An immunologic study of experimental silicosis

ESVIRONMENTAL

An

RESEARCH

1, 171-177

Immunologic HENRY

Depnrtment

(1967)

Study J.

ESBER

of Microbiology, West Morgantown, Received

of Experimental AND

ROBERT

BURRELL

Virginia University W. Va. 26506 May

Silicosis

Medical

CCII~CI

25, 1.967

Espcrimental silicosia was produced by injecting guinea pigs intratmchcally with silica suspensions. Pathological changes consistent with silicosis werr noted at autops? nine months after injection. Many serological tests such as precipitation, passive hemagglutination, complement fixation, and passive cutaneous anaphylaxis were performed on sera from these animal?, but nonr was successful in demonstrating tissue-reactive autoantibodies. It was found that the usr of latex particles sensitized with crude lung extracts could not be used to demonstrate tissue-reactive antibodies. It was shown that such particles artually detect rheumatoid factor likr antibodies (antiglobulin antibodies). d modification of the antiglobulin consumption test employing lung connective tiszuc as antigen was developed and made available to study the tissue antibodies in guinea pigs. This modification was quite sensitive in the detection of lung-reactive antibodies in the experimental animals. The development of this serological technique should permit immunological studies of an experimental model of silicosis to bc conductpd .morc easily and under more controlled conditions in the future.

Circulating antibodies in serum specimens obtained from patients with pneumoconiosis or other chronic lung diseases have been previously shown to react with crude preparations of lung connective tissue, lung fibroblast ccl1 cultures, or lung collagen (Burrell et aZ., 1964, 1966). It was shown by in vitro tests that the most significant antibody associated with pneumoconiosis was directed primarily towards collagen. These findings led to the general conclusion that an immunological mechanism may be involved in such chronic respiratory diseases. To fully assess the role of such antibodies in the pathogenesis of silicosis (Vigliani and Pernis, 1963), it is necessary to set up an experimental model of silicosis that can be studied immunologically. In this way, such studies could be carried out under more easily controlled conditions than is possible with the study of human sera alone. This paper describes the establishment of an experimental model of silicosis in laboratory animals and an immunological method specifically adapted for the demonstration of tissue-reactive antibodies in these animals. MATERIALS

Experimental

AND

METHODS

Silicosis

Two- to three-month-old guinea pigs were placed in the animal quarters for a four week acclimatization period. From these, 44 were selected for the production of silicosis. At this time the animals averaged 350 gm in weight and were kept under strict quarantine measures for the duration of the experiment.. In addition t’o daily sanitation measures, the animals also received daily ad Zibitum supplies 171

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of sulfa prophylaxis. Drinking water containing 2 ml of 12% sodium sulfamethazine/250 ml bottle was made continuously available. .4lpha-quartz crystalline silica (SiO,) having a mean particle size of 1.1 p, as det,ermined by electron microscopy, was obtained through the kind help of Mr. Robert G. Keenan and Dr. L. D. &heel of the Public Health Service’s Occupationa1 Health Research and Training Facility, Cincinnati, Ohio. These siIica particles were suspended in saline at a 50 mg/ml concentration prior to injection. A 0.5 ml injection was given intratracheally to each anesthetized guinea pig. Care was taken to insure deep penetration of each injection. The injections were then repeated three months later. Control animals received similar injections of saline. Sera Al1 animals were bled periodicahy prior to use.

and the harvested

sera were stored at -20°C

d digens Two general types of antigenic preparations were made from fresh lungs obtained from young healthy guinea pigs; one was soluble, and the other was insoluble. One preparation of a soluble lung protein fraction was patterned after Dejenian et al. (1964). Fresh lungs were washed and perfused with cold phosphate-buffered saline (PBS), pH 7.2, to remove the blood. A homogenate, one part tissue to nine parts PBS, was then made in a Waring blendor. The resulting suspension was centrifuged in the cold at 12,000g for 20 minutes and the supernate fluid collected. After extracting the protein from this mixture with 4 M ammonium sulfate, the precipitate was washed twice with a 1.6 M solution of ammonium sulfate and then dialyzed against PBS at 4°C overnight. A second soluble antigen was prepared according to the method of Balchum et al. (1965) for use in the latex fixation tests described below. The insoluble connective tissue antigens were prepared according to previously described methods (Burrell et al., 1964). Serological

Procedures

Conventional serological techniques of complement fixation, immunodiffusion, and passive cutaneous anaphylaxis were used (Burrell and Mascoli, 1966). Latex fixation tests were carried out according to the methods of Balchum et al. (1964, 1965). Homogenation of normal guinea pig lungs in saline was carried out at 4°C. This homogenate was next agitated overnight and further homogenized before the supernate fluid was collected. This preparation was diluted with 0.066 M PBS, pH 7.4, until a 40% transmission was obtained at 550 mp using a Coleman Jr. spectrophotomet8er. Latex particles (0.81 p) were then added until the light transmission was 25-30s and then incubated for 60 minutes at 37°C. Serial dilutions of the serum in saline were prepared in 0.5 ml amounts and to each was added 0.1 ml of the latex antigen. An incubation of 60 minutes at 37°C was foIlowed by 12 hours at 4°C before any readings were made. This test was also performed using salt-free guinea pig globulin to coat the latex particles instead of the lung homogenate.

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ii modification of the ant,iglobulin consumption test (AGCT) of Steffen (1960) had to be developed for the guinea-pig system. This was accomplished by adapting the tanned cell method of Mengoli et al. (1963) as an indicator system for the I1GCT. Fresh sheep erythrocytes (less than 48 hours old) collected in modified Alsever’s solution were washed three times with cold saline and adjusted to a 2% suspension. Equal volumes of these washed cells and 1: 80,066 tannic-acid solution were mixed thoroughly and incubated at room temperature for 30 minutes with intermittent shaking. The tanned cells were washed three t,imes with cold saline and resuspended to the original volume. Equal volumes of washed, tanned erythrocytes and guinea-pig globulin (Pentex Corporation, Kanakee, Ill.) at a concentration of 100 pg/ml of PBS, pH 6.4, containing 0.01 M dipotassium versenate were mixed and incubated for one hour with intermittent shaking. The cells were then washed three times in PBS, pH 7.2, and resuspended in cold rabbit diluent (1% normal rabbit serum in PBS, pH 7.4). The test was carried out by incubating 0.2 ml of the guinea-pig serum with 2.5 mg of lyophiliaed, globulin-free guinea pig lung connective tissue at 37°C for 30 minutes. After washing the lung-tissue sediment four times with PBS, pH 7.2, and eight t.imes with 1% Na,EDTA-saline, 0.5 ml of a predetermined dilution of anti-guinea-pig globulin (Hyland Laboratories, Los Angeles, California) was added and incubated for 45 minutes at room temperature. This antiglobulin reagent was then titrated against the indicator cells and the results observed after 12 hours incubation at 4°C. Each doubling dilution of inhibition of agglutination from the known titer was considered as one unit of antiglobulin consumption. In order for a test to be considered positive, two or more units are generally regarded as necessary.

Immunofluorescen

t Techniques

(PA)

Fluorescent antibody studies were carried out on frozen sections of t,he guineapig lungs by previously described methods (Burrell et al., 1966). The lungs were inflated in situ by intratracheal injection of warm 10% gelatin. The lungs were cooled at 4°C for 30 minutes before freezing. Once frozen, care was taken to insure that the lungs did not thaw prior to sectioning. RESULTS

Initial serological studies used on bleedings from the silica-injected animals consisted of passive hemagglutination, complement fixation, passive cutaneous anaphylaxis, and precipitation procedures. Various soluble extracts and homogenates from normal lung tissue served as antigens. When such preparations were used to sensitize tanned cells, results obtained from reacting these cells with the guinea-pig sera were erratic and not reproducible. Furthermore, bleedings from control animals gave similar results. Complement fixation tests, using such extracts as antigen, gave similarly unreliable results. No reactions could be obtained with any of the sera using the Dejenian antigens by means of passive cutaneous anaphylaxis or immunodiffusion techniques. The latex fixation test, using such homogenates as antigen, has been reported

174

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as successful in detecting lung-reactive antibodies. This technique was applied to sera taken from the test guinea pigs at 5 months after the initial silica injection as well as to sera from other guinea pigs one year after having received much smaller injections. The results, seen in Table I, show that all such animals developed reasonably high titers to the lung-homogenate-sensitized latex particles. However, personal experience with this type of tissue homogenate has shown that it is mostly, if not entirely, composed of serum proteins and, hence, such a positive reaction might be due to an antiglobulin reaction similar to the rheumatoid-factor demonstration. TABLE I COMPARISON OF THE LATEX AGGLUTINATION LUNG HOMOGENATE OF GUINEA PIGS

Guinea-pig number 803 90 806 819 818 815 111 113 114 105 128 109 106 107 119 121 Anti-guinea

Dose of silica (mg) 50 50 50 50 50 50 5 5 5 2.5 2.5 2.5 2.5 2.5 Control Control pig serum

Time after silica exposure (months) 5 5 5 5 5 5 12 12 12 12 12 12 12 12 12 12

TEST USING AS SENSITIZING

GLOBULIN ANTIGENS

Latex coated with guinea-pig lung homogenate 1:40 1: 2560 1:640 1: 2560 1:640 0 1:640 1:so 1:160 1:640 1:160 1:80 1:80 1:160 0 0 1:320

AND

Latex coated with guinea-pig globulin 1:160 1:5120 1:5120 1: 10240 1: 1280 1:20 Not done Not done Iiot done Kot done Not done Sot done Not done Not done

1:10340

In order to determine if this were indeed the case, these same sera were reacted with latex particles sensitized with guinea-pig gamma-globulin. The results of these tests are given in the right hand column of Table I. In all instances t,he antiglobulin titers were even higher than those obtained against guinea pig lung homogenate. In addition, an anti-guinea-pig whole serum preparation reacted strongly with each of these two latex antigens. This same serum also evidenced lines of precipitation with lung homogenate when tested by immunodiffusion. The antiglobulin consumption test (AGCT) has proven more reliable in detecting true lung-reactive antibodies in human sera (Burrell et al., 1964, 1966). An appropriate modification of this test was applied to sera taken from the treated guinea pigs at 5-, 8-, and g-month intervals after the initial silica injections. The results, presented in Table II, demonstrate that lung-reactive antibodies had indeed developed in the experimentally induced silicotic guinea pigs. Of the sera tested, 50% had shown 3-5 units of antiglobulin consumption or above

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SILICOSIS

in all bleedings following the injection of silica. However, it seemed that the reactivity decreased with time. No serum obtained from the guinea pigs prior to silica injection was positive nor was any serum taken from control guinea pigs housed under nonquarantine conditions for several months. Such control animals were thus exposed to many of the endogenous and exogenous respiratory infections that would likely have occurred during that time. Multiple stained sections of each of the guinea pigs’ lungs (taken at 9 months) were examined by conventional light as well as polarized light microscopy. Some showed patchy areas in which the alveoli were indistinguishable and were replaced by numerous macrophages and plasma cells. The lymphoid follicles had a slight to moderate hyperplasia. The alveolar walls were thickened and congested TABLE I\NTI~+LORT-1.15

COSSUMPTION INJECTED

II

TEST

I~EACTIOKS OF SERA FROM GUINEA PIC;S WITH ALPHA-QUARTZ CRYSTALLINE SILICA

EXPERIMESTAILY

TSnit,s of consumption 4

5

-

Baseline bleeding Five months silicaa Eight months silicab Siue months silica< Control guinea pigd 0 Serum h Serum c Serum d Serum the animal

specimens were specimens were specimens were specimens were quarters for 9-12

4 3 1 obtained obtained obtained obtained months.

3 -

3 4 2 -

2

-

5 months after initial S months after initial 9 months after initial from nonquarantined

0

Total

6 5 3 -

27 0 5 3 11

27 21 22 16 12

-

4 4 3

1

4 1 4 1 silica injection. silica injection. silica injection. normal guinea

pigs which

were

kept

in

with fluid, and moderate hyperemia was seen. Nodules having the appearance of granulomatous or pregranulomatous lesions predominated the microscopic findings. They were diffusely infiltrated with lymphocytes, plasma cells, and giant cells. Most of the macrophages and some of the fibroblasts surrounded these hyperplastic granulomas giving such a lesion a nodular appearance. In addition to the hematoxylin-eosin tissue sections studied, frozen sections of the guinea-pigs’ lungs were stained by the direct FA technique using rabbit anti-guinea-pig globulin conjugated with fluorescein isothiocyanate. Although the conjugated globulin was adsorbed with bone marrow, passed through a column of Stpha8dcx G-25 to remove dissociated dye, and the proper dilution was used, several difficulties were encountered. Nonspecific fluorescence was noted in all sections observed; thus, this procedure will have to await further refinements before it can be used with lung tissue. The use of molten gelatin to inflate the lungs prior to frozen sectioning proved to be helpful although it, still remained difficult, to produce high-quality sections from the frozen lung at a thickness of 5 p. Nevertheless, consistent reproducible results were hard to obtain. Specific staining was confined to mononuclear cells seen in the pregranulomatous nodules or in the acellular portions of older nodules, but technical difficulties prevented any further interpretation.

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DISCUSSION

In an attempt to produce silicosis in experimental animals, only guinea pigs were employed and these were kept quarantined and on chemotherapeutic prophylaxis throughout the entire period. This was necessary to control the upper respiratory epizootics to which these animals were known to be susceptible. That experimental silicosis was produced was confirmed by the pathological findings. Various publications regarding the use of crude tissue extracts led to an investigation of the validity of using such extracts in serological procedures. Lunghomogenate-coated latex particles were prepared after the method of Balchum et al. (1965) to test sera from silicotic animals. Positive titers were obtained in most of the experimental animals with a reading as high as 1: 2,560. However, when globulin-coated latex particles were used, similar results were obtained, but with higher titers. That an antiglobulin was being demonstrated by using such an organ extract rather t.han an antitissue antibody, was confirmed by t,he finding that the guinea-pig-lung homogenate react,ed with anti-guinea-pig globulin in the immunodiffusion procedure. It was concluded that, the only soluble material present in such a tissue homogenate was normal blood protein and that the antibodies described by Balchum et al., are actually rheumatoid-factor-like antibodies (antiglobulins) which are known to occur in silicosis (Schroeder et nl., 1962). The complement fixation studies with tissue homogenates also showed the presence of reactivity, but could not be definitely attributed to silicosis and one can not rule out the inference of rheumatoid-factor-like antibodies as being responsible for the reactions. Due to the fact that tissue homogenate did not render itself suitable fol serological testing, studies were undertaken to develop a new method by which the induced silicotic guinea-pigs’ sera could be tested for their immunological response. At the beginning of this study no such modification of the antiglobulin consumption test was available for guinea pigs due to the lack of a suitable indicator system. The modification selected for use here consisted of sensitizing tanned sheep erythrocytes with guinea-pig globulin and balancing these cells against a known anti-guinea-pig globulin. The testing procedure was essentially the same as that used for the human AGCT. When this technique was applied to the sera from the silicotic guinea pigs, most showed positive reactions. That these reactions were regarded as more specific than those obtained with soluble extracts of lung tissue was evidenced by the observation that none of the sera taken before silica injections were positive noi were any of the sera taken from control uninjected guinea pigs. It was important that the controls were animals that had been kept in the animal quarters under nonquarantine conditions for several months, since it had been observed earlier that such animals spontaneously developed nonspecific complement-fixing antibodies to lung homogenate. Also, the antigen used in the AGCT must be globulinfree in order to function properly. Hence, rheumatoid-factor-like antibodies can not be demonstrated by this technique. The development of a suitable modification of the AGCT to the experimental

IMhlUNOLOGT

OF

EXPERIMENTAL

HILICOSIS

Iii

model offers a great. advantage to the study of silicosis. Having such a model that can be studied immunologically under controlled conditions, other studies can be undertaken which arc not possible to perform with humans. Studies involving the dose of silica particles required to produce silicosis and the duration of t.imt needed for antibody production will be greatly facilitated with such a model. Having devised a method to detect antibodies against guinea-pig lung tissue, passive transfer experiments may add more information toward protective or destructive nature of the antibodies involved in the silicotic animals. Finally, the exact relationship between the fibroblast,s and collagen fibrils seen in silicotic lesions and the antibodies against these tissue components may be elucidated with such a model. REFERENCES 0. J., BUCKLEY, R., LEVEY, S., BERMLINO, J., SWANN, H., AND HALI.. T. (1961). Studies in experimental emphysema. Arch. Environ. Health 8, 132-138. BALCHUM, 0. J., BUCKLEY, R. D., SHERWIN, R., AND GARDNER, M. (1965). Sitrogcn I-lioxitl[> inhalation and lung antibodies. Arch. Environ. Health 10, 274-277. BUHRELL, R. G., WALLACE, J. P., AND ANDREWS, C. E. (1964). Ilung antibodies in ptienta with pulmonary disease. Amer. Rev. Resp. Dk. 89, 697-706. BURRELL, R. G., ESBER, H. J., HAGADORN, J. E., AND ANDREWS, C. E. (1966). Specificity of lung rcaet.ive antibodies in human sera. Amer. Rev. Resp. Dis. 94, 74%750. BURRELL, R. G., AND MASCOLI, C. C. (1966). “Experimental Immunology,” 2nd ed., BurgcPs, Minneapolis. DEJENIAN, A. Y., BEUTNER, E. H., AND WITIZBSKY, E. (1964). Tanned cell hemagglutination test for detection of antibodies in sera of patients with myasthenia gravis. J. Lab. Clin. Mrd. 63, 60-70. MENGOLI, H. F., PRUITT, J. C., AND CARPENTER, H. M. (1963). Electronic quantitation of tanned cell hemagglutination. Lab. Invest. 12, 365-377. SCHROEDER, W., FRANKLIN, E. C., AND MCEWEN, C. (1962). Rheumatoid factors with silicosis with round nodular fibrosis of the lung in the absence of rheumatoid arthritis. BALCHUM,

Arthritis

Rheum.

5, W-18.

C. (1960). Results obtained with the antiglobulin consumption test and inl-rrtig:ktions of autoantibody eluates in immunohematology. J. Lab. Clin. Med. 55, 9-28. VIGLIANI, E. C., AND PERNIS, B. (1963). Immunological aspects of silicosis. .4,ii~nrrr Trtberc. Res. 12. 230-279. STEFFEN,