Bleomycin-lnduced Interstitial Pulmonary Disease in the Nude, Athymic Mouse2 SUSAN V. SZAPIEL, NORTON A. ELSON, JACK D. FULMER, GARY W. HUNNINGHAKE, and RONALD G. CRYSTAL
SUMMARY Evidence from divergent sources suggests that some forms of interstitial pulmonary disease are associated with abnormalities of the cellular immune system. T o evaluate whether cellular immune processes are necessary determinants for the development of parenchymal alveolitis and fibrosis secondary to bleomycin, we examined the effect of bleomycin on the NIH, outbred white mouse as compared to the homozygous nude, athymic mouse on the NIH outbred background. The nude mouse has virtually no detectable cell-mediated immune function; we therefore hypothesized that if this component of the immune system were necessary for the development of bleomycin-induced interstitial disease, bleomycin would not induce the same pulmonary lesion in the nude mouse as in the white mouse. However, both white and nude mice developed alveolitis and fibrosis after intraperitoneal administration of bleomycin. Comparison of the frequency and severity of these lesions in the 2 groups revealed no significant differences. These findings suggest that the presence of an intact cell-mediated immune system is not an absolute requirement for the development of bleomycin-induced interstitial disease in the mouse. T o the extent that this model is an appropriate approximation of human bleomycin-induced pulmonary disease, these results are consistent with the hypothesis that T-lymphocyte mediated processes are not primary determinants of this lesion.
Introduction Bleomycin, a cytotoxic a n t i m i c r o b i a l d r u g currently used to treat a variety of tumors, is a k n o w n cause of interstitial p u l m o n a r y disease (1-4). Bleomycin-induced interstitial disease has received a great deal of a t t e n t i o n for 2 reasons: (1) significant n u m b e r s of patients who receive t h e r a p e u t i c courses of this d r u g develop interstitial disease (5), a n d (2) bleomycin-induced interstitial disease is easily developed in animals, a n d thus has become a widely used model of the h u m a n disease (6-9).
(Received in original form July 5, 1978 and in revised form June 12,1979) i From the Pulmonary Branch, National Heart, Lung, and Blood Institute, Bethesda, Md. 2 Requests for reprints should be addressed to Building 10, 6D06, National Institutes of Health, Bethesda, Md. 20205.
A l t h o u g h information has accumulated conc e r n i n g the morphology, physiology, a n d connective tissue chemistry of bleomycin-induced interstitial disease, there is little information available c o n c e r n i n g the pathogenesis of this lesion. O n e clue comes from sequential histologic evaluation of the lungs of animals receiving the d r u g (6, 7, 10). Initially, bleomycin appears to d a m a g e p a r e n c h y m a l cells. Soon afterward, a chronic inflammatory process ("alveolitis") develops, a n d p a r e n c h y m a l cellular disordering a n d fibrosis ensue. As with m a n y other interstitial p u l m o n a r y disorders, this progression from alveolitis to p a r e n c h y m a l disordering a n d fibrosis suggests that inflammatory processes may be critical d e t e r m i n a n t s of the pathogenesis of bleomycin-induced interstitial disease. T h e mechanism by which the alveolitis of bleomycin-induced interstitial disease is established a n d m a i n t a i n e d is unclear. I n o t h e r interstitial disorders, however, it is k n o w n that
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o n e m e a n s by which a chronic inflammatory process may be established is activation of the cellular i m m u n e system (11). T o d e t e r m i n e w h e t h e r cellular i m m u n e processes play a role in the pathogenesis of bleomycin-induced interstitial disease, we c o m p a r e d the response of norm a l mice to bleomycin a d m i n i s t r a t i o n to that of mice lacking a n intact cellular i m m u n e system. If the chronic inflammatory response in bleomycin-induced interstitial disease is maintained as a result of activation of the cellular i m m u n e system, t h e n acfministration of bleomycin to mice that lack this a r m of the i m m u n e system should n o t develop interstitial disease of the severity observed in n o r m a l animals.
Methods Two groups of mice were evaluated. Normal mice were from an outbred colony of Swiss-type mice established at the National Institutes of Health [N:NIH(S)]. Nude, athymic mice were used as a model of an absent cellular immune system. These mice were from an outbred colony with the same genetic composition as the N:NIH (S) mice, except for the presence of the homozygous nude mutation (12). Both groups of mice were obtained from the Veterinary Resources Branch of NIH. For convenience, the normal mice with the N:NIH (S) background are referred to as "white mice," and the nude, athymic mice with the NrNIH (S) background are referred to as "nude mice." Nude mice were kept in sterile, filter-topped cages, and were given sterile food and sterile, distilled water adjusted to pH 2.5 with 1 M HC1. Under these conditions, spontaneous mortality was less than 10% during 6 months of observation. White mice were kept in nonsterile cages and were given normal food and distilled water. T h e spontaneous mortality of the white mice was less than 5% during 6 months of observation. All mice used for these studies were females, 6 to 7 wk of age, weighing 20 to 25 g. Bleomycin. Bleomycin sulfate (Blenoxane®, Nippon Kayaku Co. Ltd., Tokyo, Japan) was obtained from Bristol Laboratories (Syracuse, N.Y.) as a lyophilized powder in sterile 15-unit ampules. For injection, the bleomycin was dissolved in sterile 0.9% saline. T h e drug was injected intraperitoneally with a 25-gauge needle; its concentration was adjusted to permit administration of a volume of 0.1 ml/25 g of murine body weight. Study groups. Four groups of mice were evaluated. (1) Control: 31 white mice and 30 nude mice did not receive bleomycin; they were maintained under conditions identical to their counterparts given injections. These mice were killed at 14 to 15 wk of age, 8 wk from the beginning of observation. (2) Low dose: 27 white mice and 27 nude mice each received a total of 160 units of bleomycin/kg of body weight, administered as 8 doses of 20 units/kg
twice weekly for 4 wk. Mice from this group were killed 4 wk after completion of drug administration. (3) Medium dose: 6 white and 6 nude mice each received a total of 400 units of bleomycin/kg of body weight in 8 doses of 50 units/kg 4 times/wk during 2 wk. These mice were killed 2 wk after completion of drug administration. (4) High dose: 7 white mice and 6 nude mice received a total of 1,000 units of bleomycin/kg of body weight, in doses of 100 units/kg, 5 times/wk, during 2 wk. These mice were scheduled to be killed 2 wk after completion of drug administration, but spontaneous mortality before scheduled killing was high. Specimen preparation and evaluation. Mice were killed by intraperitoneal injection of sodium pentobarbital (10 mg/mouse). T h e trachea was cannulated with a 22-gauge, 2-cm teflon catheter and secured with 3-0 silk. T h e lungs and trachea were excised en bloc and fixed in inflation for 24 h with formalin under a pressure of 25 cm H 2 0 . T h e formalin was prepared as a neutral aqueous solution of formaldehyde (10 %, vol/vol) and sodium acetate (2 % wt/vol). T h e fixed lungs were dehydrated by 2 passages through graded alcohol and xylene, and were then imbedded in paraffin. Sections 6 /*m thick were cut and stained with hematoxylin/eosin and Masson's trichrome. Full coronal and transverse sections of lungs from both the control animals and those given injections were evaluated blindly by 3 observers for the presence of alveolitis and fibrosis. Alveolitis was evaluated with the hemotoxylin and eosin-stained sections and was graded using the following criteria: none (0), no alveolitis; mild (1-f), thickening of the alveolar septum by a mononuclear cell infiltrate, with involvement limited to focal, pleural-based lesions occupying less than 20 % of the lung and with good preservation of the alveolar architecture; moderate (2+), a more widespread alveolitis involving 20 to 50 % of the lung, although still predominantly pleural based; severe (3+), a diffuse alveolitis involving more than 50 % of the lung, with occasional consolidation of air spaces by the intra-alveolar mononuclear cells and some hemorrhagic areas within the interstitium and /or alveolus. Fibrosis was evaluated using Masson's trichrome stain sections. T h e extent of fibrosis in these sections was graded using the following criteria: none (0), no evidence of fibrosis; mild (1+), focal regions of fibrosis involving less than 20 % of the lung. Fibrosis involved the pleura and the interstitium of the subpleural parenchyma with some distortion of alveolar architecture; moderate (2+), more extensive fibrosis involving 20 to 50 % of the lung and fibrotic regions mostly extending inward from the pleura and still focal; severe (3+), widespread fibrosis, involving more than 50 % of the lung. Confluent lesions with extensive derangement of parenchymal architecture, including cystic air spaces lined by cuboidal epithelium.
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BLEOMYCIN-INDUCED INTERSTITIAL DISEASE
Data analysis. To compare the severity of the alveolitis or fibrosis at each concentration of bleomycin, a mean grade for each mouse was determined by averaging the grade from each observer and rounding to the nearest integer. Differences in the severity of the lesions between white and nude mice were evaluated for significance using the standard chi-square test with the continuity correction. This analysis was performed for both low- and mediumdosage groups; insufficient data were available for evaluation of the high-dosage group. In addition to the graded analysis at each dosage, all lungs were scored for the presence or absence of alveolitis or fibrosis, and the results of white versus nude mice were compared by the 2-tailed "Student's" t test. Results A d m i n i s t r a t i o n of bleomycin resulted in p u l m o nary lesions of widely varying severity in b o t h white mice (figure 1) a n d n u d e mice (figure 2).
Although a few mice that received bleomycin r e m a i n e d n o r m a l , most exhibited focal, pleuralbased lesions consisting of p l e u r a l thickening, disorganization of alveolar architecture, m o n o nuclear interstitial infiltrates, accumulation of collagen in the alveolar a n d interstitial spaces, a n d intra-alveolar foamy macrophages. Some mice of both groups h a d lesions in which the p a r e n c h y m a l disordering was m o r e confluent, involving large areas of the l u n g b e y o n d t h e subpleural areas, a n d e x h i b i t i n g cystic air spaces lined by cuboidal e p i t h e l i u m . T h e m o n o n u c l e a r cell infiltrates associated with the bleomycininduced interstitial disease were similar in the white a n d the n u d e mice. Relative
Susceptibility Mice to
of White Bleomycin
and
Nude
Controls.
A m o n g the 31 white mice n o t given
injections, 26 h a d completely n o r m a l parenchy-
Fig. 1. Lungs of white mice. {A) Control mouse that did not receive bleomycin. (B) Mouse receiving lowdose bleomycin. There is a focal, pleural-based lesion with inflammatory cells in the interstitial and alveolar spaces. Fibrosis is demonstrable, but the over-all alveolar architecture is still recognizable. (C) Mouse receiving low-dose bleomycin. T h e lesion is more extensive than in (B), with some areas of disruption of the alveolar architecture. (D) Mouse receiving medium-dose bleomycin. T h e alveolitis and fibrosis are widespread, with involvement of the airways a n d presence of numerous cystic air spaces (Massons' trichrome stain; original magnification: X 55).
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SZAPIEL, ELSON, FULMER, HUNNINGHAKE, AND CRYSTAL
Fig. 2. Lungs of n u d e mice. (A) Control mice that did not receive bleomycin. (J3) Mouse receiving low-dose bleomycin. T h e lesion is localized and pleural based. T h e basic alveolar architecture is still recognizable despite the presence of mild alveolitis a n d fibrosis. (C) Mouse receiving medium-dose bleomycin. T h e lesion is more advanced t h a n in (B). Both pleural surfaces of one lobe are visible with the lesion extending from one surface to the other. In some areas, the normal alveolar architecture is lost. (D) Mouse receiving medium-dose bleomycin. Widespread alveolitis and fibrosis are present (Masson's trichrome stain; original magnification: X 55).
mal structures. The alveolar septums and pleura were thin, and the alveolar walls formed a fine, fishnet pattern. There was little, if any stainable collagen visible in the alveolar septums. Within the air spaces, there were a few mononuclear cells, and the septums were devoid of inflammatory cells. Four (13%) white mice not given injections had mild or moderate alveolitis manifested by a thickening of alveolar walls accompanied by an accumulation of mononuclear cells within the parenchymal structures. One white mouse had a single focal area of fibrosis. Among the 30 control nude mice, 25 had normal lungs with no evidence of alveolitis or fibrosis. During the study period, 5 of the nude mice, but none of the control mice died. Low dosage. The survival rate among 27 white and 27 nude mice that received low-dose bleomycin was 96 % (26 of 27) and 85 % (23 of 27), respectively. Of the survivors, more than 90 % of both the white and nude mice had varying de-
grees of parenchymal lesions (figure 3). Statistical analysis using a 2-by-4 contingency table revealed no significant difference between white and nude mice in the distribution of severity of either alveolitis (p > 0.9) or fibrosis (p = 0.2). Medium dosage. Six white and 6 nude mice received medium-dose bleomycin. The survival in both groups was 83 % (5 of 6 mice in each group). Although the proportion of mice exhibiting pulmonary lesions was larger than at low dosage, the results were similar to the lowdose group in that there was no significant difference between the white and nude mice, either for alveolitis (p = 0.7) or for fibrosis (p = 0.6) (figure 4). Analysis of low- and medium-dosage groups combined. Combining the mice that received low and medium dosage, a total of 33 white mice and 33 nude mice received bleomycin, with an over-all survival rate in each group of 94 %
897
BLEOMYCIN-INDUCED INTERSTITIAL DISEASE
A. ALVEOLITIS, LOW DOSE
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GRADING OF HISTOLOGIC LESION Fig. 3. Frequency distribution of the severity of pulmonary lesions induced by low-dose bleomycin in white and n u d e mice: (A) alveolitis and (B) fibrosis. Comparison of the white a n d n u d e mice demonstrated no significant differences in the frequency distribution of either alveolitis (p > 0.9) or fibrosis (p = 0.2).
and 85 %, respectively. These combined groups were compared in 2 ways. First, the distribution of severity of both alveolitis and fibrosis was compared in a manner similar to that described previously for the individual groups. Using a 2-by-4 contingency table, there was no significant difference between white and nude mice in the severity of alveolitis (p > 0.8) or the severity of fibrosis (p > 0.3) (data not shown). In a second analysis of this combined data, the mice receiving low- and medium-dose bleomycin were grouped according to presence versus absence of alveolitis or fibrosis, irrespective of severity. Using a 2-by-2 contingency table, there was no significant difference between white and nude mice in the occurrence of either alveolitis (p > 0.8) or fibrosis (p > 0.2) (figure 5). High dosage. High-dose bleomycin was unsatisfactory in both white and nude mice because of extreme wasting and high, early mortality. Among 7 white mice that received this dosage, two survived; among 6 nude mice, one survived. Because of the small number of survivors, these lesions were not evaluated statistically. In the survivors, the lungs showed severe alveolitis with a dense cellular infiltrate and little fibrosis (data not shown).
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o
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Fig. 4. Frequency distribution of the severity of pulmonary lesions induced by medium-dose bleomycin in white and n u d e mice: (A) alveolitis and (B) fibrosis. Comparison of the white and nude mice demonstrated no significant differences in the frequency distribution of either alveolitis (p = 0.7) or fibrosis (p = 0.6).
Discussion
Bleomycin sulfate (Blenoxane) is a soluble mixture of cytotoxic glycopeptide antimicrobial drugs derived from a strain of Streptomyces verticillus (13). Clinically, it is currently used alone or in combination with other chemotherapeutic agents in the treatment of neoplasms such as squamous-cell carcinoma, testicular carcinoma, and lymphoma (5). Its major side effect is pulmonary toxicity, which occurs in approximately 11% of treated patients (5). This toxicity is manifested most frequently as alveolitis, progressing to diffuse fibrosis in approximately 1% of all patients who receive the drug (2, 5). To begin to evaluate the mechanisms by which bleomycin induces pulmonary injury, we asked whether an intact cellular immune system was necessary for development of bleomycininduced alveolitis and fibrosis in animals that received this drug. Comparison of normal mice to mice of the same genetic background but with no functioning, cell-mediated immune system demonstrated that both groups were susceptible to the development of alveolitis and fibrosis
898
SZAPIEL, ELSON, FULMER, HUNNINGHAKE, AND CRYSTAL
100
ALVEOLITIS (n=28)
(n=31) 80
0.8) or fibrosis (p > 0.2).
secondary to administration of bleomycin. Thus, to the extent that this animal model is a valid representation of bleomycin-induced pulmonary disease in humans, these studies suggest that cell-mediated immune processes are not necessary components in the pathogenesis of this lesion. Validity of the model. The nude mouse was described initially as a hairless, recessive mutant, designated nu/nu (14). When it was found that the nu/nu mouse was also athymic, it was soon demonstrated that this strain lacked a cell-mediated immune system (15). Although some T-cell antigens have been described on lymphocytes in the nu/nu mutation, these cells are considered nonfunctional, and the nu/nu mutation is generally regarded as an excellent model of a profound defect of the cell-mediated immune system (16). The nu/nu mutation is most commonly available on the inbred Balb/C background. This strain is difficult to use in studies of the lung because these mice are susceptible to infection, particularly the naturally occurring Sendai virus that invades the respiratory tract and causes a chronic alveolitis (17). In an attempt to overcome the limitations of working with this strain of mice, the inbred Balb/C nude mouse was crossed with a normal, outbred Swiss-type mouse, resulting in the nude, athymic mutation on
the outbred background [N:NIH (S)] (12). The nu/nu N:NIH(S) mice live approximately 1 yr and rarely develop respiratory infections when kept under minimally sterile laboratory conditions. In addition, these mice do not have autoantibodies to nucleic acids, which have been reported in the nude Balb/C mouse (12, 18). Thus, not only is the N:NIH (S) nude mouse a good model of an absent cell-mediated immune system, but it is also particularly suitable for studies of the respiratory system. In patients who receive bleomycin, the toxicity of the drug is generally dose-related with a total dose of more than 400 units most commonly associated with the development of interstitial disease (5). Most murine studies use total doses of 140 to 240 units/kg of body weight to produce interstitial disease regularly. When this is corrected for body surface area (19), these murine doses correspond to approximately 800 to 1,400 units in a 70-kg man, the same general range known to cause interstitial disease in humans. Morphologic evaluation of bleomycin-induced pulmonary disease in the murine model suggests that the alveolitis and fibrosis produced by these doses of bleomycin are similar, if not indistinguishable, from those found in patients who receive the drug (6). In addition, bleomycin is not considered to be immunosuppressive and has little effect on bone marrow (20). Thus, the murine model of bleomycin toxicity seems to be a valid representation of bleomycin-induced pulmonary disease in humans; furthermore, its lack of suppressive effects on the immune and inflammatory systems simplifies the evaluation of the role of these systems in the development of the bleomycin-induced lesion. Role of cell-mediated immune processes in the pathogenesis of interstitial pulmonary disease. There is increasing evidence that cellmediated immune processes are important in the development of interstitial pulmonary disorders such as hypersensitivity pneumonitis (21), sarcoidosis (22), berylliosis (23), and schistosomiasis (24). In contrast, little is known of the mechanisms by which the chronic alveolitis and ensuing fibrosis that follow administration of bleomycin are established and maintained, and a possible role for cell-mediated immunity in this disease process has not been evaluated. If the results of the present study can be extrapolated to humans, it is likely that the cell-mediated immune system is not a vital link in the development of bleomycin-induced interstitial pul-
BLEOMYCIN-INDUCED INTERSTITIAL DISEASE
monary disease. It should be noted, however, that this concept is based on the evaluation of bleomycin-induced pulmonary disease using morphologic criteria only. It is conceivable that the cell-mediated system does mediate bleomycin-induced pulmonary injury on a more subtle level than can be detected by histologic methods (25). It is likely that there are multiple mechanisms by which a chronic inflammatory process may be established in the lungs. Therefore, because of the differences between bleomycin-induced pulmonary disease and many of the other interstitial lung disorders (11), it is important that the results of these studies not be extended to human interstitial disorders other than that caused by bleomycin. However, the concept of using an immune-deficient model may prove useful in evaluating the immune system in the pathogenesis of other interstitial disorders.
References 1. Luna MA, Bedrossian CWM, Lichtiger B, Salem PA. Interstitial pneumonitis associated with bleomycin therapy. Am J Clin Pathol 1972; 58: 501-10. 2. Bedrossian CWM, Luna MA, MacKay B, Lichtiger B. Ultrastructure of pulmonary bleomycin toxicity. Cancer 1973; 32:44-51. 3. Samuels ML, Johnson DE, Holoye PY, Lanzotti VJ. Large dose bleomycin therapy and pulmonary toxicity. JAMA 1976; 235:1117-20. 4. Daskal Y, Gyorkey F, Gyorkey P, Busch H. Ultrastructural study of pulmonary bleomycin toxicity. Cancer Res 1976; 36:1267-72. 5. Blum R, Carter S, Agre K. A clinical review of bleomycin—a new antineoplastic agent. Cancer 1973;31:903-13. 6. Adamson IYR, Bowden DH. The pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Am J Pathol 1974; 77:185-90. 7. Aso Y, Yoneda K, Kikkawa Y. Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab Invest 1976; 35:558-68. 8. McCullough B, Collins JF, Johanson WG. Bleomycin-induced diffuse interstitial pulmonary fibrosis in baboons. J Clin Invest 1978; 61:79-88. 9. Snider GL, Celli BR, Goldstein RH, O'Brien JJ, Lucey EC. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am Rev Respir Dis 1978; 117:289-97.
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10. Sikic BI, Young DM, Mimnaugh EG, Gram TE. Quantification of bleomycin pulmonary toxicity in mice by changes in lung hydroxyproline content and morphometric histopathology. Cancer Res 1978; 38:787-92. 11. Fulmer JD, Crystal RG. Interstitial lung disease. In: Simmons DH, ed, Current pneumonology, vol. I. Boston: Houghton Mifflin, 1979:165. 12. Gershwin ME, Merchant B, Gelfand MC, Vickers J, Steinberg AD, Hansen CT. The natural history and immunopathology of outbred athymic (nude) mice, Clin Immunol Immunopathol 1975; 4:324-40. 13. Umazawa H, Maeda K, Takeuchi T. New antibiotics, bleomycin A and B. J Antibiot (Tokyo) 1966; 19:200-9. 14. Flanagan SP. "Nude," a hairless gene with pleiotropic effects in the mouse. Genet Res 1966; 8: 295-309. 15. Pantelouris EM. Absence of thymus in a mouse mutant. Nature 1968; 217:370-1. 16. Patelouris EM. Athymic development in the mouse. Differentiation 1973; 1:437-50. 17. Ward JM, Houcheus DP, Collins MJ, Young DM, Reagan RL. Naturally occurring Sendai virus infection of athymic nude mice. Vet Pathol 1976; 13:36-44. 18. Morse HC, Steinberg AD, Schur PH, Reed ND. Spontaneous "autoimmune disease" in nude mice. J Immunol 1974; 113:688-97. 19. Freireich EJ, Gehan EA, Rail DP, Schmidt LH, Skipper HE. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemotherapy Rep 1966; 50:219-44. 20. Umezawa H, Ishizuka M, Maeda K, Takeuchi T. Studies on bleomycin. Cancer 1967; 20:891-5. 21. Fink JN. Hypersensitivity pneumonitis. In: Kirkpatrick CH, Reynolds HY, ed, Immunologic and infectious reactions in the lung. New York: Marcel Dekker, 1976:229-41. 22. Mitchell DN, Scadding JG. Sarcoidosis. Am Rev Respir Dis 1974; 110:774-802. 23. Deodhar SD, Barna B, Van Ordstrand HS. A study of the immunologic aspects of chronic berylliosis. Chest 1973; 63:309-13. 24. Boros DV. Schistosomiasis mansoni: a granulomatous disease of cell-mediated immune etiology. Ann NY Acad Sci 1976; 278:34-46. 25. Thrall RS, McCormick JR, Phan SH, Jack RH, Ward PA. The effect of antilymphocyte globulin on the development of bleomycin-induced pulmonary fibrosis in the rat. Am Rev Respir Dis 1979; 119 (Suppl 2:83).
SUMMARY Evidence from divergent sources suggests that some forms of interstitial pulmonary disease are associated with abnormalities of the cellular immune system. T o evaluate whether cellular immune processes are necessary determinants for the development of parenchymal alveolitis and fibrosis secondary to bleomycin, we examined the effect of bleomycin on the NIH, outbred white mouse as compared to the homozygous nude, athymic mouse on the NIH outbred background. The nude mouse has virtually no detectable cell-mediated immune function; we therefore hypothesized that if this component of the immune system were necessary for the development of bleomycin-induced interstitial disease, bleomycin would not induce the same pulmonary lesion in the nude mouse as in the white mouse. However, both white and nude mice developed alveolitis and fibrosis after intraperitoneal administration of bleomycin. Comparison of the frequency and severity of these lesions in the 2 groups revealed no significant differences. These findings suggest that the presence of an intact cell-mediated immune system is not an absolute requirement for the development of bleomycin-induced interstitial disease in the mouse. T o the extent that this model is an appropriate approximation of human bleomycin-induced pulmonary disease, these results are consistent with the hypothesis that T-lymphocyte mediated processes are not primary determinants of this lesion.
Introduction Bleomycin, a cytotoxic a n t i m i c r o b i a l d r u g currently used to treat a variety of tumors, is a k n o w n cause of interstitial p u l m o n a r y disease (1-4). Bleomycin-induced interstitial disease has received a great deal of a t t e n t i o n for 2 reasons: (1) significant n u m b e r s of patients who receive t h e r a p e u t i c courses of this d r u g develop interstitial disease (5), a n d (2) bleomycin-induced interstitial disease is easily developed in animals, a n d thus has become a widely used model of the h u m a n disease (6-9).
(Received in original form July 5, 1978 and in revised form June 12,1979) i From the Pulmonary Branch, National Heart, Lung, and Blood Institute, Bethesda, Md. 2 Requests for reprints should be addressed to Building 10, 6D06, National Institutes of Health, Bethesda, Md. 20205.
A l t h o u g h information has accumulated conc e r n i n g the morphology, physiology, a n d connective tissue chemistry of bleomycin-induced interstitial disease, there is little information available c o n c e r n i n g the pathogenesis of this lesion. O n e clue comes from sequential histologic evaluation of the lungs of animals receiving the d r u g (6, 7, 10). Initially, bleomycin appears to d a m a g e p a r e n c h y m a l cells. Soon afterward, a chronic inflammatory process ("alveolitis") develops, a n d p a r e n c h y m a l cellular disordering a n d fibrosis ensue. As with m a n y other interstitial p u l m o n a r y disorders, this progression from alveolitis to p a r e n c h y m a l disordering a n d fibrosis suggests that inflammatory processes may be critical d e t e r m i n a n t s of the pathogenesis of bleomycin-induced interstitial disease. T h e mechanism by which the alveolitis of bleomycin-induced interstitial disease is established a n d m a i n t a i n e d is unclear. I n o t h e r interstitial disorders, however, it is k n o w n that
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o n e m e a n s by which a chronic inflammatory process may be established is activation of the cellular i m m u n e system (11). T o d e t e r m i n e w h e t h e r cellular i m m u n e processes play a role in the pathogenesis of bleomycin-induced interstitial disease, we c o m p a r e d the response of norm a l mice to bleomycin a d m i n i s t r a t i o n to that of mice lacking a n intact cellular i m m u n e system. If the chronic inflammatory response in bleomycin-induced interstitial disease is maintained as a result of activation of the cellular i m m u n e system, t h e n acfministration of bleomycin to mice that lack this a r m of the i m m u n e system should n o t develop interstitial disease of the severity observed in n o r m a l animals.
Methods Two groups of mice were evaluated. Normal mice were from an outbred colony of Swiss-type mice established at the National Institutes of Health [N:NIH(S)]. Nude, athymic mice were used as a model of an absent cellular immune system. These mice were from an outbred colony with the same genetic composition as the N:NIH (S) mice, except for the presence of the homozygous nude mutation (12). Both groups of mice were obtained from the Veterinary Resources Branch of NIH. For convenience, the normal mice with the N:NIH (S) background are referred to as "white mice," and the nude, athymic mice with the NrNIH (S) background are referred to as "nude mice." Nude mice were kept in sterile, filter-topped cages, and were given sterile food and sterile, distilled water adjusted to pH 2.5 with 1 M HC1. Under these conditions, spontaneous mortality was less than 10% during 6 months of observation. White mice were kept in nonsterile cages and were given normal food and distilled water. T h e spontaneous mortality of the white mice was less than 5% during 6 months of observation. All mice used for these studies were females, 6 to 7 wk of age, weighing 20 to 25 g. Bleomycin. Bleomycin sulfate (Blenoxane®, Nippon Kayaku Co. Ltd., Tokyo, Japan) was obtained from Bristol Laboratories (Syracuse, N.Y.) as a lyophilized powder in sterile 15-unit ampules. For injection, the bleomycin was dissolved in sterile 0.9% saline. T h e drug was injected intraperitoneally with a 25-gauge needle; its concentration was adjusted to permit administration of a volume of 0.1 ml/25 g of murine body weight. Study groups. Four groups of mice were evaluated. (1) Control: 31 white mice and 30 nude mice did not receive bleomycin; they were maintained under conditions identical to their counterparts given injections. These mice were killed at 14 to 15 wk of age, 8 wk from the beginning of observation. (2) Low dose: 27 white mice and 27 nude mice each received a total of 160 units of bleomycin/kg of body weight, administered as 8 doses of 20 units/kg
twice weekly for 4 wk. Mice from this group were killed 4 wk after completion of drug administration. (3) Medium dose: 6 white and 6 nude mice each received a total of 400 units of bleomycin/kg of body weight in 8 doses of 50 units/kg 4 times/wk during 2 wk. These mice were killed 2 wk after completion of drug administration. (4) High dose: 7 white mice and 6 nude mice received a total of 1,000 units of bleomycin/kg of body weight, in doses of 100 units/kg, 5 times/wk, during 2 wk. These mice were scheduled to be killed 2 wk after completion of drug administration, but spontaneous mortality before scheduled killing was high. Specimen preparation and evaluation. Mice were killed by intraperitoneal injection of sodium pentobarbital (10 mg/mouse). T h e trachea was cannulated with a 22-gauge, 2-cm teflon catheter and secured with 3-0 silk. T h e lungs and trachea were excised en bloc and fixed in inflation for 24 h with formalin under a pressure of 25 cm H 2 0 . T h e formalin was prepared as a neutral aqueous solution of formaldehyde (10 %, vol/vol) and sodium acetate (2 % wt/vol). T h e fixed lungs were dehydrated by 2 passages through graded alcohol and xylene, and were then imbedded in paraffin. Sections 6 /*m thick were cut and stained with hematoxylin/eosin and Masson's trichrome. Full coronal and transverse sections of lungs from both the control animals and those given injections were evaluated blindly by 3 observers for the presence of alveolitis and fibrosis. Alveolitis was evaluated with the hemotoxylin and eosin-stained sections and was graded using the following criteria: none (0), no alveolitis; mild (1-f), thickening of the alveolar septum by a mononuclear cell infiltrate, with involvement limited to focal, pleural-based lesions occupying less than 20 % of the lung and with good preservation of the alveolar architecture; moderate (2+), a more widespread alveolitis involving 20 to 50 % of the lung, although still predominantly pleural based; severe (3+), a diffuse alveolitis involving more than 50 % of the lung, with occasional consolidation of air spaces by the intra-alveolar mononuclear cells and some hemorrhagic areas within the interstitium and /or alveolus. Fibrosis was evaluated using Masson's trichrome stain sections. T h e extent of fibrosis in these sections was graded using the following criteria: none (0), no evidence of fibrosis; mild (1+), focal regions of fibrosis involving less than 20 % of the lung. Fibrosis involved the pleura and the interstitium of the subpleural parenchyma with some distortion of alveolar architecture; moderate (2+), more extensive fibrosis involving 20 to 50 % of the lung and fibrotic regions mostly extending inward from the pleura and still focal; severe (3+), widespread fibrosis, involving more than 50 % of the lung. Confluent lesions with extensive derangement of parenchymal architecture, including cystic air spaces lined by cuboidal epithelium.
895
BLEOMYCIN-INDUCED INTERSTITIAL DISEASE
Data analysis. To compare the severity of the alveolitis or fibrosis at each concentration of bleomycin, a mean grade for each mouse was determined by averaging the grade from each observer and rounding to the nearest integer. Differences in the severity of the lesions between white and nude mice were evaluated for significance using the standard chi-square test with the continuity correction. This analysis was performed for both low- and mediumdosage groups; insufficient data were available for evaluation of the high-dosage group. In addition to the graded analysis at each dosage, all lungs were scored for the presence or absence of alveolitis or fibrosis, and the results of white versus nude mice were compared by the 2-tailed "Student's" t test. Results A d m i n i s t r a t i o n of bleomycin resulted in p u l m o nary lesions of widely varying severity in b o t h white mice (figure 1) a n d n u d e mice (figure 2).
Although a few mice that received bleomycin r e m a i n e d n o r m a l , most exhibited focal, pleuralbased lesions consisting of p l e u r a l thickening, disorganization of alveolar architecture, m o n o nuclear interstitial infiltrates, accumulation of collagen in the alveolar a n d interstitial spaces, a n d intra-alveolar foamy macrophages. Some mice of both groups h a d lesions in which the p a r e n c h y m a l disordering was m o r e confluent, involving large areas of the l u n g b e y o n d t h e subpleural areas, a n d e x h i b i t i n g cystic air spaces lined by cuboidal e p i t h e l i u m . T h e m o n o n u c l e a r cell infiltrates associated with the bleomycininduced interstitial disease were similar in the white a n d the n u d e mice. Relative
Susceptibility Mice to
of White Bleomycin
and
Nude
Controls.
A m o n g the 31 white mice n o t given
injections, 26 h a d completely n o r m a l parenchy-
Fig. 1. Lungs of white mice. {A) Control mouse that did not receive bleomycin. (B) Mouse receiving lowdose bleomycin. There is a focal, pleural-based lesion with inflammatory cells in the interstitial and alveolar spaces. Fibrosis is demonstrable, but the over-all alveolar architecture is still recognizable. (C) Mouse receiving low-dose bleomycin. T h e lesion is more extensive than in (B), with some areas of disruption of the alveolar architecture. (D) Mouse receiving medium-dose bleomycin. T h e alveolitis and fibrosis are widespread, with involvement of the airways a n d presence of numerous cystic air spaces (Massons' trichrome stain; original magnification: X 55).
896
SZAPIEL, ELSON, FULMER, HUNNINGHAKE, AND CRYSTAL
Fig. 2. Lungs of n u d e mice. (A) Control mice that did not receive bleomycin. (J3) Mouse receiving low-dose bleomycin. T h e lesion is localized and pleural based. T h e basic alveolar architecture is still recognizable despite the presence of mild alveolitis a n d fibrosis. (C) Mouse receiving medium-dose bleomycin. T h e lesion is more advanced t h a n in (B). Both pleural surfaces of one lobe are visible with the lesion extending from one surface to the other. In some areas, the normal alveolar architecture is lost. (D) Mouse receiving medium-dose bleomycin. Widespread alveolitis and fibrosis are present (Masson's trichrome stain; original magnification: X 55).
mal structures. The alveolar septums and pleura were thin, and the alveolar walls formed a fine, fishnet pattern. There was little, if any stainable collagen visible in the alveolar septums. Within the air spaces, there were a few mononuclear cells, and the septums were devoid of inflammatory cells. Four (13%) white mice not given injections had mild or moderate alveolitis manifested by a thickening of alveolar walls accompanied by an accumulation of mononuclear cells within the parenchymal structures. One white mouse had a single focal area of fibrosis. Among the 30 control nude mice, 25 had normal lungs with no evidence of alveolitis or fibrosis. During the study period, 5 of the nude mice, but none of the control mice died. Low dosage. The survival rate among 27 white and 27 nude mice that received low-dose bleomycin was 96 % (26 of 27) and 85 % (23 of 27), respectively. Of the survivors, more than 90 % of both the white and nude mice had varying de-
grees of parenchymal lesions (figure 3). Statistical analysis using a 2-by-4 contingency table revealed no significant difference between white and nude mice in the distribution of severity of either alveolitis (p > 0.9) or fibrosis (p = 0.2). Medium dosage. Six white and 6 nude mice received medium-dose bleomycin. The survival in both groups was 83 % (5 of 6 mice in each group). Although the proportion of mice exhibiting pulmonary lesions was larger than at low dosage, the results were similar to the lowdose group in that there was no significant difference between the white and nude mice, either for alveolitis (p = 0.7) or for fibrosis (p = 0.6) (figure 4). Analysis of low- and medium-dosage groups combined. Combining the mice that received low and medium dosage, a total of 33 white mice and 33 nude mice received bleomycin, with an over-all survival rate in each group of 94 %
897
BLEOMYCIN-INDUCED INTERSTITIAL DISEASE
A. ALVEOLITIS, LOW DOSE
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GRADING OF HISTOLOGIC LESION
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B. FIBROSIS, LOW DOSE
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GRADING OF HISTOLOGIC LESION Fig. 3. Frequency distribution of the severity of pulmonary lesions induced by low-dose bleomycin in white and n u d e mice: (A) alveolitis and (B) fibrosis. Comparison of the white a n d n u d e mice demonstrated no significant differences in the frequency distribution of either alveolitis (p > 0.9) or fibrosis (p = 0.2).
and 85 %, respectively. These combined groups were compared in 2 ways. First, the distribution of severity of both alveolitis and fibrosis was compared in a manner similar to that described previously for the individual groups. Using a 2-by-4 contingency table, there was no significant difference between white and nude mice in the severity of alveolitis (p > 0.8) or the severity of fibrosis (p > 0.3) (data not shown). In a second analysis of this combined data, the mice receiving low- and medium-dose bleomycin were grouped according to presence versus absence of alveolitis or fibrosis, irrespective of severity. Using a 2-by-2 contingency table, there was no significant difference between white and nude mice in the occurrence of either alveolitis (p > 0.8) or fibrosis (p > 0.2) (figure 5). High dosage. High-dose bleomycin was unsatisfactory in both white and nude mice because of extreme wasting and high, early mortality. Among 7 white mice that received this dosage, two survived; among 6 nude mice, one survived. Because of the small number of survivors, these lesions were not evaluated statistically. In the survivors, the lungs showed severe alveolitis with a dense cellular infiltrate and little fibrosis (data not shown).
UL O
80
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60 40
o
20 0
WHITE (n=5)
n
NUDE (n=5)
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0 1+ 2+ 3+ 0 1+ 2+ 3+ GRADING OF HISTOLOGIC LESION
Fig. 4. Frequency distribution of the severity of pulmonary lesions induced by medium-dose bleomycin in white and n u d e mice: (A) alveolitis and (B) fibrosis. Comparison of the white and nude mice demonstrated no significant differences in the frequency distribution of either alveolitis (p = 0.7) or fibrosis (p = 0.6).
Discussion
Bleomycin sulfate (Blenoxane) is a soluble mixture of cytotoxic glycopeptide antimicrobial drugs derived from a strain of Streptomyces verticillus (13). Clinically, it is currently used alone or in combination with other chemotherapeutic agents in the treatment of neoplasms such as squamous-cell carcinoma, testicular carcinoma, and lymphoma (5). Its major side effect is pulmonary toxicity, which occurs in approximately 11% of treated patients (5). This toxicity is manifested most frequently as alveolitis, progressing to diffuse fibrosis in approximately 1% of all patients who receive the drug (2, 5). To begin to evaluate the mechanisms by which bleomycin induces pulmonary injury, we asked whether an intact cellular immune system was necessary for development of bleomycininduced alveolitis and fibrosis in animals that received this drug. Comparison of normal mice to mice of the same genetic background but with no functioning, cell-mediated immune system demonstrated that both groups were susceptible to the development of alveolitis and fibrosis
898
SZAPIEL, ELSON, FULMER, HUNNINGHAKE, AND CRYSTAL
100
ALVEOLITIS (n=28)
(n=31) 80
0.8) or fibrosis (p > 0.2).
secondary to administration of bleomycin. Thus, to the extent that this animal model is a valid representation of bleomycin-induced pulmonary disease in humans, these studies suggest that cell-mediated immune processes are not necessary components in the pathogenesis of this lesion. Validity of the model. The nude mouse was described initially as a hairless, recessive mutant, designated nu/nu (14). When it was found that the nu/nu mouse was also athymic, it was soon demonstrated that this strain lacked a cell-mediated immune system (15). Although some T-cell antigens have been described on lymphocytes in the nu/nu mutation, these cells are considered nonfunctional, and the nu/nu mutation is generally regarded as an excellent model of a profound defect of the cell-mediated immune system (16). The nu/nu mutation is most commonly available on the inbred Balb/C background. This strain is difficult to use in studies of the lung because these mice are susceptible to infection, particularly the naturally occurring Sendai virus that invades the respiratory tract and causes a chronic alveolitis (17). In an attempt to overcome the limitations of working with this strain of mice, the inbred Balb/C nude mouse was crossed with a normal, outbred Swiss-type mouse, resulting in the nude, athymic mutation on
the outbred background [N:NIH (S)] (12). The nu/nu N:NIH(S) mice live approximately 1 yr and rarely develop respiratory infections when kept under minimally sterile laboratory conditions. In addition, these mice do not have autoantibodies to nucleic acids, which have been reported in the nude Balb/C mouse (12, 18). Thus, not only is the N:NIH (S) nude mouse a good model of an absent cell-mediated immune system, but it is also particularly suitable for studies of the respiratory system. In patients who receive bleomycin, the toxicity of the drug is generally dose-related with a total dose of more than 400 units most commonly associated with the development of interstitial disease (5). Most murine studies use total doses of 140 to 240 units/kg of body weight to produce interstitial disease regularly. When this is corrected for body surface area (19), these murine doses correspond to approximately 800 to 1,400 units in a 70-kg man, the same general range known to cause interstitial disease in humans. Morphologic evaluation of bleomycin-induced pulmonary disease in the murine model suggests that the alveolitis and fibrosis produced by these doses of bleomycin are similar, if not indistinguishable, from those found in patients who receive the drug (6). In addition, bleomycin is not considered to be immunosuppressive and has little effect on bone marrow (20). Thus, the murine model of bleomycin toxicity seems to be a valid representation of bleomycin-induced pulmonary disease in humans; furthermore, its lack of suppressive effects on the immune and inflammatory systems simplifies the evaluation of the role of these systems in the development of the bleomycin-induced lesion. Role of cell-mediated immune processes in the pathogenesis of interstitial pulmonary disease. There is increasing evidence that cellmediated immune processes are important in the development of interstitial pulmonary disorders such as hypersensitivity pneumonitis (21), sarcoidosis (22), berylliosis (23), and schistosomiasis (24). In contrast, little is known of the mechanisms by which the chronic alveolitis and ensuing fibrosis that follow administration of bleomycin are established and maintained, and a possible role for cell-mediated immunity in this disease process has not been evaluated. If the results of the present study can be extrapolated to humans, it is likely that the cell-mediated immune system is not a vital link in the development of bleomycin-induced interstitial pul-
BLEOMYCIN-INDUCED INTERSTITIAL DISEASE
monary disease. It should be noted, however, that this concept is based on the evaluation of bleomycin-induced pulmonary disease using morphologic criteria only. It is conceivable that the cell-mediated system does mediate bleomycin-induced pulmonary injury on a more subtle level than can be detected by histologic methods (25). It is likely that there are multiple mechanisms by which a chronic inflammatory process may be established in the lungs. Therefore, because of the differences between bleomycin-induced pulmonary disease and many of the other interstitial lung disorders (11), it is important that the results of these studies not be extended to human interstitial disorders other than that caused by bleomycin. However, the concept of using an immune-deficient model may prove useful in evaluating the immune system in the pathogenesis of other interstitial disorders.
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10. Sikic BI, Young DM, Mimnaugh EG, Gram TE. Quantification of bleomycin pulmonary toxicity in mice by changes in lung hydroxyproline content and morphometric histopathology. Cancer Res 1978; 38:787-92. 11. Fulmer JD, Crystal RG. Interstitial lung disease. In: Simmons DH, ed, Current pneumonology, vol. I. Boston: Houghton Mifflin, 1979:165. 12. Gershwin ME, Merchant B, Gelfand MC, Vickers J, Steinberg AD, Hansen CT. The natural history and immunopathology of outbred athymic (nude) mice, Clin Immunol Immunopathol 1975; 4:324-40. 13. Umazawa H, Maeda K, Takeuchi T. New antibiotics, bleomycin A and B. J Antibiot (Tokyo) 1966; 19:200-9. 14. Flanagan SP. "Nude," a hairless gene with pleiotropic effects in the mouse. Genet Res 1966; 8: 295-309. 15. Pantelouris EM. Absence of thymus in a mouse mutant. Nature 1968; 217:370-1. 16. Patelouris EM. Athymic development in the mouse. Differentiation 1973; 1:437-50. 17. Ward JM, Houcheus DP, Collins MJ, Young DM, Reagan RL. Naturally occurring Sendai virus infection of athymic nude mice. Vet Pathol 1976; 13:36-44. 18. Morse HC, Steinberg AD, Schur PH, Reed ND. Spontaneous "autoimmune disease" in nude mice. J Immunol 1974; 113:688-97. 19. Freireich EJ, Gehan EA, Rail DP, Schmidt LH, Skipper HE. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemotherapy Rep 1966; 50:219-44. 20. Umezawa H, Ishizuka M, Maeda K, Takeuchi T. Studies on bleomycin. Cancer 1967; 20:891-5. 21. Fink JN. Hypersensitivity pneumonitis. In: Kirkpatrick CH, Reynolds HY, ed, Immunologic and infectious reactions in the lung. New York: Marcel Dekker, 1976:229-41. 22. Mitchell DN, Scadding JG. Sarcoidosis. Am Rev Respir Dis 1974; 110:774-802. 23. Deodhar SD, Barna B, Van Ordstrand HS. A study of the immunologic aspects of chronic berylliosis. Chest 1973; 63:309-13. 24. Boros DV. Schistosomiasis mansoni: a granulomatous disease of cell-mediated immune etiology. Ann NY Acad Sci 1976; 278:34-46. 25. Thrall RS, McCormick JR, Phan SH, Jack RH, Ward PA. The effect of antilymphocyte globulin on the development of bleomycin-induced pulmonary fibrosis in the rat. Am Rev Respir Dis 1979; 119 (Suppl 2:83).
