Depression of viral interferon induction in cell monolayers by asbestos fibers

ENVIRONMENTAL

RESEARCH

11,

52-65 (1976)

Depression of Viral Interferon Induction Monolayers by Asbestos Fibers NICHOLAS

HAHON'

AND

HERBERT

L.

in Cell

ECKERT

U. S. Public Health Service. Appalachian Labaratory for Occupational Diseases. and Departments of Pediatrics and Medicirre, West Virginia School of Medicine, Morgantown. West Virginia 26505

Respiratory University

Received May 9, 1975 Studies on the induction of interferon by influenza virus revealed that this adaptive cellular response was depressed, completely or partially, in either amosite, crocidolite, anthophyllite, Canadian or Rhodesian chrysotile-treated monkey kidney (LLC-MKz) cell monolayers. Asbestos fiber concentrations of 1 mg/l .O x 10’ cells, which minimally affected cell viability, depressed interferon production by almost 90%. Increases in the virus-cell multiplicity of infection did not significantly increase interferon yields in asbestos fiber-treated cells. Chrysotile B fibers of lengths ranging from 10 to 5 pm were more effective in reducing interferon yields than those ranging from 75 to 625 Km. Maximal depression of interferon production was dependent on prior treatment of cell monolayers with asbestos fibers before the addition of viral inducer. The processes of virus integration (attachment and penetration) into and virus multiplication in cells were unaffected by the presence of asbestos fibers. however, a slightly higher level of virus replication was noted in asbestos fiber-treated cells. When cells were primed with interferon, the presence of asbestos fibers did not interfere with the ability of the primer to enhance interferon production. Neither interferon antagonists, interference with virus-cell interactions. nor adsorption by asbestos fibers accounted for the depression of interferon yields. These findings suggest that asbestos fibers act on cell membranes in a manner, as yet undetermined, to interpose in the viral inductive process of interferon synthesis.

INTRODUCTION

The interferon system is an important component of the nonimmunological defense mechanisms of the body, and it is generally believed that interferon plays a role in recovery from acute primary viral infections (Baron, 1973). The production of interferon by alveolar macrophages and the passive protection conferred by interferon to other macrophages against viral infection suggest that cell resistance mediated by interferon may be important determinants of respiratory tract defenses (Acton and Myrvik, 1966). Steroids, hormones, carcinogenic hydrocarbons, arsenicals, and a variety of agents have been reported to inhibit interferon synthesis (VilCek, 1969; Gainer, 1972). That a number of environmental factors diminish the resistance or ability of the lung to cope with introduced infectious agents has been established through the use of infectious disease-animal model systems (Ehrlich, 1966; Ehrlich and Henry, 1968; Spurgash ef al., 1968; Henry et al., 1970; Coffin, 1972). Relevant to this problem is the demonstration that nitrogen dioxide, a prominent component of air pollution, inhibited the ability of rabbit alveolar macrophages to produce interferon which resulted in a loss of cellular resistance to infection by challenge virus (Valand er al., 1970). Mineral dusts also may challenge * Address reprint requests to Nicholas Hahon, ALFORD, 26505. 52 CopyrIght 0 1976 by Academic All rights of reproduction

Press, Inc. in any form reserved

Box 4292, Morgantown,

West Virginia

INTERFERON

DEPRESSION

BY

ASBESTOS

53

the integrity and efficacy of the interferon system. Recently, studies on the induction of interferon by influenza virus revealed that this adaptive cellular response was depressed, partially or completely, in coal dust-treated human or simian cell monolayers (Hahon, 1974). The carcinogenic potential of asbestos in the environment has become one of the foremost public and industrial health concerns of our time. Exposure to asbestos dust is now widely recognized as an important etiological factor associated with pulmonary fibrosis, lung cancer, and malignant mesothelial tumors (Wagner et al., 1960); Selikoffet al., 1965; Wagneret al., 1971). Within recent years, interest in the health aspects of asbestos by the scientific community has foStered a wide variety of studies, many of which have been concerned with elucidating the underlying biological responses and mechanisms involved in asbestosis (Huff et al., 1974). A paucity of information, however, still characterizes our knowledge of the influence of asbestos dust on the host’s immune and nonimmune defense mechanisms (Burrell, 1974). In an attempt to enlarge our understanding of the effect of mineral dusts on the host’s defense system, the factors affecting the response of cell cultures treated with different asbestos fibers to the induction of interferon by influenza virus are described in this report. Viruses The Ao/PR8/8/34 influenza and parainfluenza 1 (Sendai) virus strains employed in this study were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland. Stock virus pools of each strain were prepared from chick embryonated eggs in the manner described previously (Hahon et al., 1973). Influenza and Sendai virus pools contained 1.O x lOa and 5.4 x lo9 cell-infecting units (CIU) of virus per milliliter, respectively, when assayed by the immunofluorescent cell-counting procedure (Hahon et al., 1973). Cell Cultures Rhesus monkey kidney (LLC-MKz) and human Chang conjunctiva (clone l-5c-4) cell lines obtained from ATCC were used for induction and assay of interferon, respectively. Cell lines were propagated in plastic tissue culture flasks (75 cm2) with Eagle minimum essential medium (MEM) containing 10% fetal calf serum (FCS) and maintained with MEM plus 0.5% FCS. Asbestos UICC asbestos fiber standards, amosite, anthophyllite, crocidolite, Canadian and Rhodesian chrysotiles were obtained from Duke Standards Co., Palo Alto, California. Optical microscope evaluations indicated that from 96 to 98% of the asbestos fibers were 30 Frn or less in length. UICC Chrysotile B, obtained from N.I.O.S.H., Chemical Reference Laboratory, Cincinnati, Ohio, had a fiber length range from 75 to 625 pm. Asbestos fiber samples, sterilized in an autoclave at 20 lb/in2 pressure (126” C) for 15 min, were made into w/v suspensions in either phosphate-buffered saline (PBS), pH 7.0, or maintenance medium. Interferon Induction The procedure generally employed to study the effects of asbestos fibers on interferon induction was carried out as follows; 1 to 4 mg ofasbestos suspension in a

54

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IO-ml volume was added to flasks (75 cm2) containing complete LLC-MK2 cell monolayers which were then incubated at 35°C from 4 to 16 hours. Residual medium was decanted, and 1 ml of influenza virus that had been ultraviolet (uv)-irradiated for 45 seconds was added onto cell monolayers which were then incubated at 35°C for 2 hours. The multiplicity of infection (MOI) was approximately 10. Inoculum was removed and 10 ml of maintenance medium was added to each flask. After incubation at 35” C from 22 to 24 hours, supernatant fluid was decanted and centrifuged at 100,OOOgfor 1 hour and dialyzed against HCl-KC1 buffer, pH 2.0, at 4 C for 24 hours. Dialysis was continued against two changes of PBS, pH 7.1, at 4” C for24 hours. Fluids were passed through Millex filters, 0.45 pm (Millipore Corporation, Boston, Massachusetts) to obtain sterile preparations. Samples were stored at -70” C until assayed for interferon activity. Controls consisting of cell monolayers which were not treated with asbestos fibers were handled exactly as described above. Preparations possessed the biological and physical properties ascribed to viral interferons (Lockart, 1966). Znterferon Assay An immunofluorescent cell-counting assay of interferon was employed that is similar in principle and in reproducibility to that reported previously (Kozikowski and Hahon, 1969; Hahon and Booth, 1974). Briefly, serial twofold dilutions of interferon prepared in maintenance medium were introduced in 1.0 ml volume directly into glass vials containing 19 x 65 mm diameter cover slip monolayers of clone I-SC-4 cells and incubated at 35” C from 20 to 24 hours. Assays were carried out in duplicate. After incubation, cell monolayers were washed with PBS, and then challenged with Sendai virus in 0.2 ml volume. Virus was diluted to give from one to three infected cells per microscopic field. Controls consisted of untreated cell monolayers infected with virus. Challenge virus was attached to cells by incubation at 35” C for 1 hour. Thereafter, 1.O ml of maintenance medium was added to vials which were then incubated at 35” C for approximately 22 hours. Subsequently, the cell monolayers were rinsed with PBS and fixed with acetone. The direct fluorescent antibody procedure was used to demonstrate immunofluorescence in infected cells. Details of the preparation of virus antiserum, its conjugation with fluorescein isothiocyanate, staining technique, microscope equipment, and infected-cell enumeration have been described (Hahon et al., 1973). The reciprocal of the interferon dilution that reduced the number of infected cells to 50% of the control served as the measure of interferon activity, i.e., 50% ICDDso (50% infected cell-depressing dilution). Determination of Virus Attachment and Penetration These procedures have been described in detail previously

(Hahon et al., 1973).

RESULTS

Asbestos Toxicity for Cell Cultures The cytotoxicity (cell death) of various concentrations of asbestos fibers was determined because cell survival in the presence of asbestos fibers was a requisite in succeeding interferon induction experiments. The survival of LLC-MKz cell monolayers incubated for 24 hours with varied amounts of six different asbestos

INTERFERON

DEPRESSION

BY

55

ASBESTOS

samples is shown in Table 1. Cell survival was diminished by all asbestos fibers tested at 10 mg concentrations. At concentrations of 4 mg, cells treated with Rhodesian chrysotile or anthophyllite showed the greatest decrease in viability, 35 and 22 %, respectively. With the exception of Rhodesian chrysotile, cells were minimally affected by asbestos fibers at 2 mg concentration. Generally, Rhodesian chrysotile was more detrimental to cell viability than the other tested asbestos fibers. Because all six asbestos fibers at 1 mg concentration per 1 x lo7 cells had no significant cytotoxic effect, this concentration or less was usually employed in subsequent tests. Cell monolayers incubated with various asbestos fibers in concentrations of 0.4 and 0.1 mg/l x 10’ cells for 20 hours and, subsequently, passed or redistributed into new containers, retained their ability to multiply and form new cell monolayers in the presence of asbestos fibers. When cells were cleared of the fibers by serial passage and dilution, they retained their ability to produce the same quantity of interferon as normal cell monolayers. Any subtle, toxic reaction with cells by asbestos fibers, whether temporary or permanent, did not affect interferon induction. Interferon Induction with Varied Concentrations of Asbestos and Virus Different asbestos fibers at varied concentrations were tested to determine their effect on interferon induction by influenza virus in LLC-MKz cell monolayers. Results (Table 2) show that all asbestos fibers at 1.0 mg concentration depressed virus interferon induction by almost 90%. All asbestos fibers in 0.01 mg concentrations, with the exception of amosite, were still capable of partially depressing interferon induction. The effect of different influenza virus-cell multiplicities of infection on interferon induction in asbestos-treated and untreated cell monolayers is shown in Table 3. In asbestos-treated cells, interferon production did not increase markedly with increasing concentrations of inducer virus. Interferon production in untreated cells appeared to double with tenfold increases of the virus inducer. TABLE EFFECT OF DIFFERENT

CONCENTRATIONS

OF ASBITS-IUS

I FIBERS

ON SURVIVAL

OF

LLC-MK.

CELLS’

Surviving fraction of ceW Asbestos (mid

Amosite

10 4 2 1 0.1 Control

0.77 0.83 0.87 0.98 1.00 1.00

Anthophyllite 0.78 0.78 0.97 1.00 1.00 1.00

Crocidolite 0.63 0.93 0.95 0.97 0.97 1.00

Canadian chrysotile

Rhodesian chrysotile

Chrysotile B

0.87 0.93 0.93 0.95 0.99 1.00

0.59 0.65 0.77 0.95 1.00 1.00

0.81 0.91 0.92 0.91 0.98 1.00

a Cell monolayers incubated at 35” C for 24 hours with asbestos fibers. * Percentage of living and dead cells per 75 cm* flask determined by staining with 0.5% trypan blue. Surviving fraction of cells computed by dividing percentage of living cells in asbestos-treated monolayer by percentage of living cells in control (87.8).

56

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AND

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TABLE INTERFERON

INDUCTION

BY PRS INWJENZA DIFFERENT

VIRUS

IN

CONCENTRK~IONS

2 LLC-MK,

CELL MONOLAYERS OF ASBESTOS FIBERS

TREATED WITH

Asbestos fibers Asbestos (mg)

Amosite

1.0 0.5 0.1 0.01 Control (none)

SW 110 110 350 380

Anthophyllite

Crocidolite

50 100 100 220 380

50 loo 100 170 380

Canadian chrysotile

Rhodesian chrysotile

50 90 120 180 380

50 90 120 180 380

a Interferon titer: reciprocal of 50% infected cell-depressing dilution. TABLE INTERFERON

MO1 50 10 1 0.1

3

INDUCTION WITH DIFFERENT VIRUS-CELL MuLTwLIcrrY OF INFECTION AMOSITE-TREATED AND UNTREKTED LLC-MK, CELL MONOLAYERS

Amosite”-treated

cells

(MOI) IN

Untreated cells

700 60 <50 <50

a 0.5 mg fibers in 10 ml maintenance medium added to 75 cm2 flask of LLC-MK, b Interferon titer; reciprocal of 50% infected cell-depressing dilution.

500 380 120 60 cell monolayer.

Asbestos Fiber Length and Interferon Induction To determine the influence of asbestos fiber length on viral interferon induction, cell monolayers were exposed to chrysotile B fiber preparations that ranged in length from 10 to 50 pm and from 75 to 625 pm, respectively. The short fibers were obtained by sonic treatment of the longer fibers. Results (Table 4) reveal that both short and long asbestos fibers reduced interferon production by 50% or greater, however, short fibers were more effective in depressing interferon yields at higher asbestos concentrations. Time Relationship between Asbestos and Viral Inducer on Interferon Depression The sequence of asbestos and viral inducer administration on the inhibition of interferon production was determined with three different asbestos fibers of varied concentration. Results (Table 5) indicate that prior treatment of cell monolayers with asbestos, as early as 2 hours before the addition of virus inducer, effectively depressed interferon production from 50% to approximately 90% depending on the concentration of asbestos. At time zero, when virus and asbestos were added simultaneously to cells, only higher amounts of asbestos inhibited interferon production. After the viral inducer was in contact with cells for several hours, the

INTERFERON

DEPRESSION

BY

57

ASBESTOS

addition of asbestos did not significantly alter interferon yields. The depression of interferon production appears to be time related with respect to the pretreatment interval of cell cultures with asbestos fibers. Asbestos,

Virus Multiplication,

and interferon

The growth of PR8 influenza virus concomitant with interferon production was investigated in both untreated and anthophyllite-treated (0.1 mg/3 x 105) LLCMKz cells. Equivalent numbers of virus and cells were employed to initiate infection (MO1 = 1.0). The immunofluorescent cell-counting technique was used to determine virus multiplication from growth curve samples inoculated into clone I-5c-4 cells. The rate of virus growth was similar in both untreated and TABLE EFFECT

OF Asmsros

Chrysotile (mg)

FIBER

2 2 0.2 0.2 Control’

BY PR8 INZLLIENLA

ON INTERFERON INDUCTION LLC-MK, CULL MONOLAY~KS

Range of fiber length” km)

B

4

LENGTH

Reduction Interferon (%)

Interferon titer (ICDDsoY 130 340 310 350 700

IO-50 75-625 IO-50 75-625

VIRUS

IN

of titer

81.5 51.5 55.8 50.0

D Average length by optical microscope determination. b Reciprocal of 50% infected cell-depressing dilution. (’ Normal, untreated cell monolayer.

TABLE Trhrr

REI.AT.IONSHIP

B&I’WEEN

ASBESTOS

FIBERS

INTERFERON

Hourly relation between asbestos fibers and addition of virus at zero time= -4 -2

Control

0 4 6 I6 20 (no asbestos)

5 AND

PR8 INI-LUENZA

Asbestos Chrysotile B (IO mg) 506 140 420 960 940 1100 1090 1100

VIRLX

ON DEPRESSION

OF

PRODUCTION

fibers

Amosite (2 ml 180 2.50 900 loo0 960 1050 1080 1100

Crocidolite (0.1 mg) 290 450 800 790 800 800 800 810

a Asbestos fibers suspended in 10 ml maintenance medium were added to 75 cm* flasks LLC-MK, cell monolayers at designated hours prior to or after the addition of uv-irradiated fluenza virus at 0 time. b Interferon titer: reciprocal of 50% infected cell-depressing dilution.

containing PR8 in-

58

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anthophyllite-treated cells (Fig. l), but reached a slightly higher level in cell cultures treated with anthophyllite. Interferon was first detected 8 hours after virus infection of untreated cells, and its production reached a peak at 40 hours. Minimal amounts of interferon (less than one unit) were detected in anthophyllite-treated cells throughout the 48-hour sampling period. These data suggest that the slightly higher level of virus growth attained in asbestos-treated cell cultures may be the consequence of interferon depression. Interferon Priming Asbestos-Treated Cell Cultures Pretreatment of cell cultures with small amounts of interferon “priming” before the addition of the inducer agent enhances interferon production (Ho, 1973). An experiment was carried out to determine the influence of asbestos fibers on the priming of LLC-MK2 for interferon induction by PR8 influenza virus. Prior to the introduction of the primer (100 units of monkey interferon) cell monolayers were first treated with anthophyllite fibers for 4 hours. Results (Table 6) show that with untreated cells more than a twofold increase in interferon yield occurred in primed cell monolayers as compared to that of cells that were not stimulated. With anthophyllite-treated cells, priming also resulted in a threefold increase of interferon yield when compared to similarly treated cells that were not primed. Although asbestos fibers interfered with the ability of cells to produce interferon under the direction of the viral inducer, the fibers did not interfere with the ability of cells to react to the stimulating influence of the primer.

FIG. 1. Growth curves of PR8 influenza virus concomitant and anthophyllite-treated LLC-MKz cell monolayers.

with

interferon

production

in untreated

Anthophyllite (mg)

Primer Interferon,

0.5 0.5 None None None 0.5 a Reciprocal

Inducer 100 units

None Interferon,

100 units None

Interferon.

100 units None

of 50% infected

cell-depressing

uv-PR8 uv-PR8 uv-PRX uv-PR8 None None

Interferon titer 1CDD.w 450 IS0 600 240 0 0

dilution.

VirusXell Inteructions, Interferon Blockers, and Antagonists The possibility that the depression of viral induction of interferon may be the consequence of an impediment of primary virus-cell interactions, i.e. virus attachment to or penetration into cell monolayers by the physical presence of asbestos fibers, was explored. Results (Fig. 2) show that influenza virus attached to both untreated and amosite-treated LLC-MKz cells at a similar rate and to a comparable magnitude. The presence of asbestos fibers did not impede virus attachment. The similar rates of virus penetration into untreated and amosite-treated cells (Fig. 3) indicate that this interaction also was not hindered by asbestos fibers. A test was designed to investigate the possibility that supernatant fluid from asbestos suspensions contains soluble ingredients that may be directly responsible for the observed depression of interferon production. The results (Table 7) show that interferon production by cells treated with supernatant fluid from asbestos fiber suspensions were comparable to that of untreated (control) cells. Depression of interferon production by resuspended asbestos fibers were comparable to that by the original asbestos fiber suspensions. These findings infer that the phenomenon of interferon depression in cells may be attributable to asbestos fibers per se. To obviate the circumstance that reduced interferon yields may be related to the adsorptive power of asbestos fibers for interferon, equal volumes of a known interferon preparation were mixed with 2 mg suspensions of amosite, Canadian chrysotile, crocidolite, anthophyllite or maintenance medium (control). After incubation at 35” C for 20 hours, the suspensions were clarified by centrifugation and assayed for interferon activity. Results indicated that interferon was not bound or adsorbed to asbestos fibers. The titers of interferon from asbestos suspensions were comparable to that of the control. That the presence of asbestos fibers on cell monolayers may evoke the formation of a blocker or antagonist of interferon induction or activity was investigated in a series of experiments. A test was performed to determine whether the interaction of virus with cells to induce interferon is blocked by a soluble complex resulting from an earlier cellular response to asbestos. Supernatant fluid from untreated LLCMK2 cell monolayers and from cells that were incubated at 35” C for 24 hours with anthophyllite fibers were introduced onto new cell monolayers. After incubation at

60

HAHOh’

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loj/, , , , , , 0

30

60

90

ml

I50

180

MINUTES

FIG. 2. Rates of attachment of PR8 influenza virus to untreated and amosite-treated LLC-MKz monolayers at stationary incubation. 35” C.

cell

100, 90. 80.

0

5

IO

15

20

MINUTES

FIG. 3. Rates ofpenetration ofPR8 influenza virus into untreated and amosite-treated LLC-MK2 cell monolayers.

INTERFERON

DEPRESSION

Asbestos Additives to cell Monolayer” Asbestos suspension Supernatant fluid Resuspended fibers Maintenance Medium (control)

Amosite

61

BY ASBESTOS

Rhodesian

fibers

chrysotile

Anthophyllite

30* 270 40

30 250 30

40 190 30

270

270

270

m Asbestos fiber suspensions (4 mg fibers in IO ml maintenance containing LLC-MK, cell monolayers. b Interferon titer: reciprocal of 50% infected cell-depressing

medium)

were

added to 75 cm2 flasks

dilution.

35” C for 4 hours, influenza virus was added for induction of interferon in accord with the prescribed procedure. Results of subsequent assays for interferon showed that supernatant fluids from anthophyllite-treated cell monolayers, in comparison to controls, did not significantly affect the induction of interferon in the new cell monolayers. Supernatant fluids employed in the previous experiment were examined for the presence of an antagonist of interferon activity. Dilutions of a known interferon preparation were made in both supernatant fluids from untreated and anthophyllite-treated cell cultures. The comparable interferon titers attained with test preparations indicate that the supernatant fluid from asbestos-treated cells did not contain an interferon antagonist. An additional test was performed to determine whether an interferon antagonist was present in viral induced interferon prepared from asbestos-treated cell monolayers. This could account for the low levels of interferon activity noted in these preparations. In equal volumes, maintenance medium or undiluted low-titer interferon obtained from asbestos-treated cell cultures was added to dilutions of a standard interferon preparation, and the mixtures were then assayed for interferon activity. That the titer of the standard interferon was not diminished in the presence of interferon prepared from asbestos-treated cells, indicates the absence of an antagonistic reagent. DISCUSSION

The complete or partial depression of influenza virus-induced interferon, an adaptive cellular response, by a variety of asbestos fibers was demonstrated by this study. The viability of the LLC-MKz cell monolayer used as the in vitro experimental medium was minimally affected by 1.0 mg concentrations of either amosite, anthophyllite, crocidolite, Canadian or Rhodesian chrysotiles, or chrysotile B per 1.0 x IO7 cells. Rhodesian chrysotile, in contrast to the other chrysotiles and asbestos fibers, appeared to be the most detrimental to cell viability. In studies employing peritoneal macrophages, lung fibroblasts, or red cells, chrysotiles were reported to be more cytotoxic and hemolytic than other asbestos fibers (Bey and

62

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Harington, 1971; Robock and Klosterkotter, 1971; Richards et al., 1971; Richards and Morris. 1973; Harington et ~1.. 1974); amosite and crocidolite were less toxic (Bey and Harington, 1971). In this respect, our findings using an established cell line were in general agreement. All asbestos fibers tested at 1.0 mg concentrations were capable of depressing interferon production by almost 90%. At 0.01 mg concentrations, all asbestos fibers, with the exception of amosite, still partially inhibited interferon production. That increases in the influenza virus-cell multiplicity of infection did not significantly increase interferon yields in asbestos-treated cell monolayers was generally comparable to findings reported with coal dust (Hahon, 1974). In attempting to evaluate the influence of asbestos fiber size on interferon induction, tests were limited, of necessity, to chrysotile B. Shorter fibers (10 to 50 pm) appeared to be more effective than longer fibers (75 to 625 pm) in reducing interferon yields. The more intimate contact and subsequent interaction with cell membranes afforded by shorter fibers may account for this finding. When asbestos is ground to a fiber size less than 5pm long, its tibrogenicity for the lungs disappears (Webster, 1970). To explain this enigma, recent evidence suggests that the locus of pathogenicity of asbestos fibers resides in their polylilamentous structure (Gross and Harley, 1973). Pathogenicity is reduced as the polytilamentous arrangement of asbestos fibers is replaced by a monofilamentous structure. The role of poly- and monofilamentous asbestos fibers on interferon induction may be an interesting facet for further investigation. In concurrence with results observed with another mineral dust (Hahon, 1974), prior treatment of cell monolayers with asbestos fibers before the addition of virus inducer was required also to demonstrate interferon inhibition. When asbestos fibers were introduced onto cell monolayers simultaneously with or after the virus inducer, interferon production was not significantly affected. The depression of interferon production by asbestos fibers, with respect to the virus inducer, is time related. That both cellular attachment and penetration of influenza virus were unimpaired in the presence of asbestos fibers attests to the integrity of cell membranes in relation to these early virus-cell interactions. Rates of virus growth in asbestos fiber-treated and normal cells were comparable, but a slightly higher level (twofold) of virus multiplication was attained in asbestos fiber-treated cells. Our findings suggest that this was the consequence of inhibition of interferon synthesis by the presence of asbestos fibers on cell monolayers. By using viral agents noted for their greater interferon-inducing potency, the difference between virus replication levels in asbestos fiber-treated and untreated cells could be much greater. Nevertheless, our observations indicate that asbestos fibers did not affect the processes of virus integration into or virus multiplication in cells. Interferon pretreatment of cells to enhance interferon yields, referred to as “priming,” was demonstrated in the virus-host cell system employed. Priming resulted in an increase of interferon yield, not only in normal LLC-MKz cells but also in asbestos fiber-treated cells. Whereas asbestos fibers affected the cells’ ability to produce interferon in response to the viral inducer, it is significant that asbestos fibers did not impair the potentiating effect of the primer. Similar results

INTERFERON

DEPRLSSION

BY ASBESTOS

63

were attained with coal dust (Hahon, 1974). Indirectly, the findings add support to the suggestion that antiviral and priming activities of interferon are due to different mechanisms (Stewart et al., 1971; Roszt&zy, 1974). Attempts to detect a blocker (Vilcek, 1969) or nonviral antagonist (Friedman and Sonnabend, 1970) of interferon induction or activity in either supernatant fluids from asbestos fiber suspensions or in low-titer interferon preparations from asbestos fiber-treated cells were unsuccessful. Tests performed to determine whether reduced interferon yields may be related to the adsorptive potential of asbestos fibers for interferon indicated that the protein was not bound or adsorbed to fibers. The experimental data suggest that the depression of interferon synthesis in cell monolayers was attributable to asbestos fibers per se. The mechanism by which seemingly inert asbestos fibers interact with cells to modify interferon production is not precisely known. Because asbestos fibers do not penetrate in vitro cell cultures, or for that matter in vivo cells, with the exception of macrophages (Davis, 1973), it may be assumed that the sequence of events leading to the depression of interferon production by asbestos fibers is initiated at the cell surface. Our findings show that any ensuing alteration of the cell surface by asbestos fibers neither impedes the attachment, penetration, nor multiplication of the viral inducer into the cell, but does inhibit viral induction of interferon. For the latter to occur, the association of asbestos fibers with the cell must precede the addition of viral inducer. Although the removal or addition of trace metals does not alter the pathogenicity of asbestos fibers (Gross, 1973), the studies of Harington er al. (1971) suggest that magnesium is the principal agent in hemolysis by chrysotile and that an association exists between cytotoxicity and hemolytic activity of various forms of asbestos. Depletion of magnesium by acid treatment of chrysotiles prevented the subsequent depression of interferon synthesis by these asbestos fibers (Hahon, unpublished observations). This suggests that trace elements may play a role in the ultimate depression of interferon induction. Several studies indicate that cells exposed to asbestos fibers may endure biochemical and structural alterations and survive (Allison, 1974). Studies with macrophages have demonstrated that cell and lysosomal membranes may be damaged by asbestos fibers (Parazzi et al., 1968, Allison, 1969). Rhodesian chrysotile was reported to reduce protein production in actively growing rabbit lung fibroblasts, to increase collagen production in the cell mat, and to slightly stimulate mucopolysaccharide release into the medium (Richards et al., 1971, Richards and Morris 1973). Using Rhodesian chrysotile and rabbit lung tibroblasts, Richards et al. (1971) reported a reduction in protein synthesis by measuring for total cellular protein and, specifically, for tyrosine. Because protein synthesis is a requisite of interferon production, and asbestos fibers inhibit interferon production, it may be assumed that the presence of asbestos fibers reduces protein synthesis. Our studies imply, therefore, that asbestos fibers reduce cellular protein synthesis as demonstrated by the depression of interferon production. This may be considered a selective cytotoxic manifestation of asbestos fibers in that it affects a specific cell function, that of interferon production. At which stage in the mechanism of interferon induction (Johnston and Burke, 1973) do asbestos fibers exert their adverse effect (1) the formation of the inducing molecule, (2) activation or derepression of the

64

HAHON

interferon gene, or (3) transcription RNA, is presently unknown.

AND

ECKERT

and translation

of the interferon

messenger

ACKNOWLEDGMENTS The excellent technical assistance of James A. Booth, John D. Stewart, and Janet Simpson is gratefully appreciated.

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Hahon, N. (1974). Depression of viral interferon induction in ceil monolayers by coal dust. Brit. J. Ind. Med. 31, 201-208. Hahon, N.. and Booth, J. A. (1974). Hemadsorption cell-counting assay of interferon. Arch. Ges. Virusforsch. 44, 160-163. Hahon, N., Booth, J. A., and Eckert, H. L. (1973). Cell attachment and penetration by influenza virus. lnfec.

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