Clin. exp. Immunol. (1992) 87, 352-356
Antibodies to hepatitis C virus in essential mixed cryoglobulinaemia F. DAMMACCO & D. SANSONNO Division of Internal Medicine, Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy
(Acceptedfor publication 8 November 1991)
SUMMARY
Although essential mixed cryoglobulinaemia (EMC) is recognized to be frequently associated with chronic liver disease, aetiology and pathogenesis of liver damage remain unsolved questions. The purpose of this study was to assess the possible causative role of hepatitis C virus (HCV) in the liver impairment occurring in patients with EMC. Twenty-six consecutive EMC patients were evaluated. All patients underwent percutaneous liver biopsy. Anti-HCV antibodies were assayed by ELISA and supported by a recombinant immunoblotting assay (4-RIBA). The prevalence of anti-HCV antibodies in patients with and without chronic active liver disease (CALD) was compared. AntiHCV antibodies were detected in 13 patients (50%) by ELISA and confirmed in 11 of them (42 3%) by 4-RIBA, the remaining two patients being indeterminate in the supportive assay. CALD correlated significantly with anti-HCV antibodies: indeed, 7/11 (63 6%) anti-HCV+ patients showed histological and clinical pictures of CALD, compared with 1/15 (6-6%) anti-HCV- patients (P < 0-0 1). With the exception of the patient who was found to be HBsAg+, no liver tissue expressed hepatitis B virus-related antigens in the hepatocytes. Additional histological findings included discrete lymphoid aggregates in portal tracts, siderosis, fatty changes, hyperplasia of Kupffer cells. It can be concluded that chronic liver damage in EMC is frequently associated with anti-HCV antibodies. Although the cause of EMC remains unknown, this study has obvious implications for clarifying the etiology of associated CALD and further supports the therapeutic use of interferons in this disease. Keywords chronic active liver disease cryoglobulinaemia hepatitis C virus immune complexes rheumatoid factor
markers merely reflecting the epidemiological situation of the infection [6]. Interestingly, more recent studies have suggested a possible association of EMC with chronic Epstein-Barr virus (EBV) infection [7]. The high frequency of liver involvement in EMC is well documented [8,9] and impaired liver function has been reported in approximately two patients out of three [10,11]. Whether cryoglobulins may be promoted by the liver damage itself owing to reticuloendothelial system impairment in the blood clearance of circulating immune complexes [10] remains largely a matter for speculation. The recent discovery and molecular cloning of the genome of a small, chloroform-sensitive agent, referred to as hepatitis C virus (HCV), led to the development of an enzyme-immuno assay detecting anti-HCV antibodies directed to recombinant virus-specific proteins. These antibodies are now regarded as a specific marker of HCV infection [12,13]. The present study was undertaken to assess the prevalence of anti-HCV antibodies in EMC and hence the possible aetiologic role of HCV in the associated liver disease.
INTRODUCTION Essential mixed cryoglobulinaemia (EMC) is a sometimes familial condition of unknown aetiology [1], presenting clinically as purpura, weakness and arthralgias. In addition, kidney involvement and generalized vasculitis are often found [2]. Clinical diagnosis is confirmed by the detection in the serum of cold-insoluble globulins (cryoglobulins). These are composed of at least two types of immunoglobulins, usually IgG and IgM, the latter being often monoclonal and acting as rheumatoid factor (RF). There is much evidence to suggest that cryoglobulins are antigen-antibody complexes, and EMC itself is regarded as a model of immune complex disease [3]. Signs of exposure to hepatitis B virus (HBV) can frequently be demonstrated in EMC [4,5]. The suggestion that HBV is involved in its etiology, however, has not found confirmation in large series of patients, the frequency of HBV-associated Correspondence: Franco Dammacco, MD, Patologia Medica, Policlinico, 70124 Bari, Italy.
352
HCV infection in cryoglobulinaemia
353
Table 1. Clinical and laboratory features of liver disease in 26 patients with essential mixed cryoglobulinaemia Liver biochemical testst Age, sex
Patient no. I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24
40,F 56,M 38,F 37,M 59,F 43,F 53,F 39,M 55,F 42,F 57,F 55,M 60,F 61,F 42,M 41,M 43,F 60,F 54,F 50,M 56,F 48,M 38,M 44,F
25
26 *
Hepatomegaly*
Splenomegaly*
+ -
+ -
-
-
+
+
-
-
+ +
-
+ + + -
+ -
45,F
+
+
58,F
---
-
-
+ -
Bilirubin -
-
-
+ -
-
-
-
-
-
-
-
Above normal but up to
Alkaline SGOT phosphatase
Occurrence of HBV markers HBcAbt -
+ -
+ -
-
-
+
HBsAb+ HBcAb6 --_-
HBsAgt -
_
Anti-HCV antibodies ELISA 4-RIBAt
Liver biopsy
-
R I R R R R R R R R R I R NR NR NR NR NR NR NR NR NR NR NR
-
NR
S
NR
NS-LAPT
+ + + + + + + + + + + + + -
NS-LAPTT NS-LAPT AC NS-LAPT CPH CAH CAH NS-LAPT CAH AC NS-LAPT NS-LAPT CAH NS-LAPT S Normal picture NS-LAPT NS-LAPT NS-LAPT IC NS-LAPT NS-LAPT NS-LAPT NS-LAPT
three-fingerbreath below the costal margin.
t Above normal but lower than three times normal. t R, Reactive; NR, non-reactive; I, indeterminate. ¶ NS-LAPT, non-specific lymphoid aggregates in the portal tracts; AC active cirrhosis; IC, inactive cirrhosis; S, steatosis; AH, chronic active
hepatitis; CPH, chronic persistent hepatitis. PATIENTS AND METHODS
min) in haematocrit tubes at 40C. Purified cryoprecipitates [1] by electrophoretic and immunoelectrophoretic studies using monospecific antisera to gamma, alpha, mu, kappa, and lambda chains. Serum bilirubin, glutamic oxalacetic transaminase (SGOT), and alkaline phosphatase levels were determined by standard procedures. were characterized
Patients
Seventeen
globulins
women and nine men with detectable serum cryowere studied. The average age was 49 years with a
range of 37-61 years (Table 1). The diagnosis of EMC was established in all of them on the basis of the following criteria: (i) a clinical triad of purpura, arthralgias, and weakness (with or without kidney involvement) dominating the clinical picture throughout the course of the disease in most of the patients; (ii) absence of underlying cytomegalovirus (CMV) or EBV infections, of connective tissue or lymphoproliferative disorders, as well as of alcohol and/or drug abuse; (iii) a prolonged follow up (average 4-4 years, range 3-8 years) from the time of onset of their cryoglobulin-related symptoms. After giving their informed consent, all patients underwent liver biopsy.
Antibody tests Antibodies to homologous and heterologous gammaglobulins were looked for by means of the latex agglutination test and the sheep cell agglutination test. Sera were tested for the presence of anti-nuclear (ANA), smooth muscle (SMA) and liver-kidney microsomal (LKM) antibodies at a screening dilution of 1/40 by an indirect immunofluorescence technique on unfixed cryostat sections of mouse liver, kidney, and stomach using an antihuman F(ab')2 FITC conjugate. Details of the methods employed are reported elsewhere [1].
Serology Venous blood samples were collected into pre-warmed tubes after an overnight fast and allowed to clot at 37 C. After centrifugation, sera were incubated at 4 C for 3 days. The cryocrit was evaluated by centrifugation (1400 rev/min for 10
Elisa for anti-HC V antibody Anti-HCV antibody was sought with a commercially available kit (Ortho Diagnostic Systems, Raritan, NJ). Recombinant polypeptides (C22-3, a structural-core-associated protein, and
354
F. Dammacco & D. Sansonno
C200, a non-structural antigen) coated on microtitre wells capture specific antibodies, which are then revealed by a secondary enzyme-labelled monoclonal anti-human IgG. The cut-off was calculated as the mean OD of three negative manufacturer's controls plus 0-4 (OD) units. AlII positive tests were confirmed by retesting in duplicate.
C
2-0 H
CY)
I
Recombinant immunoblotting assay (4-RIBA) Recombinant immunoblotting assay (4-RIBA) is a recently introduced assay designed as a confirmatory test (Ortho Diagnostic Systems). Briefly, the sample is incubated with nitrocellulose strips pre-coated with the recombinant peptides produced by HCV clones: 5-1 - 1, C 100-3, C33c antigens encoded by non-structural regions and C22-3. Since HCV antigens are fused to human superoxide dismutase (SOD) as part of the DNA recombinant technique, SOD is also present as a control. After three washings, the strip is incubated with a secondary enzyme-labelled goat anti-human IgG antibody. The bound antibody is revealed by an appropriate substrate. Samples are reactive if two or more bands (other than SOD) are present, indeterminate if only one is present and non-reactive if all bands are absent. Hepatitis B virus markers and HBV DNA assay All serum samples were also tested for the presence of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies against HBsAg (anti-HBs), HBeAg (anti-HBe) and hepatitis B core antigen (anti-HBc) by commercially available reagents (Abbott, North Chicago, IL). Hepatitis B virus DNA (HBV-DNA) was determined by molecular hybridization on serum spots blotted on nitrocellulose filters as described in detail elsewhere [14].
Inmunohistochemistry Cryostat sections from all biopsies were tested with immunoperoxidase for the presence of HBsAg and HBcAg, using anti-HBs from Sorin (Saluggia, Italy) and anti-HBc from Dako (Santa Barbara, CA) respectively, as described elsewhere [15]. RESULTS The amount of cryoglobulin ranged from 0-75 to 2-8 mg/ml. All cryoprecipitates contained IgM and polyclonal IgG; in addition, the IgM was monoclonal with K light chains in 20 patients (77%) and polyclonal in the remainder. No cryoprecipitate contained IgA, whereas all cryoglobulins tested had RF activity. Overt liver disease was lacking in all patients. However, occult or subclinical liver involvement was detected in a minor proportion of them by clinical and/or biochemical parameters (Table 1). Indeed, liver enlargement was noted by physical examination in five patients (19%/0) and splenomegaly was revealed in four patients (15%). Biochemical liver tests were performed several times throughout the course of the disease in all patients and only minimal abnormalities were detected. Increased levels of SGOT were found in 15%M), of bilirubin in 4%/0, of alkaline phosphatase in 1 1%. Altogether, six patients (23%) had clinical and/or biochemical evidence of liver involvement, and three of them were anti-HCV positive. Serum samples from all patients were tested for hepatitis B virus markers and HBV DNA levels. HBeAg and anti-HBe were consistently absent. All but one were HBsAg-, the only HBsAg+ patient being however anti-HCV -. IgG-type anti-HBc
I
J0
ll
-
O*
Cut-off
Il
5
10
20 Cryocrit (0)
30
Fig. 1. Comparison between anti-hepatitis C virus (HCV) antibody by ELISA and cryocrit values in 26 essential mixed cryoglobulinaemia (EMC) patients.
and anti-HBs antibodies were detected in two patients belonging to the HCV- group, and no detectable levels of serum HBV DNA were found in any of the patients. In addition, neither HBcAg in the nuclei nor HBsAg in the cytoplasm, on the plasma membrane or both were revealed when liver biopsy sections were studied by conventional immunohistochemistry. The only exception was again patient 20 (see Table 1) who, in addition to being HBsAg-seropositive, showed a weak positivity for HBsAg in the cytoplasm. Cryoglobulinaemic sera are prone to enhance non-specific binding, thus giving false positive results in solid-phase enzyme immunoassay. To overcome this problem, all sera from this series of patients were studied both in toto and deprived of cryoprecipitates. Concordant results were, however, obtained in the detection of anti-HCV antibodies with samples containing or deprived of cryoproteins both by ELISA and by RIBA. Thirteen patients were positive for anti-HCV antibodies indicating a prevalence of 50%. RIBA confirmation, however, was obtained in 11 patients (42-3%), in that the remaining two were reactive for C22-3 antigen only and were thus classified as indeterminate. Further indirect evidence of the lack of influence of the cryoglobulin levels on anti-HCV detection test is shown in Fig. 1, where no correlation was demonstrable between antiHCV antibodies and cryocrit values. To rule out interference by RF in the test, two samples with high levels of RF activity were treated with increasing amounts of heat-aggregated human IgG. A progressive decrement of RF activity was noted, without significant changes in anti-HCV OD (Fig. 2). The lack of interference by RF was also indirectly demonstrated by comparing its average reciprocal titre in the anti-HCV+ group (875; range, 40-1280) with that of the antiHCV- group (720; range, 80-1280). The liver histological findings are reported in Table 1. A significant correlation was noted between anti-HCV serum positivity and the histological picture of chronic active liver disease (CALD). The anti-HCV+ group showed a frequency of CALD of 63-60/o (7/1 1) compared with 6 7%/, (1/15) in the antiHCV- group (P < 0-01). Reciprocal titres of ANA and SMA of more than 40 were detected in one patient positive in the anti-HCV assay. Liver biopsy showed histological features of chronic active hepatitis (CAH).
HCV infection in cryoglobulinaemia 18
N
II.S
coin
(
°
Br |S
0
0 Heat -aggregated
07q
IgG, (mngml 'I
Fig. 2. Lack of influence of rheumatoid factor (RF) in detection of IgG anti-hepatitis C virus (HCV) antibody in the sera of two essential mixed cryoglobulinaemia (EMC) patients. The addition of increasing amounts of heat-aggregated human IgG results in the progressive decrease of RF titres (El El; 0 0), but with no effect on the anti-HCV activity 0). BSA, Bovin serum albumin. E; 0 (El
Finally, along with pentoxyphylline or indobuphene, 19 patients were receiving 4 mg methyl-prednisolone daily at the time of this study. However, since EMC is a typically chronic disorder, results were not analysed with respect to response/ non-response to treatment.
DISCUSSION This study demonstrates that 42 3'S, of our EMC patients had anti-HCV antibody in their serum. Results offered by the current tests for the detection of circulating anti-HCV antibodies must be accepted with caution when 'sticky' serum samples are tested in solid-phase by first generation ELISA. McFarlane et al. [16] have reported false positives in autoimmune CAH, suggesting that samples taken during its active phases may contain components interfering with the antigen coated on the solid phase. Other factors involved in non-specific reactions are found in patients with serum monoclonal components [17]. False positives have also been demonstrated in samples from particular geographical areas, probably because of a coincident high prevalence of flavivirus and pestivirus infections [1 8], or in low-risk HCV infection populations [19]. In
addition, long-stored samples have been shown to contain factors able to induce non-specific reactions [20]. Finally, false positivity due to RF has been detected in over 60, of patients with a clinical diagnosis of rheumatoid arthritis who did not have risk factors for HCV infection or evidence of liver disease [21]. Introduction of second generation HCV ELISA (C200/ C22-3) provided improved sensitivity in individuals at high risk for HCV infection and improvement in assay specificity when screening low risk populations. These results are validated by an independent, supportive assay represented by the second generation immunoblotting assay (4-RIBA) that, with the additional incorporation of C33c and C22-3 antigens, has been proposed to discriminate infectious from non-infectious ELISA-positive samples [22]. Even though cryoglobulinaemic sera are somewhat 'sticky', the current ELISA proved to be a reliable test for the following reasons: (i) although two out of 13 of our cryoglobulinaemic sera displayed poor repeatibility in this assay, second generation
355
RIBA demonstrated that their positivity could be attributed to C22-3 antigen reactivity; (ii) preliminary data (manuscript in preparation) obtained in the same sera with a novel anti-HCV assay from UBI (United Biochemical, Lake Success, NY), based entirely on synthetic HCV peptides from both the structural and non-structural regions of the HCV genome [23], indicate a very close correlation between the Ortho ELISA and RIBA on one side and the UBI screening assay on the other; (iii) both the cryocrit levels and the RF titres were shown not to act as disturbing factors in the test; (iv) consistent results were obtained in the Ortho assay when whole sera and clear supernatants were separately studied. Final confirmation of the specificity of anti-HCV positivity will obviously rest on the actual demonstration of the virus, i.e. of circulating HCV RNA. Liver involvement in EMC has been known for many years and occasionally regarded as a direct consequence of immune complex-mediated damage [9]. However, the pathogenetic role of the liver in cryoglobulinaemia remains an unsolved question. The present study strongly suggests that HCV infection is associated with the liver damage in our patients, since there was a significant difference in the frequency of CALD between the anti-HCV+ and anti-HCV- groups. Clinical and histological pictures of CALD were found in 7/11 anti-HCV+ patients, compared with only 1 15 anti-HCV patients. In the remaining patients the most frequent histological findings included discrete lymphoid aggregates in portal tracts, fatty changes, siderosis, and hyperplasia of Kupffer cells without clinical and/or biochemical signs of liver involvement. Acute and chronic liver diseases of various aetiologies are sometimes accompanied by mixed cryoglobulinaemia [24,25]. In addition, patients with liver disease and cryoglobulinaemia may exhibit extrahepatic features closely resembling the clinical manifestations of EMC. Thus, it might be argued that at least some of the patients in our series could have had primary liver disease and secondary cryoglobulinaemia, rather than EMC. This, however, seems unlikely, for the following reasons: (i) all patients presented with a distinct clinical syndrome consisting of purpura, arthralgias, weakness, and frequently kidney involvement in the absence of any apparent underlying disorder. Thus, all of them fulfilled currently accepted criteria for a diagnosis of EMC; (ii) although approximately 23Oe of the patients were shown to have clinical and/or biochemical evidence of liver impairment, none of them was suffering from symptoms ascribable to serious liver involvement. These observations are in keeping with earlier results [22], indicating underestimation of the frequency of chronic liver disease based on serological features in HCV low-risk populations; (iii) in spite of the extended follow up of the patients ranging from 3 to 8 years, no major changes were observed in clinical symptoms and/or biochemical abnormalities implying progression of liver disease or modification of the initial diagnosis of EMC. Recently a high frequency of anti-HCV antibodies has been reported in autoimmune (AI)-CAH, although important geographical and/or genetic differences seem to exist among different populations [26]. Of course, this might also apply to EMC patients, in that different factors may be involved in the development of CALD in EMC patients from areas where viral hepatitis is not endemic. In this context, one patient of our series deserves special mention. She was found to have significant titres of ANA and SMA in addition to RF activity, a histological picture of CAH at liver biopsy, anti-HCV seropositivity,
F. Dammacco & D. Sansonno
356
and obviously mixed cryoglobulinaemia. A clinical diagnosis of EMC was entertained, although type-I AI-CAH could also be considered. On the other hand, an etiologic role for HBV infection can be reasonably ruled out in our series. Only one patient was an HBsAg carrier, none had circulating HBV-DNA and in all but one liver biopsy tissues did not express hepatitis B virusassociated antigens. Nonetheless, several recent reports indicate the presence of HBV transcriptionally active genome in livers from HBsAg- chronic liver disease, as detected by the specific polymerase chain reaction [27]. This invites speculation on the possible aetiologic role of HBV in a subgroup of patients in the absence of common serologic markers, although it appears to be a low-frequency event [28] that needs to be assessed in EMC. It is rather difficult to explain this high rate of apparently genuine anti-HCV positivity among our patients with EMC. The overall prevalence of anti-HCV antibodies among blood donors in the Apulia region, where all patients came from, is approximately 2%. Sexual or vertical transmission of HCV seems to be much less common than for HBV [29]. In addition, none of our patients had been given blood or blood products, although most of them had required more frequent hospitalization. Identification of an EMC subgroup associating anti-HCV antibody in the serum obviously supports the therapeutic use of anti-viral drugs such as interferons in these patients [30].
ACKNOWLEDGMENTS The excellent secretarial assistance of Vito lacovizzi is gratefully acknowledged. This study was supported in part by a grant from the Italian Ministry for Universities and Scientific and Technological Research, group 'Liver Cirrhosis', Rome.
REFERENCES I Dammacco F, Bonomo L. Crioglobulinemia e piroglobulinemia. In: Dammacco F. Immunologia in Medicina. Milano: Edi Ermes, 1989:599-619. 2 Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N. Cryoglobulinemia. A clinical and laboratory study. Cryoglobulins with rheumatoid factor activity. Am J Med 1966; 40:837-56. 3 Gorevic DP, Kassab HJ, Levo Y, Kohn R, Meltzer M, Prose P. Frankin EC. Mixed cryoglobulinemia. Clinical aspects and long term follow-up of 40 patients. Am J Med 1980; 69:287-308. 4 Levo Y, Gorevic PD, Kassab HJ, Zucker-Franklin D, Franklin EC. The association between hepatitis B virus and essential mixed cryoglobulinemia. N Engl J Med 1977; 296:1501-4. 5 Galli M, Caredda F, D'Arminio A, Monti G, Messina K, Invernizzi F. Hepatitis B virus and essential mixed cryoglobulinemia. Lancet 1980; i: 1093. 6 Popp JW, Dienstag JL, Wands JR, Bloch KJ. Essential mixed cryoglobulinemia without evidence for hepatitis B virus infection. Ann Intern Med 1980; 92:379-83. 7 Fiorini GF, Sinico RA, Winearls C, Custode P. De Giuli-Morghen. C, D'Amico G. Persistent Epstein-Barr virus infection in patients with type II essential mixed cryoglobulinemia. Clin Immunol Immunopathol 1988; 47:262-9. 8 Levo Y, Gorevic PD, Kassab HJ, Tobias H, Franklin EC. Liver involvement in the syndrome of mixed cryoglobulinemia. Ann Intern Med 1977; 87:287-92. 9 Jori GP, Buonanno G. Chronic hepatitis and cirrhosis of the liver in cryoglobulinemia. Gut 1972; 13:610-3.
10 Zarski JP, Rougier D, Aubert H, Renverseez JC, Cordonnier D, Stroebner P, Rachail M. Association cryoglobuline et maladie hepatique: frequence, nature et caractere immuno-chimique de la cryoglobulinemie. Gastroenterol Clin Biol 1984; 8:845-50. 11 Florin-Christensen A, Roux MEB, Arana RM. Cryoglobulins in acute and chronic liver diseases. Clin Exp Immunol 1974; 16: 599-605. 12 Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244:359-62. 13 Kuo G, Choo Q-L, Alter HY et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244:362-4. 14 Sansonno D, Detomaso P, Papanice MA, Fiore G, Bufano G, Manghisi OG. Correlation between hepatitis B virus deoxyribonucleic acid and receptors for polymerized human albumin in HBV chronic infection. Digestion 1987; 37:206-1 1. 15 Sansonno D, Fiore G, Bufano G, Manghisi OG. Cytoplasmic localization of hepatitis B core antigen in hepatitis B virus infected livers. J Immunol Meth 1988; 109:245-52. 16 McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in autoimmune chronic active hepatitis: pathogenetic factor or false-positive results? Lancet 1990; 335:754-7. 17 Boudart D, Lucas JG, Muller JV, Lecarrer D, Planchon B, Harousseau JL. False positive hepatitis C virus antibody tests in paraproteinemia. Lancet 1990; 336:63. 18 Wong DC, Diwan AR, Rosen L, Gerin JL, Johnson RG, Polito A, Purcell RH. Non-specificity of anti-HCV test for seroepidemiological analysis. Lancet 1990; 336:750-1. 19 Weiner AJ, Truett MA, Rosenblatt J et al. HCV testing in low-risk population. Lancet 1990; 336:695. 20 Desmyter J, Goubau P. False positivity in Ortho anti-hepatitis C virus ELISA upon storage and handling of sera: 1st International Meeting on Hepatitis C Virus. Rome 1989;488 (abstr.). 21 Theilmann L, Blazek M, Goeser T, Gmelin K, Kommerel B, Fiehn W. False positive anti-HCV tests in rheumatoid arthritis. Lancet 1990; 335:1346. 22 Van Der Poel CL, Cuypers HTM, Reesink HW et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991; 337:317-9. 23 Hosein B, Fang CT, Popovsky MA, Ye J, Zhang ML, Wang CY. Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein. Proc Natl Acad Sci USA 1991;
88:3647-51. 24 Dienstag JL, Rhodes AR, Bhan AK, Dvorak AM, Mihm MC Jr, Wands JR. Urticaria associated with acute viral hepatitis type B: studies of pathogenesis. Ann Intern Med 1978; 89:34-9. 25 Jori GP, Buonanno G, D'Onofrio F, Tirelli A, Gonnella F, Gentile S. Incidence and immunochemical features of serum cryoglobulins in chronic liver diseases. Gut 1977; 18:245-9. 26 Lenzi M, Johnson PJ, McFarlane IG et al. Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity. Lancet 1991; 338:277-80. 27 Paterlini P. Gerken G, Nakajima E et al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. N Engl J Med 1990; 323:80-5. 28 Pontisso P. Stenico D, Diodati G et al. HBV-DNA sequences are rarely detected in the liver of patients with HBsAg-negative chronic active liver disease and hepatocellular carcinoma in Italy. Liver 1987; 7:211-5. 29 Ideo G, Bellati G, Pedraglio E, Bottelli R, Donzelli T, Putignano G. Intrafamilial transmission of hepatitis C virus. Lancet 1990; 335:353. 30 Bonomo L, Casato M, Afeltra A, Caccavo D. Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 1987; 83:726-30.
Antibodies to hepatitis C virus in essential mixed cryoglobulinaemia F. DAMMACCO & D. SANSONNO Division of Internal Medicine, Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy
(Acceptedfor publication 8 November 1991)
SUMMARY
Although essential mixed cryoglobulinaemia (EMC) is recognized to be frequently associated with chronic liver disease, aetiology and pathogenesis of liver damage remain unsolved questions. The purpose of this study was to assess the possible causative role of hepatitis C virus (HCV) in the liver impairment occurring in patients with EMC. Twenty-six consecutive EMC patients were evaluated. All patients underwent percutaneous liver biopsy. Anti-HCV antibodies were assayed by ELISA and supported by a recombinant immunoblotting assay (4-RIBA). The prevalence of anti-HCV antibodies in patients with and without chronic active liver disease (CALD) was compared. AntiHCV antibodies were detected in 13 patients (50%) by ELISA and confirmed in 11 of them (42 3%) by 4-RIBA, the remaining two patients being indeterminate in the supportive assay. CALD correlated significantly with anti-HCV antibodies: indeed, 7/11 (63 6%) anti-HCV+ patients showed histological and clinical pictures of CALD, compared with 1/15 (6-6%) anti-HCV- patients (P < 0-0 1). With the exception of the patient who was found to be HBsAg+, no liver tissue expressed hepatitis B virus-related antigens in the hepatocytes. Additional histological findings included discrete lymphoid aggregates in portal tracts, siderosis, fatty changes, hyperplasia of Kupffer cells. It can be concluded that chronic liver damage in EMC is frequently associated with anti-HCV antibodies. Although the cause of EMC remains unknown, this study has obvious implications for clarifying the etiology of associated CALD and further supports the therapeutic use of interferons in this disease. Keywords chronic active liver disease cryoglobulinaemia hepatitis C virus immune complexes rheumatoid factor
markers merely reflecting the epidemiological situation of the infection [6]. Interestingly, more recent studies have suggested a possible association of EMC with chronic Epstein-Barr virus (EBV) infection [7]. The high frequency of liver involvement in EMC is well documented [8,9] and impaired liver function has been reported in approximately two patients out of three [10,11]. Whether cryoglobulins may be promoted by the liver damage itself owing to reticuloendothelial system impairment in the blood clearance of circulating immune complexes [10] remains largely a matter for speculation. The recent discovery and molecular cloning of the genome of a small, chloroform-sensitive agent, referred to as hepatitis C virus (HCV), led to the development of an enzyme-immuno assay detecting anti-HCV antibodies directed to recombinant virus-specific proteins. These antibodies are now regarded as a specific marker of HCV infection [12,13]. The present study was undertaken to assess the prevalence of anti-HCV antibodies in EMC and hence the possible aetiologic role of HCV in the associated liver disease.
INTRODUCTION Essential mixed cryoglobulinaemia (EMC) is a sometimes familial condition of unknown aetiology [1], presenting clinically as purpura, weakness and arthralgias. In addition, kidney involvement and generalized vasculitis are often found [2]. Clinical diagnosis is confirmed by the detection in the serum of cold-insoluble globulins (cryoglobulins). These are composed of at least two types of immunoglobulins, usually IgG and IgM, the latter being often monoclonal and acting as rheumatoid factor (RF). There is much evidence to suggest that cryoglobulins are antigen-antibody complexes, and EMC itself is regarded as a model of immune complex disease [3]. Signs of exposure to hepatitis B virus (HBV) can frequently be demonstrated in EMC [4,5]. The suggestion that HBV is involved in its etiology, however, has not found confirmation in large series of patients, the frequency of HBV-associated Correspondence: Franco Dammacco, MD, Patologia Medica, Policlinico, 70124 Bari, Italy.
352
HCV infection in cryoglobulinaemia
353
Table 1. Clinical and laboratory features of liver disease in 26 patients with essential mixed cryoglobulinaemia Liver biochemical testst Age, sex
Patient no. I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24
40,F 56,M 38,F 37,M 59,F 43,F 53,F 39,M 55,F 42,F 57,F 55,M 60,F 61,F 42,M 41,M 43,F 60,F 54,F 50,M 56,F 48,M 38,M 44,F
25
26 *
Hepatomegaly*
Splenomegaly*
+ -
+ -
-
-
+
+
-
-
+ +
-
+ + + -
+ -
45,F
+
+
58,F
---
-
-
+ -
Bilirubin -
-
-
+ -
-
-
-
-
-
-
-
Above normal but up to
Alkaline SGOT phosphatase
Occurrence of HBV markers HBcAbt -
+ -
+ -
-
-
+
HBsAb+ HBcAb6 --_-
HBsAgt -
_
Anti-HCV antibodies ELISA 4-RIBAt
Liver biopsy
-
R I R R R R R R R R R I R NR NR NR NR NR NR NR NR NR NR NR
-
NR
S
NR
NS-LAPT
+ + + + + + + + + + + + + -
NS-LAPTT NS-LAPT AC NS-LAPT CPH CAH CAH NS-LAPT CAH AC NS-LAPT NS-LAPT CAH NS-LAPT S Normal picture NS-LAPT NS-LAPT NS-LAPT IC NS-LAPT NS-LAPT NS-LAPT NS-LAPT
three-fingerbreath below the costal margin.
t Above normal but lower than three times normal. t R, Reactive; NR, non-reactive; I, indeterminate. ¶ NS-LAPT, non-specific lymphoid aggregates in the portal tracts; AC active cirrhosis; IC, inactive cirrhosis; S, steatosis; AH, chronic active
hepatitis; CPH, chronic persistent hepatitis. PATIENTS AND METHODS
min) in haematocrit tubes at 40C. Purified cryoprecipitates [1] by electrophoretic and immunoelectrophoretic studies using monospecific antisera to gamma, alpha, mu, kappa, and lambda chains. Serum bilirubin, glutamic oxalacetic transaminase (SGOT), and alkaline phosphatase levels were determined by standard procedures. were characterized
Patients
Seventeen
globulins
women and nine men with detectable serum cryowere studied. The average age was 49 years with a
range of 37-61 years (Table 1). The diagnosis of EMC was established in all of them on the basis of the following criteria: (i) a clinical triad of purpura, arthralgias, and weakness (with or without kidney involvement) dominating the clinical picture throughout the course of the disease in most of the patients; (ii) absence of underlying cytomegalovirus (CMV) or EBV infections, of connective tissue or lymphoproliferative disorders, as well as of alcohol and/or drug abuse; (iii) a prolonged follow up (average 4-4 years, range 3-8 years) from the time of onset of their cryoglobulin-related symptoms. After giving their informed consent, all patients underwent liver biopsy.
Antibody tests Antibodies to homologous and heterologous gammaglobulins were looked for by means of the latex agglutination test and the sheep cell agglutination test. Sera were tested for the presence of anti-nuclear (ANA), smooth muscle (SMA) and liver-kidney microsomal (LKM) antibodies at a screening dilution of 1/40 by an indirect immunofluorescence technique on unfixed cryostat sections of mouse liver, kidney, and stomach using an antihuman F(ab')2 FITC conjugate. Details of the methods employed are reported elsewhere [1].
Serology Venous blood samples were collected into pre-warmed tubes after an overnight fast and allowed to clot at 37 C. After centrifugation, sera were incubated at 4 C for 3 days. The cryocrit was evaluated by centrifugation (1400 rev/min for 10
Elisa for anti-HC V antibody Anti-HCV antibody was sought with a commercially available kit (Ortho Diagnostic Systems, Raritan, NJ). Recombinant polypeptides (C22-3, a structural-core-associated protein, and
354
F. Dammacco & D. Sansonno
C200, a non-structural antigen) coated on microtitre wells capture specific antibodies, which are then revealed by a secondary enzyme-labelled monoclonal anti-human IgG. The cut-off was calculated as the mean OD of three negative manufacturer's controls plus 0-4 (OD) units. AlII positive tests were confirmed by retesting in duplicate.
C
2-0 H
CY)
I
Recombinant immunoblotting assay (4-RIBA) Recombinant immunoblotting assay (4-RIBA) is a recently introduced assay designed as a confirmatory test (Ortho Diagnostic Systems). Briefly, the sample is incubated with nitrocellulose strips pre-coated with the recombinant peptides produced by HCV clones: 5-1 - 1, C 100-3, C33c antigens encoded by non-structural regions and C22-3. Since HCV antigens are fused to human superoxide dismutase (SOD) as part of the DNA recombinant technique, SOD is also present as a control. After three washings, the strip is incubated with a secondary enzyme-labelled goat anti-human IgG antibody. The bound antibody is revealed by an appropriate substrate. Samples are reactive if two or more bands (other than SOD) are present, indeterminate if only one is present and non-reactive if all bands are absent. Hepatitis B virus markers and HBV DNA assay All serum samples were also tested for the presence of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies against HBsAg (anti-HBs), HBeAg (anti-HBe) and hepatitis B core antigen (anti-HBc) by commercially available reagents (Abbott, North Chicago, IL). Hepatitis B virus DNA (HBV-DNA) was determined by molecular hybridization on serum spots blotted on nitrocellulose filters as described in detail elsewhere [14].
Inmunohistochemistry Cryostat sections from all biopsies were tested with immunoperoxidase for the presence of HBsAg and HBcAg, using anti-HBs from Sorin (Saluggia, Italy) and anti-HBc from Dako (Santa Barbara, CA) respectively, as described elsewhere [15]. RESULTS The amount of cryoglobulin ranged from 0-75 to 2-8 mg/ml. All cryoprecipitates contained IgM and polyclonal IgG; in addition, the IgM was monoclonal with K light chains in 20 patients (77%) and polyclonal in the remainder. No cryoprecipitate contained IgA, whereas all cryoglobulins tested had RF activity. Overt liver disease was lacking in all patients. However, occult or subclinical liver involvement was detected in a minor proportion of them by clinical and/or biochemical parameters (Table 1). Indeed, liver enlargement was noted by physical examination in five patients (19%/0) and splenomegaly was revealed in four patients (15%). Biochemical liver tests were performed several times throughout the course of the disease in all patients and only minimal abnormalities were detected. Increased levels of SGOT were found in 15%M), of bilirubin in 4%/0, of alkaline phosphatase in 1 1%. Altogether, six patients (23%) had clinical and/or biochemical evidence of liver involvement, and three of them were anti-HCV positive. Serum samples from all patients were tested for hepatitis B virus markers and HBV DNA levels. HBeAg and anti-HBe were consistently absent. All but one were HBsAg-, the only HBsAg+ patient being however anti-HCV -. IgG-type anti-HBc
I
J0
ll
-
O*
Cut-off
Il
5
10
20 Cryocrit (0)
30
Fig. 1. Comparison between anti-hepatitis C virus (HCV) antibody by ELISA and cryocrit values in 26 essential mixed cryoglobulinaemia (EMC) patients.
and anti-HBs antibodies were detected in two patients belonging to the HCV- group, and no detectable levels of serum HBV DNA were found in any of the patients. In addition, neither HBcAg in the nuclei nor HBsAg in the cytoplasm, on the plasma membrane or both were revealed when liver biopsy sections were studied by conventional immunohistochemistry. The only exception was again patient 20 (see Table 1) who, in addition to being HBsAg-seropositive, showed a weak positivity for HBsAg in the cytoplasm. Cryoglobulinaemic sera are prone to enhance non-specific binding, thus giving false positive results in solid-phase enzyme immunoassay. To overcome this problem, all sera from this series of patients were studied both in toto and deprived of cryoprecipitates. Concordant results were, however, obtained in the detection of anti-HCV antibodies with samples containing or deprived of cryoproteins both by ELISA and by RIBA. Thirteen patients were positive for anti-HCV antibodies indicating a prevalence of 50%. RIBA confirmation, however, was obtained in 11 patients (42-3%), in that the remaining two were reactive for C22-3 antigen only and were thus classified as indeterminate. Further indirect evidence of the lack of influence of the cryoglobulin levels on anti-HCV detection test is shown in Fig. 1, where no correlation was demonstrable between antiHCV antibodies and cryocrit values. To rule out interference by RF in the test, two samples with high levels of RF activity were treated with increasing amounts of heat-aggregated human IgG. A progressive decrement of RF activity was noted, without significant changes in anti-HCV OD (Fig. 2). The lack of interference by RF was also indirectly demonstrated by comparing its average reciprocal titre in the anti-HCV+ group (875; range, 40-1280) with that of the antiHCV- group (720; range, 80-1280). The liver histological findings are reported in Table 1. A significant correlation was noted between anti-HCV serum positivity and the histological picture of chronic active liver disease (CALD). The anti-HCV+ group showed a frequency of CALD of 63-60/o (7/1 1) compared with 6 7%/, (1/15) in the antiHCV- group (P < 0-01). Reciprocal titres of ANA and SMA of more than 40 were detected in one patient positive in the anti-HCV assay. Liver biopsy showed histological features of chronic active hepatitis (CAH).
HCV infection in cryoglobulinaemia 18
N
II.S
coin
(
°
Br |S
0
0 Heat -aggregated
07q
IgG, (mngml 'I
Fig. 2. Lack of influence of rheumatoid factor (RF) in detection of IgG anti-hepatitis C virus (HCV) antibody in the sera of two essential mixed cryoglobulinaemia (EMC) patients. The addition of increasing amounts of heat-aggregated human IgG results in the progressive decrease of RF titres (El El; 0 0), but with no effect on the anti-HCV activity 0). BSA, Bovin serum albumin. E; 0 (El
Finally, along with pentoxyphylline or indobuphene, 19 patients were receiving 4 mg methyl-prednisolone daily at the time of this study. However, since EMC is a typically chronic disorder, results were not analysed with respect to response/ non-response to treatment.
DISCUSSION This study demonstrates that 42 3'S, of our EMC patients had anti-HCV antibody in their serum. Results offered by the current tests for the detection of circulating anti-HCV antibodies must be accepted with caution when 'sticky' serum samples are tested in solid-phase by first generation ELISA. McFarlane et al. [16] have reported false positives in autoimmune CAH, suggesting that samples taken during its active phases may contain components interfering with the antigen coated on the solid phase. Other factors involved in non-specific reactions are found in patients with serum monoclonal components [17]. False positives have also been demonstrated in samples from particular geographical areas, probably because of a coincident high prevalence of flavivirus and pestivirus infections [1 8], or in low-risk HCV infection populations [19]. In
addition, long-stored samples have been shown to contain factors able to induce non-specific reactions [20]. Finally, false positivity due to RF has been detected in over 60, of patients with a clinical diagnosis of rheumatoid arthritis who did not have risk factors for HCV infection or evidence of liver disease [21]. Introduction of second generation HCV ELISA (C200/ C22-3) provided improved sensitivity in individuals at high risk for HCV infection and improvement in assay specificity when screening low risk populations. These results are validated by an independent, supportive assay represented by the second generation immunoblotting assay (4-RIBA) that, with the additional incorporation of C33c and C22-3 antigens, has been proposed to discriminate infectious from non-infectious ELISA-positive samples [22]. Even though cryoglobulinaemic sera are somewhat 'sticky', the current ELISA proved to be a reliable test for the following reasons: (i) although two out of 13 of our cryoglobulinaemic sera displayed poor repeatibility in this assay, second generation
355
RIBA demonstrated that their positivity could be attributed to C22-3 antigen reactivity; (ii) preliminary data (manuscript in preparation) obtained in the same sera with a novel anti-HCV assay from UBI (United Biochemical, Lake Success, NY), based entirely on synthetic HCV peptides from both the structural and non-structural regions of the HCV genome [23], indicate a very close correlation between the Ortho ELISA and RIBA on one side and the UBI screening assay on the other; (iii) both the cryocrit levels and the RF titres were shown not to act as disturbing factors in the test; (iv) consistent results were obtained in the Ortho assay when whole sera and clear supernatants were separately studied. Final confirmation of the specificity of anti-HCV positivity will obviously rest on the actual demonstration of the virus, i.e. of circulating HCV RNA. Liver involvement in EMC has been known for many years and occasionally regarded as a direct consequence of immune complex-mediated damage [9]. However, the pathogenetic role of the liver in cryoglobulinaemia remains an unsolved question. The present study strongly suggests that HCV infection is associated with the liver damage in our patients, since there was a significant difference in the frequency of CALD between the anti-HCV+ and anti-HCV- groups. Clinical and histological pictures of CALD were found in 7/11 anti-HCV+ patients, compared with only 1 15 anti-HCV patients. In the remaining patients the most frequent histological findings included discrete lymphoid aggregates in portal tracts, fatty changes, siderosis, and hyperplasia of Kupffer cells without clinical and/or biochemical signs of liver involvement. Acute and chronic liver diseases of various aetiologies are sometimes accompanied by mixed cryoglobulinaemia [24,25]. In addition, patients with liver disease and cryoglobulinaemia may exhibit extrahepatic features closely resembling the clinical manifestations of EMC. Thus, it might be argued that at least some of the patients in our series could have had primary liver disease and secondary cryoglobulinaemia, rather than EMC. This, however, seems unlikely, for the following reasons: (i) all patients presented with a distinct clinical syndrome consisting of purpura, arthralgias, weakness, and frequently kidney involvement in the absence of any apparent underlying disorder. Thus, all of them fulfilled currently accepted criteria for a diagnosis of EMC; (ii) although approximately 23Oe of the patients were shown to have clinical and/or biochemical evidence of liver impairment, none of them was suffering from symptoms ascribable to serious liver involvement. These observations are in keeping with earlier results [22], indicating underestimation of the frequency of chronic liver disease based on serological features in HCV low-risk populations; (iii) in spite of the extended follow up of the patients ranging from 3 to 8 years, no major changes were observed in clinical symptoms and/or biochemical abnormalities implying progression of liver disease or modification of the initial diagnosis of EMC. Recently a high frequency of anti-HCV antibodies has been reported in autoimmune (AI)-CAH, although important geographical and/or genetic differences seem to exist among different populations [26]. Of course, this might also apply to EMC patients, in that different factors may be involved in the development of CALD in EMC patients from areas where viral hepatitis is not endemic. In this context, one patient of our series deserves special mention. She was found to have significant titres of ANA and SMA in addition to RF activity, a histological picture of CAH at liver biopsy, anti-HCV seropositivity,
F. Dammacco & D. Sansonno
356
and obviously mixed cryoglobulinaemia. A clinical diagnosis of EMC was entertained, although type-I AI-CAH could also be considered. On the other hand, an etiologic role for HBV infection can be reasonably ruled out in our series. Only one patient was an HBsAg carrier, none had circulating HBV-DNA and in all but one liver biopsy tissues did not express hepatitis B virusassociated antigens. Nonetheless, several recent reports indicate the presence of HBV transcriptionally active genome in livers from HBsAg- chronic liver disease, as detected by the specific polymerase chain reaction [27]. This invites speculation on the possible aetiologic role of HBV in a subgroup of patients in the absence of common serologic markers, although it appears to be a low-frequency event [28] that needs to be assessed in EMC. It is rather difficult to explain this high rate of apparently genuine anti-HCV positivity among our patients with EMC. The overall prevalence of anti-HCV antibodies among blood donors in the Apulia region, where all patients came from, is approximately 2%. Sexual or vertical transmission of HCV seems to be much less common than for HBV [29]. In addition, none of our patients had been given blood or blood products, although most of them had required more frequent hospitalization. Identification of an EMC subgroup associating anti-HCV antibody in the serum obviously supports the therapeutic use of anti-viral drugs such as interferons in these patients [30].
ACKNOWLEDGMENTS The excellent secretarial assistance of Vito lacovizzi is gratefully acknowledged. This study was supported in part by a grant from the Italian Ministry for Universities and Scientific and Technological Research, group 'Liver Cirrhosis', Rome.
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88:3647-51. 24 Dienstag JL, Rhodes AR, Bhan AK, Dvorak AM, Mihm MC Jr, Wands JR. Urticaria associated with acute viral hepatitis type B: studies of pathogenesis. Ann Intern Med 1978; 89:34-9. 25 Jori GP, Buonanno G, D'Onofrio F, Tirelli A, Gonnella F, Gentile S. Incidence and immunochemical features of serum cryoglobulins in chronic liver diseases. Gut 1977; 18:245-9. 26 Lenzi M, Johnson PJ, McFarlane IG et al. Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity. Lancet 1991; 338:277-80. 27 Paterlini P. Gerken G, Nakajima E et al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. N Engl J Med 1990; 323:80-5. 28 Pontisso P. Stenico D, Diodati G et al. HBV-DNA sequences are rarely detected in the liver of patients with HBsAg-negative chronic active liver disease and hepatocellular carcinoma in Italy. Liver 1987; 7:211-5. 29 Ideo G, Bellati G, Pedraglio E, Bottelli R, Donzelli T, Putignano G. Intrafamilial transmission of hepatitis C virus. Lancet 1990; 335:353. 30 Bonomo L, Casato M, Afeltra A, Caccavo D. Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 1987; 83:726-30.
