Azathioprineassociated Interstitial Pneumonitis CARLOS W. M. BEDROSSIAN, M.D., AND BARRY KAHAN, M.D., PH.D. JEFFREY SUSSMAN, M.D., RICHARD H. CONKLIN, PH.D., M.D. Seven renal allograft recipients taking azathioprine (Imuran ) for immunosuppression developed bilateral pulmonary infiltrates and a falling p0 2 that did not respond to antibiotic therapy. lung biopsies revealed changes ranging from diffuse alveolar damage (DAD) to usual interstitial pneumonia () culminating in pulmonary fibrosis. There was no evidence of immune deposits, eosinophilia, vasculitis, granulomas, or microorganisms by cultures and appropriate stains. Following discontinuance of Imuran, the two cases with DAD revealed a significant clearing of the lung infiltrates, whereas four of five patients with died while suffering from respiratordependent ARDS. Biopsies showing hyaline membranes, intraalveolar edema and cuboidalization of alveolar epithelium were associated with total doses from 2,850 to 4,355 mg, whereas atypical epithelial hyperplasia, reorganization of distal air spaces, and fibrosis were noted in cases receiving from 5,600 to 28,625 mg of azathioprine. Ultrastructural changes were indistinguishable from those induced by other drugs causing pulmonary toxicity. In three cases atypical epithelial cells were detected cytologically in brushing specimens and appeared identical to those noted in the lung biopsies. Our findings are consistent with the view that azathioprine should be added to the list of agents capable of causing direct, dosedependent pulmonary toxicity. Accordingly, drugassociated diffuse interstitial pulmonary disease should enter the differential diagnosis of a lung infiltrate that develops in renal transplant patients receiving Imuran. (Key words: Imuran; Drug toxicity; Interstitial pneumonitis; Pneumotoxic drugs) Am J Clin Pathol 1984; 82: 148154 AZATHIOPRINE (Imuran ; Burroughs Wellcome, Research Triangle Park, NC) is a potent immunosuppressive drug used primarily as an antiproliferative agent to prevent rejection after renal transplantation. Other indications for use of the drug include chronic inflammatory bowel diseases such as ulcerative colitis and regional enteritis and the collagen vascular diseases systemic lupus erythematosus and rheumatoid arthritis. 7 More recently, the drug also has been utilized in the treatment of chronic active hepatitis, glomerulonephritis, and a variety of hematologic and nonhematologic disorders. Complications of azathioprine therapy consist chiefly of hepatotoxicity, bone marrow suppression, and opportunistic infection. Pulmonary toxicity generally is not recognized as a side effect, even though two isolated case Received October 3, 1983; received revised manuscript and accepted for publication November 16, 1983. Address reprint requests to Dr. Bedrossian: Department of Pathology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, Missouri 63104. Department of Pathology, Vanderbilt University School/VA Medical Center, Nashville, Tennessee, and Transplant Division, Department of Surgery, University of Texas Medical School, Houston, Texas reports have implicated azathioprine in the development of interstitial lung disease. 8 " The present study reports seven cases of interstitial lung disease that developed in renal transplant recipients during immunosuppressive therapy with azathioprine. No infectious agents or other etiologic factors could be identified to account for the pulmonary changes; furthermore, the pathologic changes suggest drug toxicity. Materials and Methods All available clinical and pathologic data including chest roentgenograms were reviewed from seven renal transplant patients receiving azathioprine. An openlung biopsy was obtained in six of the seven cases from which viral, mycoplasma, Legionella, aerobic, anaerobic, AFB and fungal cultures were obtained. In the remaining case, a transbronchial biopsy was available for examination. Bronchial washings and brushings available in three cases and imprint smears from the openlung biopsies were stained with Papanicolaou, hematoxylin and eosin, Toluidine blue, AFB, and Gomori's methenamine silver stains. Formalinfixed lung tissue was embedded in paraffin, sectioned and stained with hematoxylin and eosin, periodic acidschiff, Masson's trichrome, AFB, and Gomori's methenamine silver stains. Small cubes of glutaraldehyde fixed lung tissue were postfixed in osmium tetraoxide, embedded in epoxy resin, thinsectioned in an ultramicrotome, and stained with lead citrate and uranyl acetate. Results Clinical and pathological data in seven patients who developed interstitial lung disease during immunosuppressive therapy with azathioprine are summarized in Table 1. All patients had received a cadaveric renal allograft prior to the development of lowgrade fever, an abnormal chest xray, and a falling p0 2, despite therapy with broadspectrum antibiotics. The chest xray abnormalities consisted of infiltrates that were diffuse and bi Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055 148
vol. 82.No. 2 AZATHIOPRINE PNEUMONITIS 149 Table 1. Clinical and Pathologic Data on Seven Cadaveric Renal Allograft Recipients Treated with Azathioprine Distribution of Total Prednisone Consolidation Imuran Dose at Bx Patient/Age/Sex Chest xray Dose (mg) (mg/day) Type of Bx 1/74/M 2/51/M 3/42/F 4/49/F 5/41/F 6/40/F 7/31/M Left Lower Lobe Left Lung 5600 7350 28,625 4355 2850 3400 10,675 40 35 45 30 30 No. Days After Transplant 35 68 229 107 83 30 68 22.5 Transbronchial 130 Bx Pathology DAD DAD Outcome Died two days after biopsy. Autopsy showed superimposed bronchopneumonia and cerebral hemorrhage. Died one day after biopsy. Consolidation did not clear after discontinuance of azathioprine. Consolidation did not clear following discontinuance of azathioprine. Died 30 days post biopsy. Autopsy showed disseminated Aspergillus. Consolidation cleared on discontinuance of azathioprine: alive and well y/i years later. Consolidation cleared following discontinuance of azathioprine and institution of Cytoxan; alive and well four years later. Consolidation cleared following discontinuance of azathioprine and institution of Cytoxan; died one year postbiopsy. Died two days after biopsy. lateral in five patients and diffuse but more severe on one side in the other two patients. No parenchymal cavities, pleural effusions, or focal lesions were noted in any of the cases. Total doses of azathioprine administered before the onset of the infiltrate ranged from 2,850 mg to 28.625 mg. The infiltrate developed from 35 to 229 days following transplantation and disappeared spontaneously in one patient (patient 4) when azathioprine therapy was discontinued. In two additional patients (patients 5 and 6) the infiltrate disappeared when azathioprine was discontinued and Cytoxan (Mead Johnson, Evansville, IN) was instituted. In four patients (patients 1, 2, 3, and 7) the p0 2 never improved, the infiltrates progressed, and the patients expired despite supportive therapy with a respirator. Results of cultures obtained from the openlung biopsies were negative in all cases. Histopathology lung biopsies in six of the cases revealed changes varying from diffuse alveolar damage (DAD) to usual Table 2. Pulmonary Histopathologic Changes in Seven Patients Receiving Azathioprine Patient 1 2 3 4 5 6 7 Hyaline Membranes _ Intraalveolar Edema _ Cuboidal Hyperplasia Interstitial Fibrosis Atypical Epithelial Cells _ Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055
150 BEDROSSIAN ET AL. Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055 AJ.C.P. August 1984
Vol. 82 No. 2 AZATHIOPR1NE PNEUMONITIS 151 FIG. 1 (upper). lung biopsy from 49yearold woman who received 4,355 mg azathiopnne. Note interstitial edema, prominent intraalveolar hyaline membranes, and rows of cuboidal cells lining alveoli. Hematoxylin and eosin (X400). FIG. 2 (lower). This is an openlung biopsy in a 42yearold woman who received a total of 28,625 mg of azathiopnne. There is reorganization of distal air spaces, edema,fibrosis,and proliferation of atypical epithelial cells. Hematoxylin and eosin (X400). interstitial pneumonia () (Table 2). DAD was characterized by hyaline membranes, intraalveolar edema, and cuboidalization of alveolar epithelium (Fig. 1). Features of were reorganization of distal air spaces, atypical epithelial hyperplasia, and varying degrees of fibrosis (Fig. 2). No cytoplasmic or intranuclear viral inclusions were seen in any of the cases. The special stains for microorganisms were negative in all cases. There was no evidence of eosinophilia, granulomas, or vasculitis. Acute inflammatory cellular infilrate was not prominent in any of the cases. In one patient (patient 7) transbronchial biopsy showed collapse of air spaces, atypical epithelial hyperplasia, and fibrosis, consistent with. Cytopathology Bronchial washings and brushings in three cases revealed atypical epithelial cells similar to those lining the reorganized distal air spaces in the biopsies. The cytologic characteristics of these cells were better appreciated in the imprint smears prepared from the biopsies and consisted of a crescentic shape, granular cytoplasm without pigmentation, atypical nuclei and prominent nucleoli (Fig. 3). No inclusion bodies were noted in any of the cytologic preparations. Ultrastructure Electron microscopic examination was performed on all six openlung biopsies (Table 3). Electron micrographs revealed intraalveolar edema, fibrin accumulation, air spaces lined predominantly by type II alveolar pneumonocytes, and a marked paucity of type I cells (Fig. 4). The type II cells showed bizarre shapes, prominent 1ammelar bodies, blunted microvilli, and partial extrusion of osmiophilic material. No viral particles were seen. Type FIG. 3. Imprint smear of lung biopsy from same patient as Figure 2. Note atypical cells with crescent shape, nuclei from prominent nucleoli and granular cytoplasm without inclusions. Papanicolaou (X800). Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055
152 BEDROSSIAN ET AL. AJ.C.P. August 1984 Table 3. Electron Microscopy in Six Cases of Azathioprineassociated Interstitial Pneumonitis Patient Blebs in Type I Cells 1 2 3 4 5 6 Alveolar Edema Fibrin Accumulation I cells, when present, showed minimal bleb formation, whereas endothelial cells appeared intact. The basement membranes did not contain immune deposits. The interstitium showed fibrosis, more pronounced in cases of usual interstitial pneumonia (Fig. 5). Bizarre Type II Cells Collagen/ Fibroblasts Immunopathology Material for immunofluorescence was available in three of six openlung biopies. They were stained with antisera against IgG, IgM, C3, albumin, and fibrinogen. A small ' ' i,, ",. "' FIG. 4. Electron micrograph of lung biopsy from same patient as Figures 2 and 3. Note crescentshaped Type II cells lining alveoli and free in the lumen, with prominent lamellar bodies. Lead citrate and uranyl acetate (X 12,000). Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055
AZATHIOPR1NE PNEUMONITIS Vol. 82 No. 2 153 FIG. 5. Another area from same biopsy as Figure 2. The interstitium shows increased collagen,fibroblastsand a macrophage. The basement membranes remain intact. Lead citrate and uranyl acetate (X20.000). amount of fibrinogen was noted within alveolar spaces. However, no staining was noted with the other antisera within epithelial or endothelial alveolar wall basement membranes. Discussion In the transplant patient, pulmonary infections are the most common cause of pulmonary failure and an abnormal chest xray.6 Infections can be of viral, bacterial, fungal or protozoal origin.9 The least recognized cause of respiratory failure in the allograft recipient is toxicity due to the immunosuppressive agent itself. To our knowledge, only two previous case reports have appeared in the literature relating instances of Imuran Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055 associated pulmonary toxicity. The first case was a 20yearold man who developed a reticular infiltrate on the chest xray accompanied by bloodgas evidence of restrictive lung disease.8 He had received up to 150 mg/ day of cyclophosphamide over a threemonth period prior to therapy with azathioprine. The second was a 24yearold woman who developed a similar pattern on the chest xray without prior treatment with any known pneumotoxic medication. This second case came to biopsy, which revealed interstitial pulmonary fibrosis with features very similar to those seen in our patients.'' The seven cases reported here all had clinical and pathologic features consistent with azathioprine pulmonary toxicity. All patients developed hypoxia, lowgrade fever, and abnormal chest roentograms following cadav
154 BEDROSSIAN ET AL. A.J.C.P. August 1984 eric renal allografts and histologically showed varying degrees of hyaline membranes, intraalveolar edema, atypical hyperplasia of alveolar lining cells, and interstitial fibrosis. All cases were submitted to careful microbiologic cultures and thorough histopathologic examination for viruses, bacteria, and fungi resulting in no significant positive findings. Two patients receiving the lower doses of azathioprine (patients 4, 5) were classified as having diffuse alveolar damage and had a definite better prognosis. In one patient (patient 4) radiographic consolidation cleared following discontinuance of azathioprine. In the other patient (patient 5) discontinuance of azathioprine followed by treatment with Cytoxan was accompanied by clearing of the consolidation. One patient who also received low total dose (patient 6) had on the lung biopsy but had significant clinical improvement when azathioprine was discontinued and Cytoxan instituted. This patient died one year after biopsy. No autopsy was performed. Patients receiving higher doses (patients 1,2,3 and 7) were classified as having usual interstitial pneumonia and all showed some degree of interstitial fibrosis. Three patients (patients 1, 2, and 7) died one to two days after biopsy. Only one case came to autopsy which revealed and superimposed bronchopneumonia. One patient (patient 3) died 30 days after biopsy. In this patient, consolidation did not improve following discontinuance of azathioprine, and death was due to disseminated aspergillosis, which, at autopsy was noted in addition to. In human subjects the diagnosis of druginduced pulmonary toxicity is based on clinical history of drug exposure and absence of other known causative agents. 12 Diffuse interstitial pulmonary disease is by far the most frequent type of lung pathology induced by drugs.' The lesion can be reproduced experimentally by the administration of some of the drugs clinically implicated in pulmonary toxicity. 2,5 The pulmonary alterations, as noted in tissue biopsies, appear to be mediated either by direct, dosedependent toxicity or an allergic mechanism. None of our cases revealed immune deposits as observed in examples of druginduced lung disease believed to occur on an allergic basis. 10 On the other hand, ultrastructural evidence of epithelial damage in the absence of endothelial involvement is indistinguishable from that of previous cases of dosedependent pulmonary drug toxicity examined by electron microscopy. 3 In three of our patients atypical epithelial cells were seen in cytologic specimens at the time abnormalities appeared on chest radiographs. This occurrence also has been noted previously during drug therapy accompanied by pulmonary side effects. 4 These findings suggest that cytology can be useful in monitoring patients receiving potential pneumotoxic drugs. Although the evidence is circumstantial, our findings support the view that azathioprine is capable of causing dosedependent pulmonary toxicity. Broader recognition of this complication and discontinuance of azathioprine in patients who develop acute pulmonary failure in the absence of infection could prevent progression of pulmonary damage in such cases. Addendum. Since the acceptance of our manuscript, another case report of interstitial pneumonitis secondary to azathioprine in a renal transplant patient has appeared in the literature. The lung biopsy changes described by D. J. S. Carmiachel and associates (Thorax 1983; 38:951 952) are virtually identical to the ones noted in our patients who received the higher doses of Imuran. References 1. Bedrossian CWM: Pathology of druginduced lung diseases. Sem Resp Med 1982;4:98106 2. Bedrossian CWM, Greenberg SD, Yawn D, O'Neal RM: Experimentally induced bleomycin sulphate pulmonary toxicity. Arch Pathol Lab Med 1977; 101:248254 3. Bedrossian CWM, Luna MA, MacKay B, Lichtiger B: Ultrastructure of Bleomycin pulmonary toxicity. Cancer 1973; 32:4451 4. Bedrossian CWM, Corey BJ: Abnormal sputum cytopathology during chemotherapy with Bleomycin. Acta Cytol 1978; 22:202 207 5. Gould VE, Miller S: Sclerosing alveolitis induced by ciclyphosphamide. Am J Pathol 1975; 81:513530 6. Huertas VE, Port FK, Rozas VV, Niederhuber JE: Pneumonia in recipients of renal allografts. Arch Surg 1976; 111:162166 7. Rossman M, Bestino J: Axathioprine. Ann Intern Med 1973; 79:694 700 8. Rubin G, Baume P, Vandenberg R: Azathioprine and acute restrictive lung disease. Austr NZ J Med 1972; 3:272274 9. Rubin RH, Wolfson JS, Cosimi AB, TolkorT NE: Infection in the renal transplant recipient. Am J Med 1981; 70:405411 10. Smith WR, Dearden LC, McRae DM: Deposits of immunoglobulin and complement in the pulmonary tissue of patients with "heroin lung." Chest 1978;73:471476 11. Weisenburger DD: Interstitial pneumonitis associated with azathioprine therapy. Am J Clin Pathol 1978; 69:181185 12. Weiss RR, Muggia FM: Cytotoxic druginduced pulmonary disease: Update 1980. Am J Med 1980; 68:259266 Downloaded from https://academic.oup.com/ajcp/articleabstract/82/2/148/1868055