbestos carcinogenesis. In: Biological Effects of Asbestos

The cytostatic and cytocidal effects of macrophages: are they really specific for tumour cells? In:

The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid.

The secretion of hydrolytic enzymes by mononuclear phagocytes. In: Handbook of Inflammation, Vol 2. The Cell Biology of Inflammation.

Tumour promoting diterpene esters prevent macrophage activation.

Two stage carcinogenesis studies with asbestos in Fischer 344 rats.

Interactions of asbestos-activated macrophages with an experimental fibrosarcoma

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Interactions of asbestos-activated macrophages with an experimental fibrosarcoma.

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Interactions of asbestos-activated macrophages with an experimental fibrosarcoma

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