Serum complement levels in patients with mixed (IgM–IgG) cryoglobulinaemia

Serum complement levels were determined in seven patients with mixed (IgM–IgG) cryoglobulinaemia. All seven patients had either no measurable or very low levels of C'2. C'50 haemolytic units were determined in three patients and were also found low, although less striking. The mechanism by which the blood of these patients became depleted of C'2 could not be determined.     

T cell defect in essential mixed cryoglobulinaemia.

Peripheral blood lymphocytes from untreated patients with essential cryoglobulinaemia were studied for their surface markers and for their in vitro mitogenic reactivity. No differences in lymphocyte subpopulations were observed between cryoglobulinaemic patients and normal controls. Cultures of separated lymphocytes were stimulated with different concentrations of phytohaemagglutinin, Con-A and pokeweed mitogen. Incorporation of [3H]-thymidine in patients' cultures was compared with that of normal controls. Significantly decreased reactivity to phytohaemagglutinin and Con-A, but not to pokeweed mitogen, was found in all patients studied. The depressed mitogenic reactivity to phytohaemagglutinin and Con-A might be referred to a qualitative T cell defect.     

Antibodies to hepatitis C virus in essential mixed cryoglobulinaemia.

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. Anti-HCV 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 less than 0.01). 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.     

Clonal B cell expansions in patients with essential mixed cryoglobulinaemia.

In essential mixed, type II, cryoglobulinaemia (EMC) monoclonal autoantibodies with rheumatoid factor activity are synthesized at an accelerated rate by non-malignant B lymphocytes. In order to determine if the high cryoglobulin production rate is related to a clonal B cell expansion, cell surface markers of peripheral blood lymphocytes (PBL) were analysed by flow cytometry and the rearrangement of immunoglobulin (Ig) genes was investigated by Southern blot analysis of DNA extracted from the PBL of 12 EMC patients. Clonal expansion of B cells could be detected using DNA probes specific for the c kappa, c-mu, and JH genes in four out of 12 patients, two of whom also showed specific expansions of mu heavy and kappa light chain bearing cells using flow cytometry. The rearrangement of the c-myc locus was also noted in one of the patients with detectable Ig gene rearrangements. Demonstration of clonal B cell expansions in EMC patients shows that the clonal type of Ig gene rearrangements are not unique markers of malignant lymphomas but may also occur in autoimmune lymphoproliferative disorders. Since malignant B cell lymphomas can develop in a small number of EMC cases, the follow-up of these patients should be pursued indefinitely.     

Increased frequency of antibodies to ubiquitous viruses in essential mixed cryoglobulinaemia.

Antibodies to ubiquitous Herpes viruses have been studied in 13 patients with type II essential mixed cryoglobulinemia (EMC), and in two different control groups. All the EMC patients had monoclonal IgM kappa in their cryoprecipitates. IgM antibodies to the viral capsid antigen (VCA) of Epstein-Barr virus (EBV) were found in the sera of 11 EMC patients but all the cryoprecipitates were negative. IgG antibodies were also present in the sera of all and in the cryoprecipitates of some patients. In contrast, the number of subjects with antibodies to Cytomegalovirus (CMV) and Hepatitis B Virus (HBV) was not higher than in controls. Possible correlations between EMC and EBV infection are discussed.     

The complement system in cryoglobulinaemia. Interaction with immunoglobulins and lipoproteins.

Serum from a patient with an IgM-lipoprotein cryoglobulin, both before and after removal of the cryoprecipitate at 0 degrees C, had extremely low levels of whole complement (C), C1, C4 and C2, while amounts of the remaining components were normal or only slightly reduced. The cryopredipitate, when added to fresh normal human serum, reproduced this pattern of C fixation. Separation of the patients's serum at 37 degrees C into its lipoprotein, IgG and IgM fractions revealed that the IgM alone would precipitate at 0 degrees C. This precipitation was unaffected by the patients's IgG, but was markedly enhanced by extremely small amounts of the patient's d less than 1-075 lipoprotein fraction or of homologous very low density lipoprotein (VLDL). Aggregation occurred even at 37 degrees C in the presence of VLDL. Fixation of semi-purified human C1 paralleled these results closely: it occurred with the patient's IgM alone at 0 degrees but not at 37 degrees C, while IgM in the presence of the patient's lipoprotein, or of VLDL from normal serum, fixed C1 strongly at 37 degrees as well as at 0 degrees C. Fab dimers and monomers prepared from the patient's IgM did not aggregate in the cold, even in the presence of lipoprotein, and did not inhibit the aggregation of intact IgM in the presence of VLDL, at any temperature. All three highly purified IgM cryoglobulins, and three of four IgG cryoglobulins, fixed C1 strongly. The IgG preparation which failed to fix C1 was the only one which had lost its cryoprecipitability during purification. Measurement of C3 or whole C levels may be an insensitive method for detecting C fixation in cryoglobulinaemia. It is suggested that analysis for C1, C4 or C2 should be employed instead.     

Autoantibodies from mixed cryoglobulinaemia patients bind glomerular antigens.

Mixed cryoglobulinaemia (MC) is a disorder characterized by the presence of large amounts of cryoprecipitating IgM-IgG complexes. An immune complex glomerulonephritis develops in one third of all patients, but its occurrence does not seem related to the amount of cryoglobulins in the sera, nor to their complement-fixing ability. In this study we investigated the presence of IgG antibodies reactive with kidney antigens in 33 MC patients (11 with glomerulonephritis, 22 without renal involvement). A total glomerular extract was run on a 10% acrylamide gel, blotted to nitrocellulose and probed with the patients' sera. Sera from half of the patients without renal involvement reacted with several glomerular antigens whose molecular weight ranged between 200 and 29 kD. In the group with renal involvement, sera from 7/11 patients reacted with an antigen of 50 kD, which is also expressed in thymus, but not in the heart or liver. In a follow-up study of four patients with renal involvement, the amount of serum antibody specific for the 50-kD antigen fluctuated, either spontaneously or in response to therapy. These results show that antibodies specific for glomerular antigens are detectable in MC sera. The immune response against a 50-kD antigen expressed in the kidney and thymus seems to be restricted to a subset of MC patients with renal involvement. Circulating autoantibodies specific for glomerular antigens might contribute to the induction of glomerulonephritis in MC forming immune complexes in situ.     

Impaired hepatosplenic elimination of circulating cryoglobulins in patients with essential mixed cryoglobulinaemia and hepatitis C virus (HCV) infection

The pathogenic mechanisms that lead to renal deposition of the cryoprecipitable IgM rheumatoid factor–IgG complexes in essential mixed cryoglobulinaemia (EMC) are unknown. Defective removal of cryoprecipitable complexes from the circulation has been postulated in EMC-associated nephritis. To test this hypothesis, the kinetics and fate of a trace dose of ¹²³I-radiolabelled autologous cryoglobulins were analysed in 13 patients with EMC grouped according to renal involvement. The time course of radioactivity distribution in the blood and organ uptake were measured by gamma camera scintigraphy. In blood sampled 30–300 s after injection, only a minor fraction (< 15%) of the circulating cryoglobulins bound to the erythrocytes, suggesting the elimination mechanisms are independent of binding to CR1 on erythrocytes. The overall blood disappearance curve showed a fast (≤ 1 min) and slow (> 4 h) biphasic pattern. In patients with quiescent or mild nepthritis, the liver and to a lesser extent the spleen were the major organs that mediated the rapid uptake and processing of the cryoglobulins from the circulation. In contrast, patients with active mesangiocapillary glomerulonephritis showed significantly (P< 0.001) less hepatic uptake, low liver-to-precordium ratio, and slower processing of cryoglobulins, prolonged liver mean transit time, than quiescent patients or mild nephritis patients. To elucidate the role and influence of HCV infection in the pathogenesis of EMC-nephritis, sera and cryoglobulins from all patients were assayed for HCV. None of the control group cases without nephritis showed any evidence of HCV-RNA in serum or cryoglobulin pellet. In contrast, all 10 EMC-nephritis patients' sera, and eight corresponding cryoglobulin pellets contained HCV-RNA. Collectively, these findings suggest an impaired reticuloendothelial system removal of IgM–IgG–HCV complexes may underlie their renal deposition.     

IgM and IgG antibodies to hepatitis C virus in patients with mixed cryoglobulinaemia

To assess the relationship between hepatitis C virus (HCV) infection and essential mixed cryoglobulinaemia (EMC), sera from 23 patients with EMC were tested for IgG and IgM antibodies to HCV antigens and for HCV RNA. Quantitative HCV antibody studies were performed on scrum and purified cryoglobulin fractions. HCV antibodies of both IgG and IgM class were found in 22 (96%) patients. Ten of these were also HCV-RNA positives. Higher litres of anti-HCV IgM were present in the 11 patients with evidence of liver damage. Anti-HCV IgG antibodies were shown to be concentrated in the IgG fraction of cryoglobulins in all eight patients studied. These results strongly suggest a role for HCV in the pathogenesis of EMC.     

Serum cytokine profile in hepatitis C virus carriers presenting cryoglobulinaemia and non-organ-specific autoantibodies

This work investigated the serum cytokine profile (IL-2, IL-4, IL-5, IL-10, IFN-γ and BAFF) of hepatitis C virus (HCV) carriers with autoimmunity. Forty-seven HCV carriers and 28 healthy controls were evaluated. Cytokine levels were measured by ELISA. Patients and controls presented similar levels of IL-2, IL-4, IL-5, IL-10, IFN-γ and BAFF (p > 0.05). Cryoglobulinaemic HCV carriers had increased IL-2 (p = 0.013), IL-5 (p = 0.018) and BAFF (p = 0.050). IFN-γ level was decreased in HCV carriers with rheumatoid factor in comparison with those that were RF-seronegative (p = 0.035). Patients with β2GPI IgA antibodies when were compared with those without this autoantibody, had more serum IL-2 (p = 0.009), IL-5 (p = 0.018) and BAFF (p = 0.039). Interleukin-2 was increased in HCV carriers with positive ANA when they were compared with ANA-seronegative carriers (p = 0.044). Interleukins IL-4 and IL-10 were not associated with autoimmunity (P > 0.05). In HCV carriers, IL-2 was correlated with IL-5 (p < 0.0001) and IFN-γ (p = 0.015), and IL-5 with IFN-γ (p = 0.015). We concluded that the serum profile of cytokines in HCV carriers presenting autoimmune markers may be mainly represented by increased IL-2, IL-5 and BAFF.     

Efficacy of alpha interferon treatment in patients with chronic hepatitis C associated to mixed cryoglobulinaemia

Objective. Mixed cryoglobulinaemia is closely associated with hepatitis C virus (HCV) infection. The aim of this trial was to evaluate in a prospective open study the efficacy of ?-interferon in the treatment of chronic hepatitis C virus associated to mixed cryoglobulinaemia (MC). Methods. Thirty-one consecutive patients were treated for the first time with ?-interferon at a dose of 6 MU three times a week for 12 months. All the patients presented cryoglobulins, which were responsible for clinical manifestations in 9. Results.At the end of interferon treatment, 11 patients presented complete responses (biochemical and virological), including 7 subjects who presented MC-related clinical manifestations. Cryoglobulins had disappeared in 48.4% of the patients and a clinical improvement was observed in 7 out of 9 patients. Twelve months after interferon treatment was stopped, only 25.8% of patients still had undetectable cryoglobulins and 5 subjects who presented complete responses, all with MCrelated clinical manifestations had a relapse both of HCV-related biochemical and virological indexes and of MC clinical manifestations. Conclusions. A 12-month course of ?-interferon is effective treatment for HCV-related cryoglobulinaemia, especially during therapy. However we obtained scarce results in the follow-up above all in the patients with clinical manifestations of MC.     

Lectin complement system and pattern recognition

Living organisms have strong defense mechanisms against invading microorganisms as survival strategies. One of the defense mechanisms is the complement system, composed of more than 30 serum and cell surface components. This system collaborates in recognition and elimination of pathogens as a part of both the innate and acquired immune systems. The two collagenous lectins, mannose-binding lectin (MBL) and ficolins, are pattern recognition proteins acting in innate immunity and, upon recognition of the pathogens, they trigger the activation of the lectin complement pathway through attached serine proteases (MASPs). A similar lectin-based complement system, consisting of the lectin-protease complex and C3, is present in ascidians, our closest invertebrate relatives and in lamprey, the most primitive vertebrate. Furthermore, a lamprey N-acetylglucosamine (GlcNAc)-binding lectin was identified as the orthlogue of mammalian C1q, and lamprey MASP is suggested as the prototype of MASP-2/C1r/C1s, indicating that the classical complement pathway arose as a part of the innate immune system. Thus, the complement system is one of the most highly organized innate immune systems in invertebrates and jawless vertebrates, and this system has survived in vertebrates with its core components little changed for 600-700 million years.     

Serum terminal complement component levels in hypocomplementemic glomerulonephritides

Measurements of serum C3 through C9 are reported for patients with acute poststreptococcal glomerulonephritis (AGN), membranoproliferative glomerulonephritis type I (MPGN I), MPGN II, and MPGN III. Except in MPGN II, depressed C5 levels correlated with depressed C3 levels. In MPGN II, levels of C5 and of other terminal components were normal. In MPGN III, markedly depressed levels of C7 through C9 correlated strongly with depressed levels of C3 and C5. C6 was less severely depressed. In MPGN I, terminal component levels were less often depressed than in MPGN III and in AGN, depression of terminal components was seen only when levels of C3 and C5 were extremely low. The data indicate that late terminal components are activated in MPGN III to a greater extent than in the other nephritides despite C5 activation approximately equal in extent to that in AGN and MPGN I.     

Non-enveloped HCV core protein as constitutive antigen of cold-precipitable immune complexes in type II mixed cryoglobulinaemia

Hepatitis C virus (HCV) infection has been detected in a large proportion of patients with mixed cryoglobulinaemia (MC). Circulating 'free' non-enveloped HCV core protein has been demonstrated in HCV-infected patients, and this suggests its possible involvement in the formation of cryoprecipitable immune complexes (ICs). Thirty-two anti-HCV, HCV RNA-positive patients with type II MC were evaluated. Non-enveloped HCV core protein, HCV RNA sequences, total IgM, rheumatoid factor (RF) activity, IgG and IgG subclasses, C3 and C4 fractions, C1q protein and C1q binding activity were assessed in both cryoprecipitates and supernatants. Non-enveloped HCV core protein was demonstrated in 30 of 32 (93.7%) type II MC patients. After separation of cold-precipitable material, the protein was removed completely from supernatant in 12 patients (40%), whereas it was enriched in the cryoprecipitates of the remaining 18. In addition, HCV RNA and IgM molecules with RF activity were concentrated selectively in the cryoprecipitates. Differential precipitation was found for both total IgG and IgG subclasses, as they were less represented in the cryoglobulins and no selective enrichment was noted. Immunological characterization of HCV core protein-containing cryoprecipitating ICs after chromatographic fractionation showed that the IgM monoclonal component had RF activity, whereas anti-HCV core reactivity was confined to the IgG fraction. C1q enrichment in addition to high avidity of ICs for C1q binding in the cryoprecipitates suggest that complement activation may occur through the C1q protein pathway. The present data demonstrate that non-enveloped HCV core protein is a constitutive component of cryoprecipitable ICs in type II MC patients.