Clin. exp. Immunol. (1976) 24, 218-222.

Treatment of Goodpasture syndrome with cyclophosphamide, prednisone and plasma exchange transfusions R. D. ROSSEN, J. DUFFY, K. B. McCREDIE, M. A. REISBERG, J. T. SHARP, E. M. HERSH, G. EKNOYAN & W. N. SUKI Departments of Microbiology, Immunology and Medicine, Baylor College of Medicine, Department of Developmental Therapeutics, University of Texas at Houston and Laboratories of Immunology Research at the Veterans Administration, Methodist and M. D. Anderson Hospitals, Houston, Texas 77025, U.S.A.

(Received 12 August 1975)

SUMMARY

Repeated plasma exchanges were performed in a 44-year-old man with Goodpasture syndrome, also treated with cyclophosphamide and prednisone. Improvement was observed within 3 weeks of starting the protocol, and by the 76th week, endogenous creatinine clearance had increased from 30 to 56 ml/min/1-73 M2 and serum albumin from 2-7 to 3-7 g/dl. Prior treatment with immunosuppressive drugs had not significantly influenced circulating antibody levels. But sustained suppression of antibody was achieved after the plasma exchanges were begun, suggesting that physical removal of circulating antibody combined with antiproliferative drug treatment may be a useful way to control undesirable humoral immune responses. INTRODUCTION Sudden withdrawal of antibody from the circulation stimulates a brisk compensatory increase in antibody synthesis (Uhr & M6ller, 1968; Graf & Uhr, 1969; Bystryn, Graf & Uhr, 1970) which is only partially suppressed by appropriately timed therapy with cyclophosphamide (Bystryn, Schenkein & Uhr, 1971). Nevertheless these experiments suggest that repeated exchange transfusion associated with cyclophosphamide treatment might be used to achieve a significant decrease in unwanted circulating antibodies in patients with auto-immune diseases. This hypothesis has been tested in a patient with the Goodpasture syndrome. CASE REPORT The diagnosis of Goodpasture syndrome was suggested by the occurrence of haemoptysis, pleuritic chest pain, haematuria with red cell casts, a rising creatinine and increasing dyspnoea on exertion associated with recurrent pulmonary infiltrates and anaemia in a 44-year-old male. Lung biopsy showed haemorrhagic pneumonitis with alveolar septal thickening. Renal biopsy exhibited mesangial and endothelial cell proliferation with uniform thickening of glomerular basement membranes and, by immunofluorescence, linear deposition of IgG along the glomerular basement membranes (GBM). During treatment with up to 120 mg prednisone and 200 mg azathioprine daily for 1 month, pulmonary infiltrates recurred and fever and haemoptysis continued. On transfer to the Clinical Research Center, the creatinine was 2-4 mg/dl and 24 hr protein excretion ranged between 8 and 14 g. Vital signs were within normal limits except for a blood pressure of 245/110. Optic fundi showed arteriolar narrowing without exudates, haemorrhages or Correspondence: Dr Roger D. Rossen, Veterans Administration Hospital, 2002 Holcombe Blvd., Houston, Texas 77031, U.S.A.

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219 Role for plasma exchange in immunosuppression papilloedema. The lungs were clear to auscultation, the heart was at the upper limit of normal size but was otherwise unremarkable. Except for pretibial and presacral pitting oedema, the remainder of the physical exam was within normal limits. Chest X-ray showed no active infiltrates. Fiberoptic bronchoscopy revealed small amounts of blood emanating from the most distant ramifications of the bronchial tree bilaterally. Pulmonary macrophages from bronchial washings were heavily laden with iron. Laboratory: Hgb-10 g/dl, Hct 3000, white blood cell (WBC) 14,700/mm3 with normal differential and platelet counts. Serum Fe 120 mcg/dl, TIBC 255 mcg/dl. Serum albumin was 2 7 g/dl. Urinalysis showed 50-100 red cells per high power field but no red cell casts and only a few white blood cells. Nephrotomograms showed normal size kidneys with no obstructing lesions of the collecting system. Arterial blood gases showed a Po2 of 82 mmHg, Pco2 of 34 mmHg, pH of 7-45 and 02 saturation of 96%.

METHODS (1) Experimental protocol. During the first 3 weeks, only prednisone was given (0 50-0 75 mg/kg body weight daily). On day 21, oral cyclophosphamide was added at 2 mg/kg/day and the dose adjusted to maintain a total WBC in excess of 2 5 x 103/mm3. After 8 more weeks, exchange transfusions of plasma were performed through a radial artery-to-vein fistula by means of an IBM blood cell separator (Buckner, Clift & Thomas, 1969). Approximately 2000 ml of plasma were removed in a period of 3 hr on two occasions within a 48-hr period and simultaneously replaced with compatible, hepatitis B antigen-negative plasma. Four weeks later, plasma exchange was repeated three more times over a 4-day period during which 4500 ml was removed and replaced with normal donor plasma. Thereafter, he was maintained on continuous therapy with cyclophosphamide and prednisone. He was discharged in the 18th week and readmitted for evaluation 76 weeks after beginning the protocol. (2) Immunological studies. 100 mcg keyhole limpet haemocyanin (KLH) was injected intradermally (i.d.) in the 3rd, 5th, 8th and 10th weeks (Curtis et al., 1971). Antibodies to KLH were measured by estimating the quantity of patient 125I-labelled globulin which bound to insoluble KLH in the presence of an equal quantity of 13 I-labelled globulin from normal donors not immunized with KLH (Carpenter & Reisberg, 1968). Aliquots of the mixture of patient and normal globulins were incubated in triplicate with 1 mg of insolubilized KLH. The KLH was washed, counted for radioactivity and the net quantity of patient globulin bound to the KLH was calculated by the formula: Net globulin bound (jig/mg) = pg patient globulin bound pg control globulin bound mg patient globulin added mg control gobulin added Binding of the control served as an index of non-specific binding and varied as much as 8%Y (2 s.d.). Antibodies to IgG (antiglobulins) were measured in duplicate or triplicate (a) by two-fold dilutional titration of heat-inactivated 56°C for 30 min) sera using the latex fixation test (Singer & Plotz, 1956), (b) by reverse single radial diffusion in 0 7%. agarose using heat aggregated IgG (63°for 20 min) as antigen (Agnello et al., 1971) and (c) by measuring the binding of patient globulin to immunoadsorbents coated with human Cohn fraction II by the procedure described above for measurement of KLH antibody (Carpenter & Reisberg, 1968). Antibodies to glomerular basement membrane were measured by indirect immunofluorescence using cryo-

stat sections of rat and human renal cortex (McPhaul & Dixon, 1969) and by the immunoadsorbent binding

method as described above for KLH antibody using finely suspended particles of human glomerular basement membranes as antigen (Rossen et al. 1975). Serum IgA, IgG, IgM and C3 levels were measured by single radial diffusion in agar using a commercially available kit (Meloy Laboratories, Biological Products Division, 6715 Electronic Drive, Springfield, Vir-

ginia, U.S.A.).

RESULTS Clinical Proteinuria decreased and a marginal improvement in the ECC was seen 4-6 weeks into the protocol (Fig. 1). However, cyclophosphamide was discontinued for 3 weeks because of leukopenia between weeks 11 and 14 and, as the leucocyte count rebounded, the ECC returned to the admission value. Nevertheless, the decrease in proteinuria was maintained

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FIG. 1. Renal functional measurements and serum immunoglobulin (Ig) levels (IgG, IgA,

1gM) and C3 (I,~Cl/it1A globulin) are shown in relation to changes in total leucocyte count (WBC x 10- 3/mm3) and treatment with cyclophosphamide, prednisone and plasma exchange (P).

and the plasma albumin rose from 2*7 to 3*7 g/dl during the initial hospitalization. Also, pulmonary bleeding ceased and by the 5th week of the protocol only 500/0 of the pulmonary macrophages contained stainable iron; subsequent bronchial washings were entirely normal. As a result, dyspnoea on exertion virtually disappeared. Seventy-six weeks after the start of the protocol, the ECC had increased to 56 ml/min/l 73 M2, urinary protein loss was 2*5 g/24 hr, and plasma albumin 3*7 g/dl. Renal biopsy showed some histological evidence of improvement: Basement membrane thickening was focal rather than diffuse and, although some glomeruli were hyalinized, others looked normal. Weak (1 plus on a scale of 1-3 plus) linear deposition of IgG was still evident by immunofluorescence. Pulmonary evaluation, including bronchoscopy, showed no evidence of recurrent bleeding. The patient has been followed subsequently for an additional 18 months without further deterioration.

Immunological IgG levels rose abrupty after the first plasma exchange and remained elevated thereafter. After cyclophosphamide treatment was begun, C3 levels rose within the normal range (1 a3-22 mg/ml) but they remained elevated only during the 3 weeks the patient was leukopenic (Fig. 1). Treatment with cyclophosphamide and prednisone did not prevent immunization with KLH (Fig. 2). But, after the first plasma exchanges, KLH antibodies declined and remained suppressed for the duration of the first hospital stay. Although the precipitins for aggregated IgG disappeared prior to the plasma exchanges, a decrease in antiglobulin antibodies as measured by the latex test and immunoadsorbent assay similar to that seen with KLH anti-

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FIG. 2. Circulating antibody levels. At the top, the response to keyhold limpet haemocyanin (KLH). Arrows indicate injections of KLH. The bars show the net quantity ± s.d. of patient globulin which bound to KLH immunoadsorbents after correction for non-specific binding. See text for method. P = Plasma exchange; middle panels, antibody responses to antigens of human IgG; bottom, treatment with cyclophosphamide and prednisone.

bodies occurred following the exchange transfusions (Fig. 2). Antibodies to GBM were not detectable by either assay in sera collected before and after treatment. DISCUSSION A brisk increase in antibody synthesis resulting in prompt return to or overshoot beyond antibody level present prior to treatment usually occurs after exchange transfusion in experimental animals (Bystryn et al., 1970, 1971) or after plasmaphoresis in people (Bowman, Peddle & Anderson, 1968; Powell, 1968; Clarke et al., 1970). The sustained decrease in KLH and antiglobulin levels after plasma exchange in this patient suggests that cyclophosphamide and prednisone suppressed the expected proliferation of antigen-reactive cells and/or their transformation into antibody-producing cells. The mechanism behind this effect has not yet been explained. But it has been suggested that memory cells, stimulated to divide by residual antigen may be more susceptible to the effects of immunosuppressive drugs during cell division (Bystryn et al., 1971). The impact of this treatment on the immunological response which is believed to have caused this patient's disease could not be measured directly because he, like the majority of patients with Goodpasture syndrome (McPhaul & Dixon, 1969) did not have detectable anti-GBM antibody in his serum. However, there appeared to be a close temporal relationship between treatment and clinical improvement in this patient, confirming the recent report that combined plasmaphoresis and immunosuppressive drug treatment may be beneficial in patients with this disease (Lockwood et al., 1975). Moreover, the present report suggests that once remission is induced, modest improvement may continue for some time afterwards and the remission may be maintained by continuous low dose immunosuppressive drug therapy.

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Although physical removal of circulating antibodies combined with antiproliferative drug treatment depresses circulating antibody levels better than either means alone, the apparent effect of this treatment in now two cases of Goodpasture syndrome must be accepted with the recognition that the clinical course in this disease is highly variable. Although Goodpasture syndrome is usually considered to be fatal, sometimes justifying extraordinary measures in its treatment (Nowakowski et al., 1971), spontaneous remissions may occur (Munro, Geddes & Lamb, 1967) and it may be responsive to conventional immunosuppressive therapy (Hayslett, Berte & Kashgarian, 1971; Everett et al., 1971 and Strauch et al., 1974). Indeed in the present patient considerable improvement began after cyclophosphamide was added to the treatment programme. Nevertheless, these observations appear to warrant continued trial of plasma exchange as an adjunct to immunosuppressive drug treatment in other patients with antibody-mediated hypersensitivity diseases. Supported by NIH grants numbers RR 00350, HE 05435, AM-1755 and 72-2531 and the Veterans Administration Hospital Houston, Texas 77031, U.S.A. We are grateful for the excellent technical assistance of C. Schimbor, R. Rickaway, and A. Solomon, and for the secretarial assistance of E. Bowers. REFERENCES moval of specific antibody by means of immunoAGNELLO, V., KOFFLER, D., EISENBERG, J.W., adsorption. J. exp. Med. 130,1175. WINCHESTER, R.J. & KUNKEL, H.G. (1971) Clq precipitins in the sera of patients with systemic HAYSLETi, J.P., BERTE, J.B. & KASHGARIAN, M. (1971) Successful treatment of renal failure in lupus erythematosus and other hypocomplementGoodpasture's Syndrome. Arch. intern. Med. emic states: Characterization of high and low 127,953. molecular weight types. J. exp. Med. 134, suppleLOCKWOOD, C.M., BOULTON-JONES, J.M., LOWENment 228. THAL, R.M., SIMPSON, I.J., PETERS, D.K. & BOWMAN, J.M., PEDDLE, L.J. & ANDERSON, C. (1968) WILSON, C.B. (1975) Recovery from GoodPlasmapheresis in severe Rh iso-immunization. pasture's syndrome after immunosuppressive Vox Sang. (Basel), 15,272. treatment and plasmapheresis. Brit. med. J. i, 252. BUCKNER, C.D., CLIFT, R. & THOMAS, E.D. (1969) Plasma exchange with NCI-IBM blood cell separa- MCPHAUL, J.J. & DIXON, F.J. (1969) The presence of anti-glomerular basement membrane antibodies tor. Rev. Franc ttudes clin. Biol. XIV, 803. in peripheral blood. J. Immunol. 103, 1168. BYSTRYN, J.C., GRAF, M.W. & UHR, J.W. (1970) Regulation of antibody formation by serum anti- MuNRO, J.F., GEDDES, A.M. & LAMB, W.L. (1967) Goodpasture's Syndrome: survival after acute body. II. Removal of specific antibody by means renal failure. Brit. med. J. ii, 95. of exchange transfusion. J. exp. Med. 132,1279. BYSTRYN, J.C., SCHENKEIN, I. & UHR, J.W. (1971) A NOWAKOWSKI, A., GROVE, R.B., KING, JR., L.H., ANTONOVYCH, T.T., FURTUNER, R.W., KNEISER, model for regulation of antibody synthesis by M.R., CARTER, C.B. & KNEPSHIELD, J.H. (1971) serum antibody, Progress in Immunology, volume Goodpasture's syndrome: recovery from severe 1 (ed. by B. Amos), p. 627. Academic Press, New pulmonary hemorrhage after bilaterial nephrecYork. tomy. Ann. intern. Med. 75,243. CARPENTER, R.R. & REISBERG, M.A. (1968) Cardodiimide-induced bentonite-antigen complexes: POWELL, L.C. JR. (1968) Intense plasmapheresis in the pregnant Rh-sensitized woman. Amer. J. readily prepared immunoadsorbents. J. Immunol. Obstet. Gynaec. 101, 153. 100,873. CLARKE, C.A., BRADLEY, J., ELSON, C.J., DONAHOE, RosSEN, R.D., REISBERG, M.A., SHARP, J.T., SUKI, W.N., SCHLOEDER, F.X., HILL, L.L. & EKNOYAN, W.T.A., LEHANE, D. & HUGHES-JONES, N.C. G. (1975) Antiglobulins and glomerulonephritis: (1970) Intensive plasmapheresis as a therapeutic classification of patients by the reactivity of their measure in Rhesus immunized women. Lancet, i, sera and renal tissue with aggregated and native 793. human IgG. J. clin. Invest. 56,427. CURTIS, J.E., HERSH, E.M., BUTLER, W.T. & RosSEN, R.D. (1971) Antigen dose in the human immune SINGER, J.M. & PLOTZ, G.M. (1956) The latex fixation test. I. Application to the serologic diagresponse: dose-reponse relationships in the human nosis of rheumatoid arthritis. Amer. J. Med. 21,888. immune response to keyhole limpet hemocyanin. STRAUCH, B.S., CHARNEY, A., DOCTOROUFF, S. & J. Lab. clin. Med. 78, 61. KASHGARIAN, M. (1974) Goodpasture's syndrome EVERETT, E.D., NEWCOMER, K.L., ANDERSON, J., with recovery after renal failure. J. Amer. med. BERGIN, J. & OVERHOLT, E.A. (1971) GoodAssoc. 229,444. pasture's Syndrome. Response to mercaptopurine UHR, J.W. & MOLLER, G. (1968) Regulatory effect of and prednisone. J. Amer. med. Assoc. 213,1849. antibody on the immune response. Advanc. GRAF, M.W. & UHR, J.W. (1969) Regulation of Immunol. 8, 81. antibody formation by serum antibody. I. Re-