HCV, have been correlated with 'idiopathic' pulmonary fibrosis. ... yr) with interstitial lung fibrosis and a variable number of rheumatic disorders are described.
British Journal of Rheumatology 1997;36:360–365
INTERSTITIAL LUNG FIBROSIS AND RHEUMATIC DISORDERS IN PATIENTS WITH HEPATITIS C VIRUS INFECTION C. FERRI, L. LA CIVITA, P. FAZZI,* S. SOLFANELLI,* F. LOMBARDINI, E. BEGLIOMINI,* M. MONTI,† G. LONGOMBARDO, G. PASERO and A. L. ZIGNEGO† Rheumatology Unit and *Respiratory Disease Unit, University of Pisa, Pisa and †Institute of Internal Medicine, University of Florence, Florence, Italy SUMMARY A possible aetiopathogenetic role of hepatitis C virus (HCV) has been reported in various immune-mediated disorders, such as mixed cryoglobulinaemia, which may be complicated by interstitial lung involvement; moreover, different viruses, including HCV, have been correlated with ‘idiopathic’ pulmonary fibrosis. Here, a cohort of eight HCV-positive patients (M/F = 4/4, mean age 61 2 8 .. yr) with interstitial lung fibrosis and a variable number of rheumatic disorders are described. Interstitial lung involvement appeared medially 4.5 2 3.2 .. yr after the clinical onset of chronic hepatitis. During the clinical follow-up, some rheumatic symptoms were also recorded: articular involvement (four patients); mild sicca syndrome (one patient); severe polymyositis and cranial neuropathy (one patient); serum cryoglobulins and/or autoantibodies (eight patients). In all patients, a moderate (four patients) or severe (four patients) lung fibrosis was evaluated by means of high-resolution computed tomography. The presence of parenchymal radiotracer uptake on 67Ga scan (7/7 patients) and increased percentages of neutrophils (4/4 patients) and lymphocytes (2/4) at bronchoalveolar lavage suggested an active lung involvement. Different degrees of reduction of single breath diffusing capacity for carbon monoxide (DLco) (mean value 57.6 2 15%, range 37–80) were observed in all cases, while spirometric abnormalities, consistent with a global restrictive pattern, were less frequently found. In all cases, anti-HCV antibodies and HCV viraemia were demonstrated; viral genome was also detected in peripheral lymphocytes from 4/4 subjects and in one case in lung biopsy specimens. A desquamative interstitial pneumonia pattern was demonstrated in two cases by lung biopsy. The present work supports the hypothesis that HCV chronic infection could represent a trigger factor for interstitial lung fibrosis and various rheumatic disorders. K : Lung fibrosis, Rheumatic diseases, Hepatitis C virus, Chronic hepatitis.
A its identification in 1989, hepatitis C virus (HCV) represents the major aetiologic factor in non-A, non-B chronic hepatitis worldwide [1]. HCV, being both a hepato- and lymphotropic virus, can represent a chronic stimulus for the immune system [2–5]. The HCV lymphotropism could explain the striking association between this virus and some rheumatic disorders, such as mixed cryoglobulinaemia [2, 4–6]. This is a systemic vasculitis characterized by a variable combination of clinical symptoms, more frequently chronic hepatitis, glomerulonephritis and peripheral neuropathy [6–8]; an interstitial lung involvement, generally asymptomatic, has also been described in these patients [9, 10]. Tissue injury in HCV-related mixed cryoglobulinaemia is probably due to the vascular deposition of circulating HCV-containing immune complexes [6, 11]. Similarly, in the general population of HCV-positive patients, the appearance of various organ involvement can be related to different immunological factor(s), namely various autoantibodies and immune complex production secondary to B-lymphocyte expansion [7, 8]. Among the systemic manifestations of HCV chronic infection, lung involvement has been described in only anecdotal case reports [12, 13]. Here, we describe a series of patients
with HCV infection and clinical evidence of interstitial lung disease and different rheumatic manifestations. PATIENTS AND METHODS A cohort of eight HCV-positive patients with interstitial lung involvement were consecutively recruited for the study between 1992 and 1995. These subjects belong to our whole population of 300 patients with clinically evident HCV infection, namely liver involvement and/or extrahepatic manifestations, screened for the presence of lung involvement by means of clinical symptoms and two-projection chest X-rays. The eight subjects (M/F = 4/4, mean age 61 2 8 .. yr) were Italian-born heterosexuals and had no history of drug or alcohol abuse, or known exposure to dusts, extrinsic allergens or fibrosis-inducing treatments. In no cases were episodes of asthma referred, while 5/8 patients had a history of smoking (patients 1, 2, 3, 4, 8; Table I), and only one had previous blood transfusions (patient 2). Routine blood chemistry, involvement of various organs, and associated immunological and rheumatic disorders were evaluated as previously described [2, 8, 9]. Lung involvement In all patients, interstitial lung involvement was evaluated by chest X-ray and high-resolution computed tomography (HRCT). HRCT scans were evaluated, respectively and independently, by two
Submitted 29 April 1996; revised version accepted 30 July 1996. Correspondence to: C. Ferri, Istituto Patologia Medica I, University of Pisa, Via Roma 67, 56100 Pisa, Italy.
= 1997 British Society for Rheumatology 360
FERRI ET AL.: HCV, LUNG FIBROSIS AND RHEUMATIC DISORDERS
trained chest physicians according to the previously described method [14]. In particular, the presence and the extent of nodules, air-space consolidation and parenchymal distortion, septal and non-septal lines, ground-glass and honeycombing areas were assessed in both lung fields, after subdivision into upper, middle and lower zones. In relation to the percentage of lung parenchyma with evidence of the above findings, a profusion score (1–4) was given and the scores of each zone were then summed to obtain a global profusion score for HRCT ranging from 0 to 12 [14]. The grading criteria of the interstitial lung fibrosis at HRCT were: mild fibrosis (score 1–4), moderate (score 5–8), severe (score 9–12). The patients underwent pulmonary function tests (PFTs) using the American Thoracic Society criteria [15]. Spirometry with HP 47804A included vital capacity (VC), functional residual capacity (FRC), total lung capacity (TLC), flow volume curves [forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), FEV1/FVC, etc.]. The single breath diffusing capacity for carbon monoxide (DLco) was performed with a Morgan Resparameter MK4. The results of spirometry and diffusion indexes expressed as a per cent were compared with the predicted values by Goldman et al., Paoletti et al. and Cotes et al., respectively [16–18]. A reduction of static (VC, FRC and TLC) and/or dynamic (FVC, FEV1 and FEV1/ FVC) indices defined a restrictive, an obstructive or a mixed ventilatory pattern, respectively. The reduction of DLco was considered mild for values within 61 and 80%, moderate within 41 and 60%, and severe at E40%. Samples for blood gas analysis were taken through the radial artery while the patients were breathing room air in a sitting position and were immediately analysed by a pH/blood gas analyser IL 1312, Instrumentation Laboratory. A gallium 67 (67Ga) thoracic scan was performed at 120 h after i.v. administration of 5 mCi of radiotracer. The quantitative pulmonary 67Ga uptake was evaluated by Line’s method [19] modified by adding the hilar (0–100) to the parenchymal uptake score (0–400); the resulting pulmonary 67Ga uptake total score was 0–500 [20]. In 4/8 subjects, bronchoalveolar lavage (BAL) was also carried out by instilling 100 ml of sterile 0.9% normal saline solution into the right middle lobe or in the lingula (mean recovery 57 2 9%). Total and differential cell counts in the fluid were studied by haemocytometer and Giemsa staining [21]. Normal values for BAL were: neutrophils E1% (non-smokers) and E5% (smokers); lymphocytes E12%. Finally, thoracoscopic lung biopsy was performed in patients 1 and 5. Immunological studies The presence of anti-nuclear (ANA), anti-smooth muscle (ASMA) and anti-mitochondrial (AMA) autoantibodies was detected by current techniques [8]. Sera with a titre q1:40 were considered positive. Anti-extractable nuclear antigen (ENA) antibodies, including anti-Scl70, -Sm, -RNP, -SSA/SSB, -PCNA,
361
-SL and -Jo1 specificities, were detected by counterimmunoelectrophoresis according to Bunn et al. [22]. Serum rheumatoid factor (RF), cryocrit and cryoglobulin composition, haemolytic complement activity (CH50) and C3–C4 fractions were measured as previously described [2, 8, 9]. Virological studies Antibodies against HCV (anti-HCV), Epstein–Barr virus (anti-EBV) and human immunodeficiency virus (anti-HIV), and hepatitis B virus markers (anti-HBs, -HBc, -HBe, HBsAg) were detected on serum clotted and centrifuged at 37°C and stored at −80°C using commercially available kits (Chiron ELISA and RIBA HCV, Second Generation, Emeryville, CA, USA; Hepanostika, Vironostika anti-HTLV III, Organon Teknica, Boxtel, The Netherlands; Du Pont EBV VCA-IgM, -IgG, ELISA, E. I. Du Pont de Nemours & Co. Inc., Wilmington, DE, USA). In the same blood samples (patients 1, 3, 4, 8), HCV infection of peripheral blood mononuclear cells (PBMC) was also investigated as described [2]. Total RNA extraction was carried out from 150 ml of sera, PBMC (patients 1, 3, 4, 8) or lung biopsy (patient 1) using a modified guanidium isothiocyanate method (RNAzol B, Bioprobe Systems) [2,23,26]. HCV RNA sequences were determined by means of a ‘one-tube nested’ polymerase chain reaction (PCR) assay using primers corresponding to the wellconserved 5' non-coding region of the HCV genome as described [2, 24–26]. RESULTS In 7/8 patients, the presence of chronic active hepatitis was diagnosed on the basis of a constant increase in liver enzymes and histological alterations from 1 to 17 yr before the detection of lung involvement (Table I). A number of rheumatic or immunological symptoms were recorded during the clinical follow-up in 7/8 patients: articular involvement was present in four (patients 1, 2, 6, 7; Table I); mild sicca syndrome in one (patient 7) and severe polymyositis associated with cranial neuropathy (second branch of the left trigeminal nerve; [13]) in another one (patient 3); type II (IgG-IgMk) mixed cryoglobulinaemia was detected in two (patients 4, 8), and in one case only as trace amounts (patient 7); the presence of serum RF and haemolytic complement reduction were observed in 6/8 (patients 1, 2, 4, 5, 6, 8) and 3/8 (patients 4, 6, 8) patients, respectively; finally, circulating ANA were detected in 4/8 subjects (homogeneous pattern in patients 1, 2, 3; speckled in patient 8). In 7/8 patients, the presence of lung involvement was suspected on the basis of dyspnoea with dry cough or digital clubbing (Table II); respiratory symptoms appeared medially 4.5 2 3.2 .. yr after the clinical onset of chronic hepatitis; in only one case (patient 6) was lung involvement occasionally found at chest X-ray. The interstitial lung fibrosis was quantified by means of the global profusion score at HRCT as moderate (four patients) or severe (four
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 3 TABLE I Clinical, serological and virological findings of HCV-positive patients with lung involvement Immunological findings
Patient no.
CH Lung Age and duration duration sex (yr) (yr)
1 2 3
68M 65F 47M
22 13 4
5 4 2
4 5 6 7
66M 58F 49M 66F
6 11 – 20
5 7 6 19
8
66F
9
2
HCV HCV RNA
Rheumatic symptoms non-erosive arthritis erosive arthritis polymyositis, Raynaud’s cranial neuritis mixed cryoglobulinaemia none polyarthralgias sicca syndrome polyarthralgias‡ mixed cryoglobulinaemia
Low complement† Anti-HCV
ANA
RF
Serum
PBMC
+ + +
+ + −
− − −
+ + +
+ + +
+/+* nd +/+
− − − −
+ + + −
+ − + −
+ + + +
+ + + +
+/+ nd nd nd
+
+
+
+
+
+/+
CH, chronic hepatitis; ANA, antinuclear antibodies; RF, rheumatoid factor; PBMC, peripheral blood mononuclear cells, fresh/cultured; HCV, hepatitis C virus; nd, not done. *Presence of HCV RNA also in lung biopsy specimens. †Haemolytic complement activity. ‡Trace amounts of cryoglobulins in the serum.
patients). Spirometric parameter reduction, consistent with restrictive syndrome, was less frequently found, while different degrees of DLco reduction (mean value 57.6 2 15%, range 37–80) were observed in all. The severity of lung fibrosis, expressed by the global profusion score for HRCT, correlated with the reduction of DLco (r = 0.8; P Q 0.03). Different degrees of active lung involvement were detected in 7/7 patients by parenchymal 67Ga uptake. BAL showed an increased percentage of neutrophils in 4/4 patients (light in patients 5 and 6), and a mild increase of lymphocytes in two (Table II). Lung biopsy showing a
desquamative interstitial pneumonia pattern was carried out in two subjects (patients 1 and 5). Severe reduction of pO2 at blood gas was present in patient 2. In this subject, lung involvement progressed rapidly to respiratory failure and death in spite of the therapeutic attempts (steroids, alpha-interferon) in 1 yr follow-up (Fig. 1). Lung involvement remained stable in five patients (patients 3, 4, 5, 6, 7) and slowly worsened in the other two (patients 1, 8) over a mean follow-up of 4.9 2 5.6 .. yr. In three cases (patients 1, 2, 3), alpha-interferon treatment (9 MU/week for 1 month,
TABLE II Lung involvement in eight HCV-positive patients Patient no. 1
Symptoms Fibrosis VC and signs score* (%)
FVC FEV1 FRC (%) (%) (%)
TLC (%)
DLco (%)
dyspnoea clubbing dyspnoea cough
10†
114
92
98
61
90
37
12
49
50
61
66
60
nd
3
dyspnoea clubbing
5
82
75
81
85
78
65
4
9
72
67
81
84
84
40
9†
69
52
61
72
64
58
6
dyspnoea clubbing dyspnoea cough none
5
94
78
74
97
94
80
7
dyspnoea
5
83
66
58
91
92
65
8
dyspnoea cough
5
100
80
83
86
100
58
2
5
BAL Ga scan uptake score T ( × 106 ) M% L% 67
parenchymal 300 nd hilar– parenchymal 100 parenchymal 120 parenchymal 200 parenchymal 100 hilar– parenchymal 50 hilar– parenchymal 175
Clinical follow-up
N%
E%
nd
5.2 slow progression 0.7 respiratory failure death stable
nd
stable
9
73
1.8
20
12.7
70.4
3.4
25.5
10.6
78
12.4
6.8
3.0 stable
16.5
74.6
15.8
6.7
2.9 stable
nd
stable
nd
slow progression
VC, vital capacity (% of predicted); FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; FRC, functional residual capacity; TLC, total lung capacity; DLco, single-breath lung diffusing capacity; 67Ga scan score, see the text; BAL, bronchoalveolar lavage (T, total cells; M, macrophages; L, lymphocytes; N, neutrophils; E, eosinophils); nd, not done. *Fibrosis score (1–4: mild; 5–8: moderate; 9–12: severe) is referred to the global profusion score of high-resolution computed tomography. †Histological pattern of desquamative interstitial pneumonia.
FERRI ET AL.: HCV, LUNG FIBROSIS AND RHEUMATIC DISORDERS
(a)
363
(b)
F. 1.—PA chest X-ray (a) and one HRCT thoracic section (b) of patient no. 2. (a) Chest X-ray shows a diffuse interstitial involvement prevalent in the left lung. (b) HRCT scan demonstrates cystic spaces and patchy areas of ground-glass attenuation in a predominantly left posterior distribution.
then 6 MU/week) was carried out for a period varying from 1 to 8 months. This treatment was scarcely useful for liver involvement and ineffective for dyspnoea; moreover, in patient 3 a marked worsening of the polymyositis and left trigeminal neuropathy were also recorded during alpha-interferon therapy. Virological studies demonstrated the presence of serum anti-HCV and HCV RNA in all patients; moreover, in 4/4 subjects an ongoing viral infection was also detected in peripheral lymphocytes by the PCR technique (Table I). Finally, in patient 1, HCV genomic sequences were also demonstrated in lung biopsy specimens. On the contrary, other viral markers, namely HBsAg, anti-HBV, anti-EBV, and anti-HIV antibodies, were never detected. DISCUSSION The present work suggests that interstitial lung fibrosis could represent another extrahepatic manifestation in patients with chronic HCV infection [27–29]. Since only patients with clinically evident lung involvement were included in the study, it is likely that the actual prevalence of such a complication is underestimated. In this respect, the detection of subclinical alveolitis in a surprisingly high number of individuals with HCV-related mixed cryoglobulinaemia is of interest [10]. The presence of HCV infection in some rheumatic or immunological disorders has been demonstrated [27–28], in particular mixed cryoglobulinaemia could represent a model of virus-induced systemic vasculitis [6, 8, 27–29]. The association between HCV, pulmonary fibrosis and anti-Jo-1 antibody-positive polymyositis has been recently described in a case report, suggesting a possible role of HCV in the pathogenesis of both lung and muscle involvement [12]. In patients with polymyositis, serum anti-Jo-1 antibody is considered a predictive marker for complicating interstitial lung fibrosis [30]. In our patient (no. 3) with liver, nerve and
muscle involvement, HCV could be the trigger factor for a systemic immunological disorder including lung fibrosis [13]. A large body of clinical investigations pointed out the possible role of viral infections in different rheumatic or immune-mediated disorders [31], among which is polymyositis [12, 13, 32], as well as in ‘idiopathic’ pulmonary fibrosis [33, 34]. This latter possibility seems to be reinforced by the detection of anti-HCV antibodies in 29% of ‘idiopathic’ pulmonary fibrosis, using the first-generation tests [35]. In ‘idiopathic’ interstitial lung disease, which includes the usual interstitial pneumonia and desquamative interstitial pneumonia, the possible detection of serological abnormalities, namely ANA or rheumatoid factor, had suggested the presence of a subclinical immunological disorder [34]. In this light, the appearance of interstitial lung involvement during the clinical course of some rheumatic diseases is not surprising [9, 12, 13, 30, 34]; on the other hand, the possible causative role of viral agents has been suggested in the same disorders [33, 34]. Taken together, all the above considerations indirectly support the hypothesis of a pathogenetic role for HCV in both pulmonary and rheumatic manifestations observed in our patients. This hypothesis is mainly based on the lymphotropism of HCV [2–5] which can be responsible for a chronic activation of both T and B lymphocytes, leading to a complex immune system disorder. In particular, in HCVpositive patients with mixed cryoglobulinaemia or autoimmune hepatitis, B-cell proliferation with a variety of autoantibodies, among which is the rheumatoid factor, and immune complex production has been demonstrated [6, 37–39]. HCV genomic sequences and/or HCV-related antigens can be directly involved in the composition of cryoprecipitable immune complexes, which are responsible for the inflammatory tissue alterations of mixed cryoglobulinaemia [11]. Independently of the aetiologic agents, various serum autoantibodies, circulating and/or in situ
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 3
immune-complex formation [11, 6, 37] can be important pathogenetic factors for different organ damage, including lung involvement. In our patients, the presence of ongoing HCV replication in both fresh and mitogen-stimulated B lymphocytes could represent the ‘remote’ event leading to a complex immunological disorder [2, 4, 23, 26]. In addition, the presence of HCV genome in circulating cryoglobulins [11] and in lung biopsy specimens of one patient of the present series (patient 1) could suggest a direct involvement of this virus in the tissue damage. In three patients, the ineffectiveness of alpha-interferon, an antiviral drug, is difficult to explain. It is possible that interferon was unable to improve lung involvement because of the severity of fibrosis in two cases (patients 1, 2), while it could be responsible for additional damage to the lung in patient 3 [40]. In conclusion, HCV seems to be involved in the pathogenesis of lung fibrosis in a number of chronically infected subjects. Further studies are necessary to ascertain the exact role of this virus in lung fibrosis and the possible contribution of other environmental and/or infectious agents, and not secondarily a genetically driven host reactivity. Finally, because of the increasing diffusion of HCV infection worldwide, prospective clinical studies are necessary to confirm the present observations as well as the actual prevalence of HCV infection in ‘idiopathic’ interstitial lung disease. A This work was in part supported by the National Research Council (CNR) Targeted Project ‘Prevention and Control Disease Factors’, Sub project ‘SP3’, Contract No. 91.00168; Progetto epatite virale, I.S.S; and Associazione Italiana Ricerca Cancro.
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