266 17. Schumacher JH, O'Garra A, Shrader B, et al. The characterization of four monoclonal antibodies specific for mouse IL-S and development of mouse and human IL-S enzyme-linked immunosorbent. J Immunol 1988; 141:1576-81. 18. Coffman RL, Seymour BWP, Hudak S, Jackson J, Rennick D. Antibody to interleukin-S inhibits helminth-induced eosinophilia in mice. Science 1989; 245:308-10.

19. Giles KW, Myer A. An improved dipheny-

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lamine method for the estimation of deoxyribonucleic acid. Nature 1%5; 206:93. 20. Strath M, Warren OJ, Sanderson CJ. Detection of eosinophils using an eosinophil peroxidase assay. Its use as an assay for eosinophil differentiating factors. J Immunol Methods 1985; 83: 209-15.

21. Yamaguchi Y, Suda T, Suda J, et al. Purified interleukin 5 supports the terminal differentiation and proliferation of murine eosinophilic precur-

sors. J Exp Med 1988; 167:43-56. 22. Wang JM, Rambaldi A, Biondi A, et al. Recombinant human interleukin 5 is a selectiveeosinophil chemoattraetant. Eur J Immuno11989; 19: 701-5.

23. Johnson HG, Stout BK. Ascaris suum ovainduced bronchoconstriction, eosinophilia and 19E antibody responses in experimentally infected primates did not lead to histamine hyperreactivity. Am Rev Respir Dis 1989; 139:710-4.

Idiopathic Pulmonary Fibrosis and High Prevalence of Serum Antibodies to Hepatitis C Virus 1 •2

TAKASHI UEDA, KEN OHTA, NAOHITO SUZUKI, MASAO YAMAGUCHI, KOICHI HIRAI, TADASHI HORIUCHI, JUNNOSUKE WATANABE, TERUMASA MIYAMOTO, and KOJI ITO

In 1944, Hamman and Rich (1) predicted the involvement of viral infection in the pathogenesis of idiopathic pulmonary fibrosis (lPF). Since then, several investigators have reported various observations supporting or denying the involvement of viruses in IPF (2-5). We think the relationship between viruses and IPF should be studied more systematically and thoroughly despite the accumulated reports in the past. As the first step, we have studied serum antibody titers in 98 patients with IPF against 33 various viruses and were not able to find any viruses showing significantly high prevalence in IPF (6). Hepatitis C virus (HCV) is the newest virus discovered by Choo and coworkers (7) in 1989, and it wasnot included in our last study. Because HCV is known to cause fibrotic changes in the liver as a part of pathologic manifestations, we studied the association between IPF and HCV. The prevalence of serum antibodies to HCV was assessed by using the Ortho/Chiron enzyme-linked immunosorbent assay (ELISA) system (8), detecting antibody against Cl00-3 antigen of HCV. Additionally, we employed the Chiron recombinant immunoblotting assay (RIBA) system (9) to confirm the results obtained from the ELISA system. We report here the results of the high prevalence of anti-HCV antibodies in the sera of patients with IPF.

Serum samples from 66 patients (46 male and 20 female) with IPF werecollected for this study from the public hospitals in Japan in 1988 without any intentional selection. In these patients clinical diagnoses were made by well-trained pulmonologists using the following criteria: (1) symptoms of progressive dyspnea and cough, (2) audible fine crackles, (3) chest radiographs that show diffuse reticular and reticulonodular appearances, (4) pulmonary function tests that show restrictive patterns with a decrease in diffusing capacity, and (5) no manifestations compatible with other interstitial

SUMMARY The prwalence of serum antibodies to hepatitis C virus (HCV) was assessed by an enzyme-linked Immunosorbent allll8Y (ELISA) In 66 patients (46 male and 20 female; mean age ± SEM, 61.5 ± 10.1 yr) with Idiopathic pulmonary fibrosis (IPF). Nineteen (28.8%) were positive for this test. The frequency of HCV positiveness was significantly higher In the patients with IPF than In the 9,464 control sUbJects,whose eges ware comparable with those of the patients (3.88%, p < 0.05). Importantly, 12 of the 19 patients with IPF and positive ELISA results (83.2%) had positive results on the Chi ron recombinant Immunoblottlng allll8Y (RIBA), which Is known to be more specific for HCV. We Judged the 12 perceptible reactions as eight reactive and four Indetermlnats. When wa examined liver function retrospectively, only two of eight patients who tested positive for the HCV haclilver dysfunction, suggesting that antl·HCV positivity In IPF was not observed as 8 result of liver dl...... These results lead us to speculate thst HCV Infection may play an Important role In the pathogenesis of IPF,or that the sera of patients with IPF may contain some antibody sgslnst an unknown epltope and cross·react with the antl·HCV allll8Y. AM REV RESPIR DIS 1l1l12; 148:268-268

lung diseases such as collagen vascular disease, sarcoidosis, pneumoconiosis, and hypersensitivity pneumonitis. At presentation, the ages of the patients ranged from 32 to 82 yr (mean ± SEM, 61.5 ± 10.1). In 20 of the 66 patients (30.3010) IPF was proved histologically by means of transbronchial or open lung biopsies. Forty of the 66 patients were treated with corticosteroids, and IS of the 20 patients with lung biopsies underwent similar therapy with corticosteroids. Moreover, we received the medical histories of 33 patients regarding transfusion of blood products, parameters relating to liver function such as hepatitis B virus surface antigen and antibody, serum IgG levels, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The control subjects were 9,464 normal healthy volunteers from all over Japan, without anemia and histories of hepatitis, who intended to be blood donors. Their ages range from 55 to 64 yr, comparable with the ages of the patients with IPF in this study. The patients' serum samples were tested using the Ortho/Chiron HCV antibody ELISA test system purchased from Chiron (Emerville, CA). Yellow-coloredend-products were read by a microwell reader at 490 ± 2 nm. After calculation of negative control mean (NCx), using 210 negative control samples, the cutoff value was established

at an optical density of NCx + 0.400 by following the manufacturer's instructions. This is 6 SO above the mean value of the negative control values. Weconfirmed serum positiveness to HCV by applying the positive sera to the Chiron RIBA system, which has been shown to be highly specific for HCV. The results were assessed by following the classification provided with the test kit, i.e.,sera reacting with two or more HCV recombinant antigens as reactive and with only one antigen as indeterminate. Sera reacting to superoxide dismutase (SOD) alone or to no antigen are classified as nonreactive. The chi-square test was used for statistical analysis.

(Received in original form April 24, 1991 and in revised form February 12, 1992) 1 From the Department of Medicine and Physical Therapy, University of Tokyo School of Medicine, the Japanese Red Cross Central Blood Center, and the National Sagamihara Hospital, Thkyo, Japan. . 1 Correspondence and requests for reprints should be addressed to 'Iakashi Ueda, M.D., Department of Medicine and Physical Therapy, University of Thkyo, School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Thkyo, Japan.

267

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Nineteen ofthe 66 patients with IPF (28.8010) were found to be positive in the anti-HCV ELISA test, whereas 346of 9,464healthy control subjects (only 3.66%) were shown to be positive. Statistically, the prevalence of HCV positiveness was significantly higher in the patients than in the control subjects (p < 0.05). It should be noted that even in limiting the experiment to 20 patients in whom diagnoses were made histologically, the frequency of HCV positivity was 20.0% (four of 20), which is comparable with the one obtained from all 66 patients with IPF and issignificantly higher than that in normal control subjects (p < 0.05), as shown in table I. The sera obtained from the 19patients with IPF positive for HCV-ELISA were retested with the RIBA HCV test. Twelve of them (63.2%) showed perceptible reactions to RIBA and weclassified eight as reactive (42.1%) and four as indeterminate (21.1 %). Three of the four ELISA-positive patients in whom IPF was diagnosed histologically were also positive in the RIBA (two reactives and two indeterminates). Importantly, no sera reacted with SOD. Among the 33 patients with IPF from whom weretrospectively receivedmedical histories regarding the parameters of liver function, eight werepositive for anti-HCV ELISA. However, as shown in table 2, only two of the eight patients with IPF showed liver dysfunction, and none of the 33 patients had a history of blood transfusion. Patient 4, posi-

tive for HBs antigen and suffering from hepatocellular carcinoma, showed obvious elevation of AST, ALT, and IgG. Patient 6 had a minute elevation of ALT with normal AST and IgG as wellas negative HBs antigen and antibody, although he had extremelyhigh OD value in HCV-ELISA. The parameters relating to liver function in the other six patients were all within normal limits, suggesting that anti-HCV positivity in IPF was not caused by liver disease but could be caused by IPF itself.

••• In the present study, we found a strikingly high prevalence of anti-HCV antibodies in patients with IPF. Nineteen of the 66 (28.8%) were found to be positive for HCV-ELISA, whereas 346of the 9,464 healthy age-matched control subjects (only 3.66%) were positive for HCV; the difference was statistically significant (p < 0.05) (table I). In addition, we also found a significantly high prevalence of anti-HCV antibodies (20%) in 20 patients in whom IPF was histologically diagnosed (p < 0.05) (table 1). It should be emphasized that this is the first observation showing abnormally high prevaIenceof antibodies to a certain virus, namely, the hepatitis C virus, in IPF. The high prevalence of serum antibodies in IPF to Cl00-3 antigen, a polypeptide containing 363 amino acids of HCV, can be interpreted as follows: (1) HCV itself is a pathogen of IPF, (2) antibodies recognizing some

TABLE 1 THE PREVALENCE OF POSITIVE HCV·ELlSA AND RIBA RESULTS IN PATIENTS WITH tPF AND IN NORMAL CONTROL SUBJECTS Total (n) Patients All· Histology ( + ) Control SUbjects 55-64 yr

20

19 4

9,464

346

66

RIBA·Positive

Positive

HCV(+) (n)

RIBA·Reactive

(%)

(n)

(%)

(n)

(%)

28.8 20.0

12 3

18.2 15.0

8 2

12.2 10.0

NEt

3.66

NEt

• Ages ranged from 32 10 82 yr (mean ± SEM,61.5 ± 10.1).

t Not examined. TABLE 2 SUMMARY OF EIGHT HCV·POSITIVE PATIENTS IN HCV-ELISA Patient No. 1 2 3 4 5 6 7 8

Age

(rt)

Sex

79

M

68

M

65 82 69

M M M M

65 65 62

F

M

CS

+

+ +

00 in ELISA 1.542 2.059 0.900 0.627 0.467 Overt 0.460 0.510

HBsAg

+

HBsAb

AST

ALT

(lUlL)

(lUlL)

16 32 19 110 17 30 6 17

6 23 13

50 8 48 9 10

tgG (mgld/) 3,920 3,305 3,210 2,700 1,930 1,517 1,268 1,097

BT

RIBA·

10 N N

10 N R R N

Deflnillon of ebbrevl.lions: CS • treatmentw~h corticosteroid; AST • aspartate aminotransferase; ALT. alanineamlnotranaferue; BT • blood transfusion in Ihe paBI. • Therhub of RIBAwere_ d by following the cfaaaificalion as fo/Iowa: serareactingwith two or moreHeV recombinant antIgens as reactive(R),with onlyone HCVanllgenas Indeterminate (10), and with superoxlde dillmutaaa (SOD)aloneor no antigen 811 rlonreactlve (N). t The 00 value Is greater than 3.000.

epitope that mimics ClOO-3 antigen of HCV are involved in the pathogenesis of IPF, (3) the ELISA is detecting some antibodies against SOD to which ClOO-3 antigen is fused as part of the recombinant DNA technology, and (4) the high prevalence of HCV is accompanied by chance with IPF, and HCV does not playa role in IPF. Van der Poel and coworkers (10) reported that an anti-HCV ELISA ratio (the OD value/cutoff) > 2 was associated with HCY infection. In our study, the percentage of patients showing an anti-HCY ELISA ratio> 2 reached 52.6% (10 of 19 HCY-positive patients). In order to confirm the results obtained from the ELISA, we retested the sera positive for HCY-ELISA by means of the RIBA test system, which is more specific for HCY. It should be noted that 12 of the 19 ELISA-positive samples (63.2%) showed perceptible reactions to RIBA, and the remaining seven samples were found to be nonreactive. Importantly, eight of the 12 RIBApositive patients were defined to be reactive or positive for two or more H CY recombinant antigens. What should be emphasized is that no sera reacted with SOD, suggesting that the third interpretation is unlikely. These observations may suggest that the majority of the patients with positive results for HCY-ELISA in IPF really have serum antibodies against HCV, and that the first or second interpretation is possible, although the fourth one is not excluded. We collected data relating to liver function from 33patients with IPF retrospectively,and found that eight of the 33 were positive for the HCY-ELISA test. It should be noted that only two of these eight were accompanied by liver dysfunction (table 2). This result suggests that anti-HCY positivity in IPF cannot be assumed as a result of ordinary HCY infection. There are two possibilities that can result in the discrepancy between the HCY antibody and liver disease. Firstly, the target for HCY, which was discovered as a pathogen of non-A, non-B viral hepatitis, could be the lung in a certain population of people where HCV may induce pulmonary fibrosis. Secondly, as described in the second interpretation, the antibodies detected in IPF by either the HCY-ELISA or RIBA system are not against real antigen(s) derived from HCY but some epitope that mimics a certain HCV antigen. The epitope could bederived from some other viruses or cellular surface molecules of the lung tissue. We are further studying if the patients who show positive results for the HCY-ELISA system really have HCY itself by performing polymerase chain reaction with the 5'-terminal sequence of the HCY genome, which was newly determined and is known to be its structural genes (ll), as the primer. At any rate, our discovery of the high prevalence of serum antibodies to Cl00-3 antigen will open a new array of approaches to the pathogenesis of IPF. Acknowledgment The writers thank the doctors at the hospitals list-

268 ed below for providing sera from the patients with IPF and Dr. Kusuya Nishioka at the Japanese Red Cross Central Blood Center for helpful comments. List oj hospitals: Department of Internal Medicine, The Research Institute for Tuberculosis and Cancer, Tohoku University; The First Department of Internal Medicine, Shinshu University School of Medicine; The Third Department of Internal Medicine, Sapporo Medical College; The Second Department of Internal Medicine, Hiroshima University School of Medicine; The First Department of Internal Medicine, Nihon University School of Medicine; The First Department of Internal Medicine, Tokyo Women's Medical College; The Second Department of Internal Medicine, Nara Medical University; The Second Department of Internal Medicine, Okayama University School of Medicine; Department of Respiratory Diseases, Tokyo Teishin Hospital; Sapporo Hospital, Hokkaido, Japan Railway Company; National Sanatorium, Nishiniigata Hospital; National Sanatorium, Toneyama Hospital; The First Department of Internal Medicine, Hokkaido University School of Medicine; The Third Department of Internal Medi-

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cine, Tokushima University School of Medicine; Department of Medicine and Clinical Immunology, Chest Disease Research Institute, Kyoto University; The Second Department of Internal Medicine, Nagoya City University Medical School; The First Department of Internal Medicine, Dokkyo University School of Medicine; National Sanatorium, Kinki Chuo Hospital.

References 1. Hamman L, Rich AR. Acute diffuse interstitial fibrosis of the lungs. Bull John Hopkins Hosp 1944; 4:177-212. 2. Liebow AA, Steer A, BillingsleyJG. Desquamative interstitial pneumonia. Am J Med 1965; 39:369-404. 3. Gransler EA, Goff AM, Prowse CM. Desquamative interstitial pneumonia. N Engl J Med 1966; 274:113-26. 4. O'Shea PA, Yardley JH. The Hamman-Rich syndrome in infancy: report of a case with viruslike particles by electron microscopy. Johns Hopkins Med J 1970; 126:320-43.

5. Kawai T, Fujiwara A, Kageyama K. Diffuse interstitial fibrosing pneumonitis and adenovirus infection. Chest 1976; 69:692-4. 6. Ohta K, Kobayashi N, Ishii A, Thkizawa H, Miyamoto T. Serum antibody titers against various viruses in idiopathic interstitial pneumonia. Jpn J Thorac Dis 1989; 27:604-8. 7. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244:359-61. 8. Kuo G, Choo QL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244:362-4. 9. Choo QL, Weiner AJ, Overby LR. Hepatitis C virus: the major causative agent of viral non-A, non-B hepatitis. Br Med Bull 1990; 46:423-41. to. Van der Poel CL, Reesink HW, Schaasberg W, et al. Infectivity of blood seropositive for hepatitis C virus antibodies. Lancet 1990; 335:558-60. u. Okamoto H, Okada S, Sugiyama Y, et al. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990; 60:167-77.

Idiopathic pulmonary fibrosis and high prevalence of serum antibodies to hepatitis C virus.

The prevalence of serum antibodies to hepatitis C virus (HCV) was assessed by an enzyme-linked immunosorbent assay (ELISA) in 66 patients (46 male and...
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