Silicate minerals and the interferon system

ENVIRONMENTAL RESEARCH 43, 395-409 (1987)

Silicate Minerals and the Interferon System N I C H O L A S H A H O N * ' t A AND JAMES A . B O O T H *

*National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, and ?Department of Pediatrics, West Virginia University School of Medicine, Morgantown, West Virginia 26505 Received June 6, 1986 Natural-occurring minerals representative of six silicate classes were examined for their influence on interferon induction by influenza virus in Rhesus monkey kidney (LLC-MK/) cell monolayers. Minerals within the classes nesosilicate, sorosilicate, cyclosilicate, and inosilicate exhibited either little or marked (50% or greater) inhibition of interferon induction. Within the inosilicate class, however, minerals of the pyroxenoid group (wollastonite, pectolite, and rhodonite) all significantly showed a two- to threefold increase in interferon production. Silicate materials in the phyllosilicate and tectosilicate classes all showed inhibitory activity for the induction process. When silicate minerals were coated with the polymer poly(4-vinylpyridine-N-oxide), the inhibitory activity of silicates on viral interferon induction was counteracted. Of nine randomly selected silicate minerals, which inhibited viral interferon induction, none adversely affected the ability of exogenous interferon to confer antiviral cellular resistance. Increased levels of influenza virus multiplication concomitant with decreased levels of interferon occurred in cell monolayers pretreated with silicates. The findings of this study demonstrate the diverse effects of minerals representative of different silicate classes on the interferon system and indicate that certain silicates in compromising the viral interferon induction process may increase susceptibility to viral infection. © 1987AcademicPress, Inc.

INTRODUCTION

The major complication of silicosis, the association of silicates with a high incidence of pulmonary tuberculosis, among affected miners was an early indication that silicious dusts are potential industrial health hazards. The enhancing effect of silica on mycobacterial disease and subsequent studies pursued in an attempt to explain this entity have been concisely reviewed (Zaidi, 1969; Snider, 1978). Increased susceptibility to infection by bacterial (Friedman and Moon, 1977; Takeya et al., 1977), parasitic (Trischman et al., 1978), and viral agents (Zisman et al., 1970; Selgrade and Osborn, 1974; Morahan et al., 1977; Halstead et al., 1977; Probert et al., 1977) in silica-treated animals has been reported as well as opportunistic mycotic infections occurring in patients with acute silicosis (Bailey et al., 1974). That infectious diseases of the lungs may enhance the onset and development of silicosis is yet another alternate facet associated with this disease. More cases of severe silicosis in miners with past pulmonary infections were reported than in miners with no past history of respiratory disease (Norvitt, 1964; Palkin, 1966). This is supported further by experimental evidence of infectious agents a Author to whom reprint requests should be addressed.

395 0013-9351/87 $3.00 Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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HAHON AND BOOTH

together with dust leading to severe silicosis (Gernez-Rieux et al., 1963; Chiappino and Vigliani, 1982; Higgins et al., 1985). The immunologic aspects of silica, its effect on various components of the immune system, and its relationship to silicotic disease have been critically reviewed (Burrell, 1981). Clinical and experimental studies on this defense system have revealed that silica exposure can compromise host immunocompetence by altering both humoral and cellular immune responses (Jones et al., 1976; Cocarla et al., 1977; Scheuchenzuber et al., 1985; Zarkower et al., 1986). That exposure to silica induces corresponding immune responses which play an active role in the pathogenesis of silicosis and susceptibility to infection is highly probable. A paucity of information characterizes our knowledge of the relationship between silica and another defense mechanism, the interferon system, of which the latter's role, if any, in silicotic disease is unclear. Interferons are a closely related group of proteins which have anticellular, antiviral, and immunomodulatory functions (Taylor-Papadimitriou, 1980). Within recent years, evidence has accumulated to show that the interferon induction process is selective and highly sensitive to the effects of insidious chemicals and particulates of public health concern (Hahon and Eckert, 1976; Sonnenfeld et al., 1983, 1984; Hahon, 1983, 1985; Hahon et al., 1983; Hahon and Booth, 1984). Of the available information relevant to silica, viral infection, and interferon, protection against herpes simplex virus was reported to be markedly decreased in mice injected with silica (Storch et al., 1984). Stebbing et al. (1978) also reported that protection by macrophage interferon against encephalomyocarditis virus was abolished in mice by silica administration and that macrophage interferon was more effective than fibroblast interferon. Silica treatment of animals also decreased interferon induction by polyriboinosinic acid:polyribocytidylic acid. Activation of macrophages and natural killer (NK) cells by interferon plays a crucial antiviral defense role (Schultz, 1980; Storch et al., 1984). The toxicity of silica for both these cell types results in less interferon production, at least in the case of macrophages, and subsequent inability to activate macrophages and NK cells is believed to be responsible for reduced antiviral protection under these combined circumstances. In most studies, for reasons conducive to experimental design purposes, crystalline silica standards, i.e., D6rentrup and Min-U-Sil, have been commonly used. In an effort to achieve a more realistic assessment of silicate minerals on the interferon system, silicates, as they occur in nature and representative of major silicate classes, were employed in a controlled mammalian cell culture environment. This report describes the effect of different pristine silicates on (1) viral interferon induction, (2) interferon-mediated antiviral cellular resistance, and (3) multiplication of influenza virus along with ancillary observations. MATERIALS AND METHODS Viruses and cell cultures. Virus strains and cell lines used in this study were obtained from the American Type Culture Collection (Rockville, MD). The Ao/PR/8/34 influenza and parainfluenza (Sendal) viruses, used for interferon induction and assay, respectively, were prepared from embryonated chicken eggs and assayed for virus infectivity by the immunofluorescent cell-counting tech-

SILICATES AND THE INTERFERON SYSTEM

397

nique (Hahon et al., 1973). Rhesus monkey kidney (LLC-MK z) and human Chang conjunctival (clone 1-5c-4) cell lines were used for induction and assay of interferon, respectively. Cell lines were propagated in plastic tissue culture flasks (75 cm z) with Eagle's minimum essential medium fortified with 100 x Essential Vitamin Mixture (10 ml/liter) and 200 IxM solution L-glutamine (10 ml/liter), to which was added sodium bicarbonate (2.2 g/liter) and 10% fetal bovine serum. Cells were maintained with the aforementioned medium containing 0.5% fetal bovine serum. Silicate minerals and chemicals. Thirty-two. silicate mineral samples, representative of the six silicate classes categorized by nature of bonding (Strunz, 1966), were obtained from Geoscience Resources (Burlington, NC) (Table 1). European and American silicate standards, DQ-12 (D6rentrup quartz) and Min-U-Sil (pulverized Oriskany sandstone), were gifts from Dr. I. M. Reisner (Essen, West Germany) and Pennsylvania Sand and Glass Corp. (Pittsburgh, PA), respectively. Silicate minerals were ground in a Microjet 5 Spectromill (Micro Materials Corp., Westbury, NY) to a size of approximately 1.0 Ixm diameter as determined by a model 2B Coulter counter (Coulter Counter Electronics, Inc., Hialeah, FL). Stock suspensions of different silicate minerals (w/v) were each prepared in phosphate-buffered saline (PBS), pH 7.1, and sterilized in an autoclave at a pressure of 15 lb/in, z (121°C) for 15 min. A stock preparation of poly(4-vinylpyridine-Noxide) (PVPNO), obtained from Polysciences, Inc. (Warrington, PA), was also made in PBS and sterilized in the same manner. Interferon induction. Duplicate experiments were performed, and the procedure used to study the effect of different silicate minerals on viral interferon inTABLE 1 SILICATE MINERAL CLASSIFICATION BY NATURE OF BONDING

Class

SiO4 tetrahedra arrangement

Si/O ratio

Nesosilicates

Isolated

1:4

Sorosilicates Cyclosilicates Inosilicates

Double Rings Chains (single)

2:7 1:3 1:3

Phyllosilicates

Chains (double) Sheets

4:11 2:5

Tectosilicates

Frameworks

1:2

Groups Olivine Phenacite. , Garnet Zircon AlzSiO5 Chondrodite Epidote Beryl Pyroxenoid Pyroxene Amphibole

SiO2 Feldspar Feldspathoid Scapolite Zeolite

Mineral example Forsterite Willemite ' Pyrope Zircon Sillimanite Chondrodite AUanite Beryl Wollastonite Diopside Tremolite Talc Muscovite Quartz Microcline Sodalite Marialite Stilbite

398

HAHON A N D B O O T H

duction was carried out as follows: from 0.! to 1.0 ml (1.0 to l0 mg) silicate suspension in 10 ml vol of maintenance medium was added to 75-cm 2 plastic flasks containing complete LLC-MK2 cell monolayers (2 x 107 cells), which were then incubated at 35°C for 24 hr. Residual medium was decanted and 2 ml of influenza virus, which had been inactivated by ultraviolet irradiation for 45 sec at a distance of 76.2 mm and wavelength of 253.7 nm, was added onto cell monolayers that were then incubated at 35°C for 2 hr. The ratio of virus to cells, multi"plicity of infection (m.o.i.), was approximately 2.0. Inoculum was removed and 10 ml of maintenance medium was added to each flask. After incubation at 35°C for 20 hr, supernatant fluid was decanted and centrifuged at 100,000g for 1 hr and dialyzed against HC1-KC1 buffer, pH 2.0, at 4°C for 24 hr. Dialysis was continued against two changes of PBS, pH 7.1, at 4°C for 24 hr. Fluids were passed through Millex filters GV, 0.22 p~m (Millipore Corp., Bedford, Mass.), to obtain sterile preparations. Samples were stored at -80°C until they were assayed for interferon activity. Preparations with antiviral activity possessed the biological and physical properties ascribed to viral interferons (Lockart, 1973). Controls consisting of cell monolayers which were not treated with silicates were handled exactly as described above. Neutralization tests with antihuman oL and [3 interferon antisera (Nutritional Biochemicals, Cleveland, OH), using constant serum and varied interferon concentrations (WHO, 1984), indicated that viral-induced interferon from cell cultures was a mixture of ~ and [3 interferons. Interferon assay. An immunofluorescent cell-counting assay of interferon that had been described previously was used to determine the interferon potency of test samples (Hahon, 1981). Interferon-treated 1-5c-4 cell monolayers were challenged with 10 4 cell-infecting units of Sendai virus, and infected cells were visualized by direct fluorescent antibody staining. 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% infected cell-depressing dilution (ICDDs0). With this assay system, 0.89 interferon unit corresponded to 1.0 unit of National Institutes of Health reference standard Hu interferon [3 (G-023-902-527). A twofold decrease (50%) or increase (100%) of interferon production by silicates from the control, which exceeds 98% confidence limits of the assay (Hahon et al., 1975), was considered significant. Influenza virus multiplication. Virus growth concomitant with interferon production was measured in untreated and silicate-treated LLC-MK 2 monolayers (2 × 10 7 cells) in 75-cm z flasks that had been exposed to silicates for 24 hr previously. Following adsorption of virus to cells, m.o.i, of 1.0, at 35°C for 2 hr on a platform rocker (Bellco Glass Inc., Vineland, NJ), cell monolayers were rinsed with PBS and incubated at 35°C with 10 ml of maintenance medium. At designated time intervals, from 0 to 48 hr, flasks were removed and stored at -80°C. Thereafter, each flask was thawed (25°C) and frozen (-80°C) twice to disrupt cells, lightly centrifuged to sediment debris, and the supernatant fluid divided in aliquots. One portion was assayed for virus content on 1-5c-4 cell monolayers (Hahon et al., 1973) and the other processed for interferon assessment as described earlier.

SILICATES AND THE INTERFERON SYSTEM

399

RESULTS

Silicate and Interferon Induction Preliminary experiments were done with each of the 34 silicate minerals to determine the maximal quantity that LLC-MK2 cells could tolerate without loss of viability. Monolayers of nondividing cells (2 x 107) were incubated at 35°C for 24 hr with varied amounts of silicates and estimates of cell viability were determined by using the trypan blue dye-exclusion procedure. Depending on the silicate mineral, the maximal quantity that cells could tolerate with 95% or greater •¢iability ranged from !.0 to 10 mg. Consequently, different amounts of each silicate were used in succeeding experiments. The effect of silicates representative of the different classes of these minerals on viral interferon induction (Table 2) indicates varied reactions on this process ranging from inhibition to enhancement. Of the nesosilicate class, silicates of the phenacite and olivine groups were inhibitory for viral interferon induction (>75%) while garnet and chondrodite groups were ineffectual. Within the AlzSiO 5 group, sillimanite depressed interferon induction whereas kyanite was inactive. Lawsonite, of the sorosilicate class, also had no effect on interferon induction, however, silicate members of the epidote group were significantly inhibitory. The two minerals tested of the cyclosilicate class, beryl and cordierite, were also markedly inhibitory to interferon induction. Among and within the mineral groups that comprise the inosilicate class, the effect by representative silicates on viral interferon induction was highly varied. In the pyroxene group, hypersthene and diopside showed minimal (19%) and significant (65.5%) depression of interferon induction, respectively. Similarly, tremolite and actinolite of the amphibole group exhibited varied effects with only the former mineral decreasing interferon induction (45%). However, wollastonite, pectolite, and rhodonite, members of the pyroxenoid group, all markedly enhanced interferon production by two- to threefold as compared to the control. Both silicates, muscovite and pyrophyllite, of the phyllosilicate class were inhibitory to interferon induction. The minerals of the tectosilicate class comprise a major part of the earth's rocky crust and are built about a framework of linked S i O 4 tetrahedra. All of the silicates representative of this class and within the corresponding four groups significantly inhibited interferon induction, with the exception of cristobalite and orthoclase, which showed marginally significant interferon inhibition of 43 and 41%, respectively. The most consistent adverse effect on interferon induction inhibition was displayed by the 12 minerals tested in the tectosilicate class. The varied dose-response relationships among selected silicates and interferon induction inhibition are a reflection not only of mineral classification but also of limitations imposed by their cytocidal activity (Fig. 1).

Silicates and Interferon-Mediated Antiviral Activity Ten randomly selected silicate minerals were tested for their influence on interferon-mediated cellular resistance to virus infection. This was determined in two

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TABLE 2 EFFECT OF SILICATEMINERALSON INTERFERON(IFN) INDUCTIONBY INFLUENZAVIRUSIN LLC-MK2 CELL MONOLAYERS

Silicate Nesosilicates Phenacite GP Willemite Olivine Gp Olivine Garnet Gp Garnet Zircon Gp Zircon AIzSiO5 Gp Sillimanite Kyanite Chondrodite Gp Chondrodite Sorosilicates Lawsonite Epidote Gp Allanite Epidote Cyclosilicates Beryl Gp Beryl Cordierite Inosilicates Pyroxene Gp Hypersthene Diopside Pyroxenoid Gp Wollastonite Wollastonite Pectolite Rhodonite Amphibole Gp Tremolite Actinolite Phyllosilicates Muscovite Pyrophyllite Tectosilicates SiOz Gp Milky quartz Cristobalite DQ-12a Min-U-Sile Feldspar Gp K series Microcline Orthoclase

Quantity (rag)

IFN yield (ICDDso/ml) a

Franklin, NJ

5

66

67.0

Daybrook, NC

5

47

76.5

Martinsville, VA

10

200

0.0

Eureka Tunnel, CO

10

180

10.0

Manitouwadqe, Ontario Adirondack Mts., NY

10 10

97 180

51.5 10.0

Amity, NY

10

179

10.5

Danoche, CA

10

240

0.0

Otterlake, Quebec Banner Elk, NC

10 10

76 96

62.0 52.0

Beryl Mt., NH Ontario

10 5

55 88

22.5 55.6

Wollastonite Twp., Ontario Cascadeville, NY

10 10

162 69

19.0 65.5

Willsboro, NY Mexico Lake Nipigon, Ontario San Juan Mr., CO

10 2 2 2

733 512 426 466

0.0 0.0 0.0 0.0

Balmat, NY Sonora City, CA

10 10

110 240

45.0 0.0

5 5

81 34

59.3 83.0

Ingalls, NC Pachuca, Mexico Essen, West Germany Pittsburgh, PA

10 4 1.0 1.0

91 114 83 82

54.5 43.0 58.5 59.0

Rosman, NC Concord, NC

10 10

91 118

54.5 41.0

Geographical source

Rocky Mount, VA Asheboro, NC

IFN inhibition b (%)

(+3.6) c

(+2.5) (+2.3) (+2.3)

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SILICATES AND THE INTERFERON SYSTEM

TABLE 2--Continued EFFECT OF SILICATE MINERALS ON INTERFERON (IFN) INDUCTION BY INFLUENZA VIRUS IN LLC-MK2 CELL MONOLAYERS

Silicate N a - C a series Albite Oligoclase Feldspathoid Gp Sodalite Nepheline Scapolite series Wernerite Zeolite Gp Stilbite Control

Quantity (nag)

IFN yield (ICDDs0/ml)"

10 5

82 96

59.0 52.0

Nephton, Ontario Red Hill, NH

5 5

100 96

50.0 57.0

Bancroft, Ontario

5

74

63.0

Northeast, NY

5 0

55 200

72.5 0.0

Geographical source Anner Elk, NC Stone Mt., GA

IFN inhibitionb (%)

a Reciprocal of 50% infected cell-depressing dilution; mean of two determinations. b Reciprocal of ICDDs0/ml of IFN yield/control ICDDs0/ml) - 1.0 x 100. c Fold interferon increase of control. a European silica standard, Drrentrup quartz. e American silica standard.

ways: (1) an interferon preparation of known potency was assayed in the usual manner using 1-5c-4 cell monolayers that have been exposed to silicates for 20 hr previously and (2) by pretreating LLC-MK 2 cell monolayers with silicates (20 hr), then with interferon (20 hr), and, subsequently, infecting cells with live influenza virus. After incubation at 35°C for 20 hr, cell culture medium was assayed for virus content. Results (Table 3) show that assessment of interferon in cell cultures pretreated with any of the 10 silicate minerals did not significantly alter interferon's antiviral protective potency as indicated by interferon titers attained, which were comparable to that of the control. Influenza virus yields from all cell cultures pretreated with silicates and interferon were comparable to that of cells treated only with interferon. In cell cultures that were not pretreated with either silicates or interferon, the virus yield was eightfold higher. That the ability of exogenous interferon to confer antiviral cellular resistance was unaffected by earlier exposure of cells to silicates is evident by these findings. Virus Replication in Silicate-Treated Cells Influenza virus multiplication and concomitant interferon production were determined in normal LLC-MK2 cell monolayers and in those pretreated for 20 hr earlier with three arbitrarily selected silicates, DQ-12, stilbite, and pyrophyllite. Virus multiplication in normal and silicate-treated cells was similar to the extent that peak levels of growth were attained and maintained from 16 to 24 hr (Fig. 2). However, virus growth in silicate-treated cells reached a level that was approximately twofold higher than that noted in normal cell cultures. Thereafter, virus declined more precipitously in control than in silicate-treated cell culture.

402

HAHON AND BOOTH PERCENTAGE

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5 I

INTERFERON

10 15 20 I

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[

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50 60 70 8 0 8 5 90

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6.5

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FIG. h Dose-response relationshipbetween silicate mineralsand inhibitionof interferoninduction by influenzavirus in LLC-MK2cell monolayers.

Whereas the rate of interferon production appeared similar for the first 20 hr in normal and silicate-treated cells, interferon production attained a level at 20 hr that was twofold higher in normal cells than in silicate-exposed cell cultures (Fig. 3). This suggests that the higher level of virus growth attained in cell cultures pretreated with silicates than in normal cells may be the consequence of partial suppression of interferon synthesis by silicates.

Antagonism of P V P N O on Silicate Activity on Interferon Induction Eight silicate minerals, previously noted to inhibit interferon induction, and wollastonite, shown to enhance interferon production, were pretreated with PVPNO to determine whether the polymer could abrogate their respective activi-

403

SILICATES AND THE INTERFERON SYSTEM TABLE 3 EFFECT OF DIFFERENT SILICATES ON ANTIVIRALACTIVITY OF INTERFERON (IFN) Silicate cell treatment prior to IFN exposure (20 hr) Milky quartz DQ-12 Stilbite Pyrophyllite Diopside Beryl Olivine Allanite Tremolite Wollastonite MM c (control) MM (control)

IFN antiviral assessment Silicate (mg)

IFN exposure (20 hr)

Influenza virus yielda (CIU × 103/ml)

IFN titration b (ICDDs0/ml)

10 1 5 5 10 10 5 10 1 10 0 0

+ + + + + + + + + + + -

1.1 0.95 1.2 0.95 1.1 1.3 1.4 1.0 1.2 0.88 1.2 8.4

120 120 150 150 120 150 130 150 170 140 150 0

a After pretreatment of LLC-MK z cell monolayers with silicates and then 10 ml of interferon (18 ICDDs0/ml), virus was introduced, and after incubation at 35°C for 20 hr, culture medium was assayed for virus expressed as cell-infecting units. b All monolayers (1-5c-4) were exposed to silicates (20 hr) then serial dilutions of interferon (20 hr) and challenged with Sendai virus. Interferon titers are expressed as reciprocal of 50% infected celldepressing dilution; mean of two determinations. c Maintenance medium.

ties on the viral interferon induction process. Results (Table 4) indicate that marked suppression of the inhibitory activities of the eight silicates as well as the enhancing effect of wollastonite occurred in these minerals when pretreated with PVPNO. With untreated silicates, viral interferon induction inhibition occurred similar to that noted earlier. Untreated wollastonite also enhanced interferon induction by almost threefold. The findings indicate the effectiveness of PVPNO in altering the biologic activities of silicates with regard to viral interferon induction. DISCUSSION The findings reported herein demonstrate that natural-occurring silicate minerals affected viral interferon induction in varied magnitudes ranging from inhibition to enhancement. This was evident with member minerals and groups comprising the silicate classes nesosilicate, sorosilicate, cyclosilicate, and inosilicate, in which little or marked depression of the process occurred. The few minerals in the phyllosilicate class and a larger number tested in the tectosilicate class all showed inhibitory activity for viral interferon induction. Within the inosilicate class, however, minerals of the pyroxenoid group, wollastonite, pectolite, and rhodonite, were unique from other silicate minerals in that they significantly enhanced the viral production of interferon. That wollastonite per se did not induce interferon but only enhanced the induction process in conjunction with a viral inducer has been reported previously (Hahon et al., 1980). Although the pyrox-

404

HAHON AND BOOTH

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Control

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Stilbite x Pyrophyllite

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Fla. 2. Growth curves of influenza virus in LLC-MK2 cell monolayers untreated (control) and pretreated with silicate minerals.

enoid and the pyroxene silicates have the same ratio of Si:O = 1:3, they do not have the same structure. The single chains of linked SiO 4 tetrahedra that in the pyroxenes extend indefinitely in the c direction are not present in the pyroxenoids, e.g., wotlastonite (Hurlbut, 1971). Whether this is the determinant factor to account for viral interferon induction enhancement by these silicates remains to be resolved. Also unexplainable are the varied grades of interferon inhibitory activity or inactivity associated with minerals within and between silicate classes. This may be a reflection of structural configuration, chemical composition of the silicates, or both. Findings contrary to the depressive role of mineral dust on interferon produc-

405

SILICATES AND THE INTERFERON SYSTEM 3.0-

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Stilbite x Pyrophyllite

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Ld CA

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I0

20

30

40

50

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FIG. 3. Concomitant interferon production following influenza, virus infection of LLC-MK2 cell monolayers untreated (control) and pretreated with silicateminerals.

tion have been reported (Schlipk6ter and Brockhaus, 1960). When silica was injected intravenously into mice to determine its effect on interferon induction by statolon and by Newcastle disease virus, the mineral had no inhibitory effect on interferon production by these inducers. It was suggested that subversion of interferon production does not play a significant role in the potentiation of virus infection in silica-treated animals. When various asbestos fibers or coal dust were treated with animal sera, the usual inhibitory activity of these mineral dusts on interferon production was abrogated (N. Hahon, unpublished data). Silica is known to adsorb serum proteins (Jones et al., 1972). The lack of a subversive effect by silica on interferon production may be explained by the mode of animal

406

HAHON AND BOOTH TABLE 4 ANTAGONISTICEFFECTOF POLY(4-VINYLPYRIDINE-N-OXIDE)(PVPNO) ON INHIBITIONOF ]INFLUENZAVIRUSINDUCTIONOF INTERFERON(IFN) BYDIFFERENTSILICATES Pretreatment of silicates + PVPNO

- PVPNO

Silicate (mg)

IFN yield (ICDDs0/ml)a

IFN inhibitionb (%)

IFN yield (ICDDso/ml)

IFN inhibition (%)

DQ-12 (1)c Olivine (5) Allanite (10) Beryl (10) Muscovite (5) Stilbite (5) Milky quartz (10) Sodalite (5) Wollastonite (5) Control (0)

185 220 220 180 185 130 160 166 185 180

0.0 0.0 0.0 0.0 0.0 27.8 11.2 7.8 0.0 0.0

65 78 95 75 94 84 86 80 520 180

63.9 56.7 47.3 58.4 47.8 53.4 52.3 55.6 0.0 0.0

a Reciprocal of 50% infected cell-depressing dilution; mean of two determinations. b (Reciprocal of ICDDs0/ml of interferon yield/control ICDDs0ml) - 1.0 x 100. c Designated amounts of silicate were mixed with an aqueous suspension of PVPNO (100 mg/ml) and shaken for 3 days at 24°C. After centrifugation, silicate pellets were suspended in 10 ml maintenance medium and added onto LLC-MKz cell monolayers and incubated at 35°C for 20 hr.

inoculation, i.e., injection of the mineral directly into the bloodstream, which resulted in the coating of silica with serum proteins. O f the representative silicates, which exhibited inhibitory activity to viral interferon induction and w e r e used to determine their effect on interferon-mediated antiviral cellular resistance, no impairment of this latter p h a s e of the defense m e c h a n i s m was evident. This is similar to findings with other particulates ( H a h o n et al., 1980; H a h o n , 1983; H a h o n and Booth, 1984). It would appear, therefore, that no detrimental effect on p r e f o r m e d interferon (a/J3) or the r e q u i r e m e n t for protein synthesis (an integral part of interferon's ability to confer antiviral cellular resistance) occurred in cell cultures pretreated with silicates. In cell m o n o l a y e r s p r e t r e a t e d with selected silicates, DQ-12, stilbite, or pyrophyllite, the influenza virus growth levels attained were a p p r o x i m a t e l y twofold higher than in untreated cell cultures. Conversely, that the interferon level in untreated cells was at least twofold higher than in silicate-treated cells suggests that the higher virus growth levels obtained are a reflection of partial suppression o f interferon synthesis b y silicates. This poses the possibility that silicates, in compromising the interferon induction process, m a y increase host susceptibility to viral infection. T h e s e data also imply that neither the early stages of virus inducer-cell interaction (attachment and penetration) nor cell protein and nucleic acid synthesis required for virus replication were affected. H o w e v e r , it is evident that silicates m a y selectively impede protein synthesis or responsible precursors. The viral interferon induction process requires protein synthesis and this could possibly be impeded through either transcription of interferon m e s s e n g e r R N A or

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its translation on membrane-bound polysomes in the process of forming "mature" interferon glycoproteins (Stewart, 1979). The use of silica antagonists to prevent the initiation of silicosis is based on the position that it is the silica surface that interacts with cell membranes and components. Any masking or chemical modification of the surface may reduce adsorption of silica to cell membranes. Most studies in this area have involved the use of PVPNO, a water-soluble "relatively" inert organic polymer. The successful use of PVPNO in experimental studies to prevent silicosis has been succinctly reviewed (Iler, 1979). Of the eight silicate minerals tested which inhibit interferon induction, pretreatment with PVPNO in most instances completely abolished this adverse action on interferon induction as well as the interferon-enhancing activity of wollastonite. The counteraction of PVPNO on the inhibition of viral interferon induction by coal dust and asbestos fibers has also been noted (Hahon, 1976; Hahon et al., 1977). The mechanisms of PVPNO action, based largely on studies with silica (Holt, 1971), indicate that N-oxide is the dominant reactive group that adsorbs onto silica through hydrogen bonding. Part or all of the mechanisms that may be involved in the protection by PVPNO are the following: (1) coating the silica surface, (2) interaction with monosilicic acid formed by solution of silica in the cell, and (3) stabilization of cellular or subcellular membranes. It was not the scope of this study to resolve definitively the interplay between silicates and infectious agents on respective disease processes but rather to examine the interaction of silicate minerals representative of major silicate classes on a mammalian cellular defense mechanism. We have no knowledge of similar studies with specific minerals, inclusive of all silicate classes, pertaining to resultant disease states in either humans or experimental hosts or affecting other biologic mechanisms. REFERENCES Bailey, W. C., Brown, M., Buechner, H. A., Weill, H., Ichinose, H., and Ziskind, M. (1974). Silicomycobacterial disease in sandblasters. Amer. Rev. Respir. Dis. 110, 115-125. Burrell, R. (1981). Immunological aspects of silica. In "Health Effects of Synthetic Silica Particulates" (D. D. Dunnom. Ed.), pp. 82-93. American Society for Testing and Materials, Philadelphia. Chiappino, G., and Vigliani, E. C. (1982). Role of infective immunological, and chronic irritative factors in the development of silicosis. Brit. J. Ind. Med. 39, 253-258. Cocarla, A. A., Gabor, S., Milea, M., and Suciu, I. (1977). Serum immunoglobulins in workers exposed to silicogenic risk with tuberculin-negative test and hyperergia. J. Hyg. Epidemiol. Microbiol. Immunol. 21, 374-380. Friedman, R. L., and Moon, R. J. (1977). Hepatic clearance of Salmonella typhimurium in silicatreated mice. Infect. Immun. 16, 1005-1012. Gernez-Rieux, C., Tacquet, A., Collet, A., Macquet, V., Martin, J. C., and Poucard, A. (1963). Etude experimentale de l'influence de l'empoussierage du paumon sur son infection par les mycobacteries atipyques. C.R. Acad. Sci. 257, 3103-3109. Hahon, N. (1976). Counteraction of poly(4-vinylpyridine-N-oxide) on the depression of viral interferon induction by coal dust. Infect. Immun. 13, 1334-1342. Hahon, N. (1981). Hemadsorption and fluorescence determinations for assay of virus-yield reduction of interferon. In "Methods in Enzymology" (S. Pestka, Ed.), Vol. 78, "Interferons," Pt. A, pp. 373-381. Academic Press, New York. Hahon, N. (1983). Effect of coal rank on the interferon system. Environ. Res. 30, 72-79.

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