Clin. exp. Immunol. (1979) 36, 266-27 1.
The effect of cortisone acetate on pulmonary lesions in a rabbit model of hypersensitivity pneumonitis (cortisone and hypersensitivity pneumonitis) FRANCES M. CASHNER, M. R. SCHUYLER* & J. E. SALVAGGIO Department of Medicine, Tulane University Medical Center, New Orleans, Louisiana, USA
(Accepted for publication 15 May 1978) SUMMARY
Rabbits were sensitized with Micropolyspora faeni by intratracheal inoculations and later challenged with the same antigen either with or without parenteral administration of cortisone acetate prior to challenge. Animals developed anti-M. faeni serum precipitins, M. faeni-induced alveolar macrophage migration inhibition, and positive 48-hr skin reactivity to M. faeni. Sensitized animals also demonstrated an augmented pulmonary histopathological response following respiratory challenge with M. faeni when compared to non-sensitized controls. Cortisone acetate abrogated this augmented pulmonary histopathological response following challenge with M.faeni. Cortisone acetate also abolished the positive alveolar macrophage migration inhibition found in sensitized animals.
INTRODUCTION Human hypersensitivity pneumonitides are caused by a variety of inhaled organic dusts. Type II, Type III and Type IV hypersensitivity reactions (Kawai et al., 1973; Pepys, 1969; Wenzel, Emanuel & Gray, 1971; Wilkie, Pauli & Gygax, 1973) have all been implicated in disease pathogenesis but the relative importance of each mechanism is not well understood. Pulmonary lesions similar to those found in human hypersensitivity pneumonitis can be induced in the rabbit by intratracheal administration of particulate Micropolyspora faeni antigen, the principal antigen involved in Farmer's lung disease (Kawai et al., 1972). Although the development of the lesions may be non-immunologically mediated, immunological events are correlated with such lesions, including local delayed hypersensitivity in the lung as measured by macrophage migration inhibition (MMI) of alveolar wash cells, positive delayed skin reactivity, specific serum and bronchial wash precipitating antibody, and the appearance of activated macrophages in the alveolar wash cell populations (Harris, Bice & Salvaggio, 1976; Kawai et al., 1973; Salvaggio et al., 1975). Corticosteroids are known to be lympholytic in certain species (rabbit, mouse, rat and hamster) (Batra, Elrod & Schrek, 1966) and to interfere with the activity of pre-formed lymphokines on macrophages in steroid resistant species such as the guinea-pig (Gaumer et al., 1974). Moore & Myrvik (1973) found that cortisone acetate eradicated the pulmonary response to antigen challenge in bacille CalmetteGuerin (BCG) sensitized rabbits. This study was undertaken to determine if previous pulmonary exposure to M. faeni antigen heightened the pulmonary histologic response to a later challenge with the same antigen and to determine the effect of cortisone acetate on development of such heightened lesions. Cleveland VA Hospital, 10701 East Boulevard, Cleveland, Ohio 44106, USA. Correspondence: Dr F. Cashner, Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70122, USA. 00007-1048/78/0050-0266 $02.00 C) 1979 Blackwell Scientific Publications * Present address:
266
Cortisone acetate on pulmonary lesions
267
MATERIALS AND METHODS Antigens. Micropolyspora faeni was grown in trvpticase soy broth for 4 days in a shaker water bath at 50 C. Growth was centrifuged, washed twice with deionized water, disrupted with glass beads (0 45-0 50 mm) in a MSK Braun homogenizer (Bronwill Scientific Co., Rochester, New York) at 4000 cycles/min for 3 min, and lyophilized. Crude growth was reconstituted in sterile physiological saline for intratracheal inoculation or extracted for skin testing, macrophage migration inhibition (MMI) assays, and precipitin detection. Crude growth was extracted at 20 mg/ml with 0-018 M NaCl overnight at 4 C using a teflon stirring-bar rotating at 100 rpm. The suspension was centrifuged at 2000 rpm for 20 min, and the plug discarded. The supernatant was dialysed for 5 days against deionized water and relyophilized, followed by reconstitution in sterile Dulbecco's phosphate-buffered saline. This semi-purified preparation was 800) protein by weight as determined by the technique of Lowry et al. (1951). Skin tests. Skin tests were performed on shaved areas with 100 pg extracted dialysed Al. faeni antigen in 0 1 ml of Dulbecco's phosphate-buffered saline. Tests were read at 6, 24 and 48 hr. The extent of induration, oedema and erythema was recorded in mm. MMI assays. Direct assays were performed according to the method of George & Vaughan (1962) as modified by David (1965). Alveolar wash cells were obtained according to the technique of Myrvik, Leake & Fariss (1961) using Hank's balanced salt solution (Grand Island Biological Company, Grand Island, New York). Washings were adjusted to contain 30 to 60 x 106 cells/ml (85-98% macrophages, 2-500, lymphocytes) and were packed into 14-1-6 O.D. capillary tubes, placed in Sykes-Moore chambers containing RPMI-1640 (Grand Island Biological Co.) and 1000 normal heat-inactivated rabbit serum. MMI assays were carried out in duplicate chambers using three concentrations of dialysed soluble M. faeni antigen (10 jig/ml, 100 pg/ml, and 500 jsg/ml) or no antigen. Macrophages from normal rabbits showed migration inhibition at 500 jg/ml of antigen, and only one sensitized animal's macrophages were significantly inhibited at 10 ig/ml, therefore, only data for 100 jig/ml are reported. Extent of migration was recorded after 24 hr of incubation at 37 C. The percentage of macrophage migration inhibition was expressed by calculating the area of migration in the presence of antigen as a percentage of that in control chambers without antigen and subtracting this percentage from 100%. Precipitin determination. Precipitin assays for anti-Al. faeni antibody were performed by the Ouchterlony technique (040o agarose in PBS) using extracted M. faeni antigen (1 mg/ml in PBS) and undiluted rabbit sera. Morphological studies. Immediately after the animals were killed by air embolism under nembutal anesthesia, the lungs were removed and fixed with intratracheal 100/ formalin under 20 cm water pressure. A single section was cut through the largest area of each of the six lobes in a standard fashion disregarding the location of gross lesions. The two larger lower lobes were divided in half and mounted on two slides, resulting in a total of eight slides per rabbit. Sections were embedded in paraffin and stained with hematoxylin and eosin. The complete section was histologically evaluated in blind fashion, and graded on a scale of one to five as follows: 0: normal; 1: interstitial infiltrates present, but involving less than 20% of the section; 2: infiltrates diffuse and involving 20% or more of the section; 3: infiltrates involving more than 50% of the section; 4: infiltrates encompassing more than 900o of the section; 5: areas of consolidation. A 'lesion index' consisting of the mean numerical value of all eight sections was calculated for each animal. Animals and method of sensitization. New Zealand white rabbits, weighing 2-3 kg, were sensitized by intratracheal injection of 21 mg of particulate M. faeni antigen at 4 mg/ml in sterile saline (or plain saline as a control) in three equal doses at 4-day intervals. Animals were divided into seven groups (Table 1). Experiments were set up with eight rabbits at a time for convenience, each experiment including animals from two or more different groups so that controls were done simultaneously with experimentals. Groups 1-4 were challenged intratracheally with 3.5 mg particulate Al. faeni antigen at 4 mg/ml in saline (or plain saline as a control) 3 weeks after the first immunizing inoculation, and sacrified for histologic study 4 days later. This schedule was chosen to conform to that used by Moore & Myrvik (1973) in the induction and expression of granulomatous lung disease induced by intravenous injection of BCG. In addition, preliminary experiments indicated that at TABLE 1.
Group Number
Number of animals
1
10 10 10
2 3
4
6
5
6
6 7
8 6
Micropolyspora faeni sensitization
Skin test
4t
+
Micropolyspora
Jaeni
-
Cortisone
challenge
Histology
MMI
268
Frances M. Cashner, Al. R. Schuyler 65. E. Salveggio
4 days there was a predominant mononuclear cell pulmonary infiltrate in both sensitized and non-sensitized animals. Group 1 consisted of ten control animals given saline without antigen and challenged with M. faeni. Groups 2 (n = 10) and 3 ( - 10) were sensitized and challenged, but at 1 and 3 days prior to challenge, Group 3 received 100 mg of cortisone acetate intraperitoneally (total 200 mg at 50 mg/ml), the same dosage as that given by Moore & Myrvik (1973). Group 4 (n = 6) was inoculated with M. faeni, but challenged with saline. Groups 2-4 were skin tested 2 weeks following the first inoculation, and all four groups were bled at that time and when the animals were killed. Serum was separated and frozen at -20'C for later precipitin determination. The animals in groups 5, 6 and 7 were killed without challenge 2 weeks following the initial inoculation. The lungs were removed and bronchoalveolar cells obtained for MMI assays. Group 5 (n 6) received no inoculations prior to MMI assay, Group 6 (n = 8) received M. faeni inoculations alone and Group 7 (n 6) received M. faeni inoculations plus 100 mg of cortisone acetate 1 and 3 days prior to being killed.
RESULTS Fig. 1 illustrates typical pulmonary histopathology. The interstitium contained many cells, most of which were mononuclear, including macrophages, lymphocytes and occasional plasma cells. Although a rare giant cell was present, and an increased number of cells with the morphology of macrophages were noted within the alveoli, there xere no true organized granulomas. There were clear-cut quantitative, but no qualitative differences in pulmonary pathology between groups. Fig. 2 shows the average lesion index and standard error of the mean for each group. Micropolyspora faeni induced pulmonary lesions cleared in most cases within 3 weeks of the first inoculation. The mean lesion index for rabbits given M. faeni immunizations and saline challenge (Group 4) was 0-2 (range 000-04). M. faeni sensitized animals challenged With antigen (Group 2) developed a mean lesion index of 1-73 (s.e.m. 0.15, n = 10). Non-sensitized animals challenged with M. faeni (Group 1) developed a mean lesion index of 0 93 (s.e.m. 0-16, n = 10) significantly less (P < 0 005 by Student's t-test) than that seen in sensitized
FIG. 1. Pulmonary histopathology 4 days after challenge of an immunized rabbit. There is an extensive interstitial infiltration with mononuclear cells, centered around respiratory bronchioles.
Cortisone acetate on pulmonary lesions
269
2
-C
0
Group Sensitization
Saline
Challenge
M.faeni
Group 2
Group 3
Group 4
M. fceni M. faeni M. faeni cortisone M. faeni M. faeni Saline
FIG. 2. Extent of histopathology 4 days after challenge (average lesion index and standard error for each group). All animals were challenged on day 21 after three inoculations (days 0, 4 and 8). The third group was treated with 100 mg cortisone acetate 1 and 3 days prior to challenge. Results from the sensitized and challenged animals (Group 2) were significantly (P< 0-005) higher than results from the cortisone-treated (Group 3) and from the saline-sensitized and Micropolyspora faeni-challenged animals (Group 1).
60 40 0
20
-201 Group 5 Non - sensitized
I Group 6 Group 7 Sensitized Cortisone-treated
FIG. 3. Macrophage migration inhibition employing bronchoalveolar cells derived from non-sensitized, sensitized and cortisone-treated rabbits using 100 jig/ml Micropolyspora faeni antigen (average percentage inhibition and standard error for each group). Animals were killed on day 14 after three inoculations (days 0, 4 and 8). Cortisone-treated animals were given 100 mg cortisone acetate 1 and 3 days prior to being killed.
rabbits. Sensitized animals given cortisone acetate prior to challenge (Group 3) developed a similar mean lesion index (0.84, s.e.m. 0-24, n = 10) to that observed in non-sensitized challenge rabbits, and also significantly less (P < 0-01 by Student's t-test) than the lesion index of non-treated sensitized animals (Group 2). Delayed skin reactions performed 2 weeks after initial inoculation in all M. faeni-inoculated groups were positive (48 hr) in eighteen of twenty-two tested animals (induration and/or erythema > 10 mm diam). Skin reactions in ten control non-immunize rabbits were negative at 6, 24 and 48 hr. Specific anti-M. faeni serum precipitins in sensitized animals (Groups 2, 3 and 4) were uniformly positive when the animals were killed but were negative in all nine animals tested of the salineinoculated, M. faeni-challenged group (Group 1). Fig. 3 illustrates average percentages and standard errors of macrophage migration inhibition of alveolar wash cells in Groups 5, 6 and 7. Although the degree of inhibition was not marked, greater than
270
Frances M. Cashner, M. R. Schuyler &dJ. E. Salvaggio
20% inhibition occurred in five of the eight animals of the M. faeni inoculated group (Group 6). None of the unsensitized rabbits (Group 5), nor sensitized cortisone-treated animals (Group 7) showed greater than 20% migration inhibition in the presence of M. faeni antigen. DISCUSSION Our results suggest that intratracheally administered particulate Micropolyspora faeni can sensitize rabbits as demonstrated by development of an enhanced mononuclear cell pulmonary infiltration following a later intratracheal challenge with specific antigen. Although inoculation with M. faeni induced a histological response in non-sensitized animals, these infiltrates were significantly less intense than those produced by the same challenge in animals previously exposed to M.faeni. M. faeni contains a complex mixture of antigens with enzymatic activity which are known to be capable of inducing acute inflammation in non-sensitized animals (Zaidi et al., 1970). The mechanisms involved in production of this acute inflammatory response are not known, but it has been shown that M. faeni particulate antigen can activate complement via the alternative pathway (Edwards, 1976). Other substances which activate the alternative pathway, such as mouldy hay dust and zymosan, when inoculated intracheally in mg quantities can induce both acute lesions and mononuclear cell infiltrates at 4 days similar to those seen in this study (Edwards, Wagner & Seal, 1976). Thus, it is possible that the response seen in nonsensitized animals was mediated through the alternative pathway of complement. However, other mechanisms may also be important in development of these lesions in non-sensitized animals. Among these are the possible presence of non-complement dependent chemotactic factors in crude actinomycete antigens, and the generation of such factors by classical pathway activation, leading to recruitment of neutrophils and macrophages. The known non-specific adjuvant properties of certain actinomycetes such as M.faeni might also be important in initiating such an inflammatory response. The augmented histological response following challenge of sensitized rabbits was not due to a simple additive effect of initial residual lesions plus those induced by challenge since the lesion index in Group 4 (sensitized and challenged with saline) was quite low. Thus, prior exposure to M. Jaeni antigen seemed to specifically accelerate the later response to challenge. This altered reactivity provides evidence of the immunologic basis of these M.faeni-produced lesions. The administration of cortisone acetate prior to challenge (Group 3) reduced the histological response of the sensitized animals to that noted in unsensitized rabbits (Group 1). Corticosteriods have varied anti-inflammatory and immunological effects in different animal species (Batra et al., 1966). Rabbits and mice are relatively corticosteroid sensitive, unlike other species such as guinea-pig and man (Claman, 1972). In a murine system using Listeria monocytogenes, North (1971) showed that cortisone acetate eliminated a short-lived lymphocyte population which was committed to the response to Listeria. A similar direct effect on committed lymphocytes could cause the results observed in this study. However, other corticosteroid effects on the inflammatory response or macrophage function could also be important (Claman, 1972). In this regard, the decrease in alveolar macrophage inhibition observed in our cortisonetreated sensitized animals correlated with the loss of the accelerated histopathologic response in sensitized cortisone-treated animals. It is possible that cortisone affected the composition of the alveolar macrophage population or the response of these cells to macrophage inhibition factor. Recent work indicates that systemic corticosteroids cause subtle functional changes in alveolar macrophages (Hunninghake & Fauci, 1977) and that they interfere with the action of pre-formed lymphokines on alveolar macrophage activity (Gaumer et al., 1974). It is of interest that the pulmonary histological response and presence of alveolar macrophage migration inhibition paralleled each other in these studies. If macrophage migration inhibition is considered to be an in vitro correlate of cell-mediated hypersensitivity, this finding would further support a role for this mechanism in the pathogenesis of actinomycete-induced experimental hypersensitivity pneumonititis. This work was supported byNIAID Grant No. AI-13401, U.S. National Institutes of Health.
Cortisone acetate on pulmonary lesions
271
REFERENCES BATRA, K., ELROD, L. & SCHREK, R. (1966) Species differences in the in vitro sensitivity of lymphocytes to prednisolone and x-rays. J. Pharmacol. exp. Ther. 152, 525. CLAMAN, H. (1972) Corticosteroids and lymphoid cells. N. Eng. J. Med. 287, 388. DAVID, J. (1965) Suppression of delayed hypersensitivity in vitro by inhibition of protein synthesis. ]. exp. Med. 122, 1125. EDWARDS, J.H. (1976) A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes. Clin. Allergy, 6, 19. EDWARDS, J.H., WAGNER, J.C. & SEAL, R.M.E. (1976) Pulmonary responses to particulate materials capable of activating the alternative pathway of complement. Clin. Allergy, 6, 155. GAUMER, H.R., SALVAGGIO, J.E., WESTON, W.L. & CLAMAN, H.N. (1974) Cortisol inhibition of immunologic activity in guinea-pig alveolar cells. Int. Arch. Allergy appl. Immunol. 47, 54. GEORGE, M. & VAUGHAN, J. (1962) Immunological unresponsiveness produced in adult guinea pigs by parenteral introduction of minute quantities of hapten or protein antigen. Proc. Soc. exp. Biol. Med. 3, 514. HARRIS, J.O., BICE, D. & SALVAGGIO, J.E. (1976) Cellular and humoral broncho-pulmonary immune response of rabbits immunized with thermophilic actinomyces antigen. Amer. Rev. resp. Dis. 114, 29. HUNNINGHAKE, G.W. & FAUCI, A.S. (1977) Immunologic reactivity of the lung. III. Effects of corticosteroids on alveolar macrophage cytotoxic effector cell function. J. Immunol. 118, 146. KAWAI, T., SALVAGGIO, J.E., HARRIS, J.O. & ARQUEMBOURG, P. (1973) Alveolar macrophage migration inhibition in animals immunized with thermophilic actinomycete
antigen. Clin. exp. Immunol. 155, 123. KAWAI, T., SALVAGGIO, J., LAKE, W. & HARRIS, J.O. (1972) Experimental production of hypersensitivity pneumonitis with bagasse and thermophilic actinomycete antigen. J. Allergy c/in. Immunol. 50, 276. LOWRY, O.H., RoSEBOUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurement with the Folin phenol reagent. j. biol. Chem. 193, 265. MOORE, V.L. & MYRVIK, Q.N. (1973) Relationship of BCG-induced pulmonary delayed hypersensitivity to accelerated granuloma formation in rabbit lungs: effect of cortisone acetate. Infect. Immunity, 7, 764. MYRVIK, Q.N., LEAKE, E.S. & FARISS, B. (1961) Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to produce them in a high state of purity. .7. Immunol. 86, 128. NORTH, R. J. (1971) The action of cortisone acetate on cellmediated immunity to infection: suppression of host cell proliferation and alteration of cellular composition of infective foci. _. exp. Med. 134, 1485. PEPYS, J. (1969) Hypersensitivity diseases of the lung due to fungi and organic dusts. Monogr. Allergy, 4, 1. SALVAGGIO, J., PHANUPHAK, P., STANFORD, R., BICE, D. & CLAMAN, H. (1975) Experimental production of granulomatous pneumonitis. 5. Allergy c/in. Immunol. 56, 364. WENZEL, F.J., EMANUEL, D.A. & GRAY, R.L. (1971) Immunoflorescent studies in patients with Farmer's lung. 7. Allergy c/in. Immunol. 48, 224. WILKIE, B., PAULI, B. & GYGAX, M. (1973) Hypersensitivity pneumonitis: experimental production in guinea pigs with antigens of Micropolyspora faeni. Path. Microbiol. 39, 393. ZAIDI, S.H., DOGRA, K.S. SHAKER, R. & CHANDRA, S.V. (1970) Experimental Farmer's lung in guinea pigs. 5. Path. 105, 41.
The effect of cortisone acetate on pulmonary lesions in a rabbit model of hypersensitivity pneumonitis (cortisone and hypersensitivity pneumonitis) FRANCES M. CASHNER, M. R. SCHUYLER* & J. E. SALVAGGIO Department of Medicine, Tulane University Medical Center, New Orleans, Louisiana, USA
(Accepted for publication 15 May 1978) SUMMARY
Rabbits were sensitized with Micropolyspora faeni by intratracheal inoculations and later challenged with the same antigen either with or without parenteral administration of cortisone acetate prior to challenge. Animals developed anti-M. faeni serum precipitins, M. faeni-induced alveolar macrophage migration inhibition, and positive 48-hr skin reactivity to M. faeni. Sensitized animals also demonstrated an augmented pulmonary histopathological response following respiratory challenge with M. faeni when compared to non-sensitized controls. Cortisone acetate abrogated this augmented pulmonary histopathological response following challenge with M.faeni. Cortisone acetate also abolished the positive alveolar macrophage migration inhibition found in sensitized animals.
INTRODUCTION Human hypersensitivity pneumonitides are caused by a variety of inhaled organic dusts. Type II, Type III and Type IV hypersensitivity reactions (Kawai et al., 1973; Pepys, 1969; Wenzel, Emanuel & Gray, 1971; Wilkie, Pauli & Gygax, 1973) have all been implicated in disease pathogenesis but the relative importance of each mechanism is not well understood. Pulmonary lesions similar to those found in human hypersensitivity pneumonitis can be induced in the rabbit by intratracheal administration of particulate Micropolyspora faeni antigen, the principal antigen involved in Farmer's lung disease (Kawai et al., 1972). Although the development of the lesions may be non-immunologically mediated, immunological events are correlated with such lesions, including local delayed hypersensitivity in the lung as measured by macrophage migration inhibition (MMI) of alveolar wash cells, positive delayed skin reactivity, specific serum and bronchial wash precipitating antibody, and the appearance of activated macrophages in the alveolar wash cell populations (Harris, Bice & Salvaggio, 1976; Kawai et al., 1973; Salvaggio et al., 1975). Corticosteroids are known to be lympholytic in certain species (rabbit, mouse, rat and hamster) (Batra, Elrod & Schrek, 1966) and to interfere with the activity of pre-formed lymphokines on macrophages in steroid resistant species such as the guinea-pig (Gaumer et al., 1974). Moore & Myrvik (1973) found that cortisone acetate eradicated the pulmonary response to antigen challenge in bacille CalmetteGuerin (BCG) sensitized rabbits. This study was undertaken to determine if previous pulmonary exposure to M. faeni antigen heightened the pulmonary histologic response to a later challenge with the same antigen and to determine the effect of cortisone acetate on development of such heightened lesions. Cleveland VA Hospital, 10701 East Boulevard, Cleveland, Ohio 44106, USA. Correspondence: Dr F. Cashner, Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70122, USA. 00007-1048/78/0050-0266 $02.00 C) 1979 Blackwell Scientific Publications * Present address:
266
Cortisone acetate on pulmonary lesions
267
MATERIALS AND METHODS Antigens. Micropolyspora faeni was grown in trvpticase soy broth for 4 days in a shaker water bath at 50 C. Growth was centrifuged, washed twice with deionized water, disrupted with glass beads (0 45-0 50 mm) in a MSK Braun homogenizer (Bronwill Scientific Co., Rochester, New York) at 4000 cycles/min for 3 min, and lyophilized. Crude growth was reconstituted in sterile physiological saline for intratracheal inoculation or extracted for skin testing, macrophage migration inhibition (MMI) assays, and precipitin detection. Crude growth was extracted at 20 mg/ml with 0-018 M NaCl overnight at 4 C using a teflon stirring-bar rotating at 100 rpm. The suspension was centrifuged at 2000 rpm for 20 min, and the plug discarded. The supernatant was dialysed for 5 days against deionized water and relyophilized, followed by reconstitution in sterile Dulbecco's phosphate-buffered saline. This semi-purified preparation was 800) protein by weight as determined by the technique of Lowry et al. (1951). Skin tests. Skin tests were performed on shaved areas with 100 pg extracted dialysed Al. faeni antigen in 0 1 ml of Dulbecco's phosphate-buffered saline. Tests were read at 6, 24 and 48 hr. The extent of induration, oedema and erythema was recorded in mm. MMI assays. Direct assays were performed according to the method of George & Vaughan (1962) as modified by David (1965). Alveolar wash cells were obtained according to the technique of Myrvik, Leake & Fariss (1961) using Hank's balanced salt solution (Grand Island Biological Company, Grand Island, New York). Washings were adjusted to contain 30 to 60 x 106 cells/ml (85-98% macrophages, 2-500, lymphocytes) and were packed into 14-1-6 O.D. capillary tubes, placed in Sykes-Moore chambers containing RPMI-1640 (Grand Island Biological Co.) and 1000 normal heat-inactivated rabbit serum. MMI assays were carried out in duplicate chambers using three concentrations of dialysed soluble M. faeni antigen (10 jig/ml, 100 pg/ml, and 500 jsg/ml) or no antigen. Macrophages from normal rabbits showed migration inhibition at 500 jg/ml of antigen, and only one sensitized animal's macrophages were significantly inhibited at 10 ig/ml, therefore, only data for 100 jig/ml are reported. Extent of migration was recorded after 24 hr of incubation at 37 C. The percentage of macrophage migration inhibition was expressed by calculating the area of migration in the presence of antigen as a percentage of that in control chambers without antigen and subtracting this percentage from 100%. Precipitin determination. Precipitin assays for anti-Al. faeni antibody were performed by the Ouchterlony technique (040o agarose in PBS) using extracted M. faeni antigen (1 mg/ml in PBS) and undiluted rabbit sera. Morphological studies. Immediately after the animals were killed by air embolism under nembutal anesthesia, the lungs were removed and fixed with intratracheal 100/ formalin under 20 cm water pressure. A single section was cut through the largest area of each of the six lobes in a standard fashion disregarding the location of gross lesions. The two larger lower lobes were divided in half and mounted on two slides, resulting in a total of eight slides per rabbit. Sections were embedded in paraffin and stained with hematoxylin and eosin. The complete section was histologically evaluated in blind fashion, and graded on a scale of one to five as follows: 0: normal; 1: interstitial infiltrates present, but involving less than 20% of the section; 2: infiltrates diffuse and involving 20% or more of the section; 3: infiltrates involving more than 50% of the section; 4: infiltrates encompassing more than 900o of the section; 5: areas of consolidation. A 'lesion index' consisting of the mean numerical value of all eight sections was calculated for each animal. Animals and method of sensitization. New Zealand white rabbits, weighing 2-3 kg, were sensitized by intratracheal injection of 21 mg of particulate M. faeni antigen at 4 mg/ml in sterile saline (or plain saline as a control) in three equal doses at 4-day intervals. Animals were divided into seven groups (Table 1). Experiments were set up with eight rabbits at a time for convenience, each experiment including animals from two or more different groups so that controls were done simultaneously with experimentals. Groups 1-4 were challenged intratracheally with 3.5 mg particulate Al. faeni antigen at 4 mg/ml in saline (or plain saline as a control) 3 weeks after the first immunizing inoculation, and sacrified for histologic study 4 days later. This schedule was chosen to conform to that used by Moore & Myrvik (1973) in the induction and expression of granulomatous lung disease induced by intravenous injection of BCG. In addition, preliminary experiments indicated that at TABLE 1.
Group Number
Number of animals
1
10 10 10
2 3
4
6
5
6
6 7
8 6
Micropolyspora faeni sensitization
Skin test
4t
+
Micropolyspora
Jaeni
-
Cortisone
challenge
Histology
MMI
268
Frances M. Cashner, Al. R. Schuyler 65. E. Salveggio
4 days there was a predominant mononuclear cell pulmonary infiltrate in both sensitized and non-sensitized animals. Group 1 consisted of ten control animals given saline without antigen and challenged with M. faeni. Groups 2 (n = 10) and 3 ( - 10) were sensitized and challenged, but at 1 and 3 days prior to challenge, Group 3 received 100 mg of cortisone acetate intraperitoneally (total 200 mg at 50 mg/ml), the same dosage as that given by Moore & Myrvik (1973). Group 4 (n = 6) was inoculated with M. faeni, but challenged with saline. Groups 2-4 were skin tested 2 weeks following the first inoculation, and all four groups were bled at that time and when the animals were killed. Serum was separated and frozen at -20'C for later precipitin determination. The animals in groups 5, 6 and 7 were killed without challenge 2 weeks following the initial inoculation. The lungs were removed and bronchoalveolar cells obtained for MMI assays. Group 5 (n 6) received no inoculations prior to MMI assay, Group 6 (n = 8) received M. faeni inoculations alone and Group 7 (n 6) received M. faeni inoculations plus 100 mg of cortisone acetate 1 and 3 days prior to being killed.
RESULTS Fig. 1 illustrates typical pulmonary histopathology. The interstitium contained many cells, most of which were mononuclear, including macrophages, lymphocytes and occasional plasma cells. Although a rare giant cell was present, and an increased number of cells with the morphology of macrophages were noted within the alveoli, there xere no true organized granulomas. There were clear-cut quantitative, but no qualitative differences in pulmonary pathology between groups. Fig. 2 shows the average lesion index and standard error of the mean for each group. Micropolyspora faeni induced pulmonary lesions cleared in most cases within 3 weeks of the first inoculation. The mean lesion index for rabbits given M. faeni immunizations and saline challenge (Group 4) was 0-2 (range 000-04). M. faeni sensitized animals challenged With antigen (Group 2) developed a mean lesion index of 1-73 (s.e.m. 0.15, n = 10). Non-sensitized animals challenged with M. faeni (Group 1) developed a mean lesion index of 0 93 (s.e.m. 0-16, n = 10) significantly less (P < 0 005 by Student's t-test) than that seen in sensitized
FIG. 1. Pulmonary histopathology 4 days after challenge of an immunized rabbit. There is an extensive interstitial infiltration with mononuclear cells, centered around respiratory bronchioles.
Cortisone acetate on pulmonary lesions
269
2
-C
0
Group Sensitization
Saline
Challenge
M.faeni
Group 2
Group 3
Group 4
M. fceni M. faeni M. faeni cortisone M. faeni M. faeni Saline
FIG. 2. Extent of histopathology 4 days after challenge (average lesion index and standard error for each group). All animals were challenged on day 21 after three inoculations (days 0, 4 and 8). The third group was treated with 100 mg cortisone acetate 1 and 3 days prior to challenge. Results from the sensitized and challenged animals (Group 2) were significantly (P< 0-005) higher than results from the cortisone-treated (Group 3) and from the saline-sensitized and Micropolyspora faeni-challenged animals (Group 1).
60 40 0
20
-201 Group 5 Non - sensitized
I Group 6 Group 7 Sensitized Cortisone-treated
FIG. 3. Macrophage migration inhibition employing bronchoalveolar cells derived from non-sensitized, sensitized and cortisone-treated rabbits using 100 jig/ml Micropolyspora faeni antigen (average percentage inhibition and standard error for each group). Animals were killed on day 14 after three inoculations (days 0, 4 and 8). Cortisone-treated animals were given 100 mg cortisone acetate 1 and 3 days prior to being killed.
rabbits. Sensitized animals given cortisone acetate prior to challenge (Group 3) developed a similar mean lesion index (0.84, s.e.m. 0-24, n = 10) to that observed in non-sensitized challenge rabbits, and also significantly less (P < 0-01 by Student's t-test) than the lesion index of non-treated sensitized animals (Group 2). Delayed skin reactions performed 2 weeks after initial inoculation in all M. faeni-inoculated groups were positive (48 hr) in eighteen of twenty-two tested animals (induration and/or erythema > 10 mm diam). Skin reactions in ten control non-immunize rabbits were negative at 6, 24 and 48 hr. Specific anti-M. faeni serum precipitins in sensitized animals (Groups 2, 3 and 4) were uniformly positive when the animals were killed but were negative in all nine animals tested of the salineinoculated, M. faeni-challenged group (Group 1). Fig. 3 illustrates average percentages and standard errors of macrophage migration inhibition of alveolar wash cells in Groups 5, 6 and 7. Although the degree of inhibition was not marked, greater than
270
Frances M. Cashner, M. R. Schuyler &dJ. E. Salvaggio
20% inhibition occurred in five of the eight animals of the M. faeni inoculated group (Group 6). None of the unsensitized rabbits (Group 5), nor sensitized cortisone-treated animals (Group 7) showed greater than 20% migration inhibition in the presence of M. faeni antigen. DISCUSSION Our results suggest that intratracheally administered particulate Micropolyspora faeni can sensitize rabbits as demonstrated by development of an enhanced mononuclear cell pulmonary infiltration following a later intratracheal challenge with specific antigen. Although inoculation with M. faeni induced a histological response in non-sensitized animals, these infiltrates were significantly less intense than those produced by the same challenge in animals previously exposed to M.faeni. M. faeni contains a complex mixture of antigens with enzymatic activity which are known to be capable of inducing acute inflammation in non-sensitized animals (Zaidi et al., 1970). The mechanisms involved in production of this acute inflammatory response are not known, but it has been shown that M. faeni particulate antigen can activate complement via the alternative pathway (Edwards, 1976). Other substances which activate the alternative pathway, such as mouldy hay dust and zymosan, when inoculated intracheally in mg quantities can induce both acute lesions and mononuclear cell infiltrates at 4 days similar to those seen in this study (Edwards, Wagner & Seal, 1976). Thus, it is possible that the response seen in nonsensitized animals was mediated through the alternative pathway of complement. However, other mechanisms may also be important in development of these lesions in non-sensitized animals. Among these are the possible presence of non-complement dependent chemotactic factors in crude actinomycete antigens, and the generation of such factors by classical pathway activation, leading to recruitment of neutrophils and macrophages. The known non-specific adjuvant properties of certain actinomycetes such as M.faeni might also be important in initiating such an inflammatory response. The augmented histological response following challenge of sensitized rabbits was not due to a simple additive effect of initial residual lesions plus those induced by challenge since the lesion index in Group 4 (sensitized and challenged with saline) was quite low. Thus, prior exposure to M. Jaeni antigen seemed to specifically accelerate the later response to challenge. This altered reactivity provides evidence of the immunologic basis of these M.faeni-produced lesions. The administration of cortisone acetate prior to challenge (Group 3) reduced the histological response of the sensitized animals to that noted in unsensitized rabbits (Group 1). Corticosteriods have varied anti-inflammatory and immunological effects in different animal species (Batra et al., 1966). Rabbits and mice are relatively corticosteroid sensitive, unlike other species such as guinea-pig and man (Claman, 1972). In a murine system using Listeria monocytogenes, North (1971) showed that cortisone acetate eliminated a short-lived lymphocyte population which was committed to the response to Listeria. A similar direct effect on committed lymphocytes could cause the results observed in this study. However, other corticosteroid effects on the inflammatory response or macrophage function could also be important (Claman, 1972). In this regard, the decrease in alveolar macrophage inhibition observed in our cortisonetreated sensitized animals correlated with the loss of the accelerated histopathologic response in sensitized cortisone-treated animals. It is possible that cortisone affected the composition of the alveolar macrophage population or the response of these cells to macrophage inhibition factor. Recent work indicates that systemic corticosteroids cause subtle functional changes in alveolar macrophages (Hunninghake & Fauci, 1977) and that they interfere with the action of pre-formed lymphokines on alveolar macrophage activity (Gaumer et al., 1974). It is of interest that the pulmonary histological response and presence of alveolar macrophage migration inhibition paralleled each other in these studies. If macrophage migration inhibition is considered to be an in vitro correlate of cell-mediated hypersensitivity, this finding would further support a role for this mechanism in the pathogenesis of actinomycete-induced experimental hypersensitivity pneumonititis. This work was supported byNIAID Grant No. AI-13401, U.S. National Institutes of Health.
Cortisone acetate on pulmonary lesions
271
REFERENCES BATRA, K., ELROD, L. & SCHREK, R. (1966) Species differences in the in vitro sensitivity of lymphocytes to prednisolone and x-rays. J. Pharmacol. exp. Ther. 152, 525. CLAMAN, H. (1972) Corticosteroids and lymphoid cells. N. Eng. J. Med. 287, 388. DAVID, J. (1965) Suppression of delayed hypersensitivity in vitro by inhibition of protein synthesis. ]. exp. Med. 122, 1125. EDWARDS, J.H. (1976) A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes. Clin. Allergy, 6, 19. EDWARDS, J.H., WAGNER, J.C. & SEAL, R.M.E. (1976) Pulmonary responses to particulate materials capable of activating the alternative pathway of complement. Clin. Allergy, 6, 155. GAUMER, H.R., SALVAGGIO, J.E., WESTON, W.L. & CLAMAN, H.N. (1974) Cortisol inhibition of immunologic activity in guinea-pig alveolar cells. Int. Arch. Allergy appl. Immunol. 47, 54. GEORGE, M. & VAUGHAN, J. (1962) Immunological unresponsiveness produced in adult guinea pigs by parenteral introduction of minute quantities of hapten or protein antigen. Proc. Soc. exp. Biol. Med. 3, 514. HARRIS, J.O., BICE, D. & SALVAGGIO, J.E. (1976) Cellular and humoral broncho-pulmonary immune response of rabbits immunized with thermophilic actinomyces antigen. Amer. Rev. resp. Dis. 114, 29. HUNNINGHAKE, G.W. & FAUCI, A.S. (1977) Immunologic reactivity of the lung. III. Effects of corticosteroids on alveolar macrophage cytotoxic effector cell function. J. Immunol. 118, 146. KAWAI, T., SALVAGGIO, J.E., HARRIS, J.O. & ARQUEMBOURG, P. (1973) Alveolar macrophage migration inhibition in animals immunized with thermophilic actinomycete
antigen. Clin. exp. Immunol. 155, 123. KAWAI, T., SALVAGGIO, J., LAKE, W. & HARRIS, J.O. (1972) Experimental production of hypersensitivity pneumonitis with bagasse and thermophilic actinomycete antigen. J. Allergy c/in. Immunol. 50, 276. LOWRY, O.H., RoSEBOUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurement with the Folin phenol reagent. j. biol. Chem. 193, 265. MOORE, V.L. & MYRVIK, Q.N. (1973) Relationship of BCG-induced pulmonary delayed hypersensitivity to accelerated granuloma formation in rabbit lungs: effect of cortisone acetate. Infect. Immunity, 7, 764. MYRVIK, Q.N., LEAKE, E.S. & FARISS, B. (1961) Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to produce them in a high state of purity. .7. Immunol. 86, 128. NORTH, R. J. (1971) The action of cortisone acetate on cellmediated immunity to infection: suppression of host cell proliferation and alteration of cellular composition of infective foci. _. exp. Med. 134, 1485. PEPYS, J. (1969) Hypersensitivity diseases of the lung due to fungi and organic dusts. Monogr. Allergy, 4, 1. SALVAGGIO, J., PHANUPHAK, P., STANFORD, R., BICE, D. & CLAMAN, H. (1975) Experimental production of granulomatous pneumonitis. 5. Allergy c/in. Immunol. 56, 364. WENZEL, F.J., EMANUEL, D.A. & GRAY, R.L. (1971) Immunoflorescent studies in patients with Farmer's lung. 7. Allergy c/in. Immunol. 48, 224. WILKIE, B., PAULI, B. & GYGAX, M. (1973) Hypersensitivity pneumonitis: experimental production in guinea pigs with antigens of Micropolyspora faeni. Path. Microbiol. 39, 393. ZAIDI, S.H., DOGRA, K.S. SHAKER, R. & CHANDRA, S.V. (1970) Experimental Farmer's lung in guinea pigs. 5. Path. 105, 41.
