Available online at www.sciencedirect.com

(2007) 114–120www.elsevier.com/locate/autrev

Autoimmunity Reviews 7

B-cells and mixed cryoglobulinemia

Clodoveo Ferri a,⁎, Alessandro Antonelli b, Maria Teresa Mascia a, Marco Sebastiani a,Poupak Fallahi b, Daniela Ferrari a, Marco Giunti a,

Stefano A. Pileri c, Anna Linda Zignego d

a Chair and Rheumatology Unit, Department of Internal Medicine, University of Modena & Reggio E., Medical School, Modena, Italyb Department Internal Medicine, University of Pisa, Medical School, Pisa, Italy

c Chair of Pathology, Unit of Hematopathology, Bologna University School of Medicine, Bologna, Italyd Department of Internal Medicine, University of Florence, Medical School, Florence, Italy

Available online 28 March 2007

Abstract

Mixed cryoglobulinemia (MC) is a systemic small-vessel vasculitis; B-cell expansion is the biological substrate of thedisease. It can be regarded as benign lymphoproliferative condition that may evolve to frank lymphoma. HCV infection is themain causative factor of MC, as well as of other overlapping disorders, through multifactorial and multistep pathogeneticprocess. HCV-related B-cell proliferation represents an important model of virus-driven autoimmune/neoplastic disorder. Theterm HCV syndrome is referred to a wide spectrum of both hepatic and extrahepatic disorders.

The present review analyzes the complex virological, clinico-pathological, and therapeutic implications of B-cellproliferation, with or without HCV infection, in MC patients.© 2007 Elsevier B.V. All rights reserved.

Keywords: B-cells; Cryoglobulinemia; Autoimmunity; HCV; Lymphoma; Thyroid; Diabetes

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142. Mixed cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1153. B-cell proliferation and cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164. B-cell and HCV syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185. HCV-negative cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186. Therapeutic strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

115

⁎ Corresponding author. Reumatologia, Università di Modena e Reggio Emilia, Policlinico di Modena, Via del Pozzo, 71, 41100 Modena Italy.Tel.: +39 059 4224199; fax: +39 059 4224178.

E-mail address: clferri@unimore.it (C. Ferri).

1568-9972/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.autrev.2007.02.019

115C. Ferri et al. / Autoimmunity Reviews 7 (2007) 114–120

1. Introduction

Cryoglobulinemia is defined as the presence in theserum of one (monoclonal cryoimmunoglobulinemia) ormore immunoglobulins (mixed cryoglobulinemia),which precipitate at temperatures below 37 °C andredissolve on re-warming [1–4].

According to immunoglobulin composition, cryoglo-bulinemia is traditionally classified into three subgroups[2]: a) Type I, composed by single monoclonal immu-noglobulin, usually a paraprotein; b) Type II, and c) TypeIII mixed cryoglobulinemia: these are immune com-plexes composed of polyclonal IgGs, the autoantigens,and mono- or polyclonal IgMs, respectively. The IgMsare the corresponding autoantibodies with rheumatoidfactor (RF) activity. Cryoglobulinemia type I is mainlyfound in patients with overt lymphoid tumors, i.e. im-munocytoma/Waldenstrom's macroglobulinemia, mul-tiple myeloma, etc. [1–4]. Mixed cryoglobulinemia(MC) type II and III can be associated with variousinfectious, immunological or neoplastic diseases [1–4].The analysis of cryoprecipitates is generally carried outby means of immunoelectrophoresis or immunofixation.Using more sensitive methodologies, i.e. immunoblot-ting or two-dimensional polyacrylamide gel electropho-resis, type II MC may show a microheterogeneouscomposition [4]. This serological subset, termed type II-III MC (oligoclonal IgMs or a mixture of polyclonal andmonoclonal IgMs), could represent an intermediate state,with the possible evolution from type III to type II. Thecomposition of this particular serological subset seems to

Fig. 1. B-cell proliferation represents the biological substrate of mixed cryoglclinical immunological manifestations and ultimately to frank B-cell non-HoHCV is the main triggering agent; it represents a chronic stimulus through theon B-cell surface. But other infectious, environmental, and genetic factorslymphoproliferative disorder of undetermined significance.

reflect the most recent molecular studies showing thepresence of oligoclonal B-lymphocyte proliferation inliver and bone marrow biopsies in the majority ofpatients with type II MC [5]. The present review focus onthe B-cell proliferation, which represents the biologicalhallmark of the MC, and its relationship with virologicaland clinico-pathological findings of the disease.

2. Mixed cryoglobulinemia

Variable amounts of circulating mixed cryoglobulinsare commonly detected in a great number of infectious,immunological or neoplastic disorders [1–4]; while theso-called ‘essential’ MC represents a distinct disorder[3]. This condition is characterized by circulating mixedcryoglobulins and low complement, mainly C4 com-ponent, along with a typical clinical triad – purpura,arthralgias, and weakness – and frequent multiple organinvolvement [1–4]. These latter may include: chronichepatitis with or without cirrhosis, glomerulonephritis,peripheral neuropathy, sicca syndrome, endocrine glanddysfunction (thyroiditis, diabetes, erectile dysfunction),widespread vasculitis, and neoplasias [3,4,6–12]. B-cellnon-Hodgkin's lymphoma (B-NHL) represents the mostfrequent neoplastic complication of MC. Other malig-nancies, i.e. hepatocellular carcinoma and papillary thy-roid cancer, are less frequently observed, often as latemanifestations of the MC syndrome [4,6,8,9]. Becauseof the possible appearance of cancer during its clinicalcourse, the MC can be regarded as a pre-neoplasticdisorder [4,6,8,9].

obulinemia (MC). The evolution from a simple serological alteration todgkin's lymphoma (B-NHL) is a multistep and multifactorial process.interaction of HCV-E2 antigen (and/or other viral antigens) with CD81are probably involved. RF: rheumatoid factor; MLDUS: monotypic

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One or more serum autoantibodies can be detected inover half patients, more frequently low titer anti-nuclear(ANA) and/or anti-mitochondrial (AMA), and/or anti-smooth muscle (ASMA), without any relationship withother clinico-serological parameters [6].

During the last decade, a large body of clinico-epidemiological and laboratory investigations definitelyestablished the pathogenic role of hepatitis C virus(HCV) infection in MC syndrome [4,6–10]. Severalimmunohistochemical and molecular biology studies,including HCV RNA detection by in situ hybridization,demonstrated a direct involvement of HCV antigens inimmune-complex-mediated cryoglobulinemic vasculi-tis, as well as in different organ alterations [4,6,8,9].Therefore, the term ‘essential’ no longer seems to beappropriate for the majority of MC patients.

Immune-complex-mediated leukocytoclastic vascu-litis, involving small-sized vessels (arterioles, capillar-ies, and venues), is the histopathological hallmark ofMC [4,6–10]. It is a necrotizing vasculitis characterizedby extensive fibrinoid necrosis of the vessel wall withpermeation of the wall by disintegrating neutrophils.

Fig. 2. Etiopathogenesis of mixed cryoglobulinemia (MC) and other HCV-immune-system; this remote event may involve a number of both viral and h‘toxic’ agents. The main consequence is a benign B-cell lymphoproliferation pand cryo- and non-cryoprecipitable immune–complexes (IC). These serologorgan- and non-organ-specific immunological disorders, includingMC syndroB-cell survival (see also Fig. 1). Other genetic aberrations are necessary for thminority of cases. There is a clinico-serologic and pathologic overlap amonrepresents a crossing road between autoimmune and neoplastic disorders.

3. B-cell proliferation and cryoglobulinemia

Several epidemiological and clinico-pathologicalobservations suggest that MC is the result of a multi-factorial and multistep pathogenetic process (Fig. 1,[4,6,8,9]). While the immune-complex-mediated vascu-litis is the final step of this complex process (Fig. 2), B-lymphocyte expansion [1] may represent the remotedisorder responsible for autoantibodies and immune-complex production and in some instances for malignantlymphomas. Besides HCV infection, other possibletriggering agents, and/or environmental, and/or geneticco-factors remain still unknown (Figs. 1 and 2); on theother hand, the exact role of HCV in the lymphoproli-feration is not completely clarified [4,6–10].

Given the well-known HCV lymphotropism [4,6–10,13,14] a direct role of this virus in the B-cell ex-pansion has been initially hypothesized on the basis ofthe high frequency of HCV-RNA positive lymphocytesin peripheral blood and bone marrow of cryoglo-bulinemic patients, along with the significant percent-age of individuals developing malignant lymphomas

related disorders. HCV infection may exert a chronic stimulus on theost factors, with the possible contribution of unknown environmentalroducing a variety of autoantibodies, including rheumatoid factor (RF),ical alterations may explain, at least in part, the appearance of variousme. The activation of Bcl2 proto-oncogene is responsible for prolongede development of frank B-cell lymphomas and other malignancies in ag different HCV-related diseases; mixed cryoglobulinemia syndrome

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[4,6–10,13,14]. Moreover, in patients with chronic HCVinfection and B-NHL the presence of viral genomicsequences within pathological tissue was demonstratedby means of RT-PCR (reverse transcription–polymerasechain reaction), in situ hybridization and immunohisto-chemical techniques [4,6–10,13–15]. In type II MCcomplicated by B-NHL, the analysis of lymph nodesections showed that viral proteins were detectable in thecytoplasm of lymphoid cells in a quarter of cases.Moreover, in low-grade B-NHL, the HCV-positive cellswere localized in the lymph node cortex or in the tumoraltissue, whereas frankly anaplastic cells were negative.These findings suggest that in patients with type II MCthe B-cell infection precedes tumoral transformation,possibly playing a major role in the lymphomagenesis;while completely anaplastic cells are no longer permis-sive to HCV replication [15].

Since HCV is an RNA virus without reverse trascrip-tase activity, viral genome cannot integrate in the hostgenome. Thus, the integration of viral genome in theproximity of genes important for the regulation of cellreplication and/or survival may not be invoked. Proba-bly, HCV may exert its oncogenic potential, indirectly,through viral proteins; particular attention has been paidto the possible role of HCV-codified proteins in thelymphomagenesis, particularly the core protein [16]. In astudy, however, the expression of core protein did notappear to significantly modify the main intracellularsignaling transduction pathways [17].

Several studies outlined the relevance of an indirectpathogenetic role exerted by HCV infection; in partic-ular, the chronic stimulation of the lymphatic systemmight be exerted through viral epitopes, autoantigenproduction, and/or molecular mimicry mechanism [4,6–10,13,14]. This latter has been suggested by thepresence, in HCV-positive patients, of anti-GOR anti-bodies, which are cross-reactive autoantibodies directedto both HCV core and a nuclear antigen named GOR [4].However, the pathogenetic role played by these anti-bodies, if any, is unknown.

Another hypothesis suggests that HCV, in associationwith very low-density lipoprotein (VLDL), would in-duce a T-independent primordial B-cell populationproducing monoclonal immunoglobulin with WA idio-type [18]. The RF activity of WA clones would be aconsequence of somatic mutations induced after thestimulation by HCV–VLDL complexes. In this context,the possible evolution to B-NHL might be the con-sequence of the accumulation of stochastic geneticaberrations [18].

Laboratory investigations in HCV-positive type IIMC suggest the importance of a chronic stimulation of

the B-cell by HCVepitopes; in this context, some B-cellsubpopulations with favorable and/or dominant geneticcharacteristics will emerge [4,6–9]. This hypothesisrecalls the pathogenetic role of Helicobacter pylori inMALT lymphoma of the stomach, for which differentevolutive phases are requested [19].

The observation that, in vitro, HCV E2 protein is ableto bind to CD81 molecule (a quite ubiquitary tetra-spannin that is well represented on the surface of B-cells)has suggested the hypothesis that this interaction mayrepresent one of the causes of the strong and sustainedpolyclonal stimulation of the B-cell compartment [20].However, a pathogenetic role of HCV E2/CD81 inter-action in the pathogenesis of MC and its malignantevolution seems to be unlikely, as suggested by the lackof sequence homologies between mFR WA, the mainmRF in HCV-related type II MC, and anti-HCV E2antibody [18].

In the multistep process of HCV-related lymphopro-liferation, of interest is the significantly high frequencyof t(14,18) translocation or Bcl-2 rearrangement ob-served in B-cells [21]. The consequence is the ab-normally elevated expression of Bcl-2 protein withconsequent inhibition of apoptosis and abnormal B-cellsurvival. Interestingly, the relevant prevalence of t(14,18) translocation in patients with only type C hep-atitis (about 37–38%), become particularly high inpatients with HCV-related cryoglobulinemic syndrome,ranging 85% in type II MC [21]. These data have beenconfirmed by several studies in patients’ populationsfrom different countries, utilizing similar or comple-mentary techniques [4,6–9]. In addition, it has beenobserved that the overexpression of antiapoptotic Bcl-2protein in peripheral B-cells, with an imbalance of Bcl-2/Bax ratio and clonal expansion of these cells, actuallycorrespond to this genetic event [21]. Some interestingobservations in HCV-positive patients undergoing anti-viral treatment suggest that the clone of expanded cellsneeds viral replication to be maintained [21,22]. In ad-dition, the antiviral treatment generally utilized for HCV-related liver diseases, might also play a role in theprevention of the development of MC and probably ofdifferent B-cell lymphoproliferative disorders as well. Inconclusion, it is possible to hypothesize that duringchronic HCV infection, several factors, including theinteraction between HCV E2 protein and CD81molecule, the high viral variability, and the persistentinfection of both hepatic and lymphatic cells, may favora sustained and strong B-cell activation (Fig. 1). Thislatter may in turn favors the apparition of t(14;18)translocation with Bcl-2 protein overexpression respon-sible for abnormally prolonged B cell survival. Some

Fig. 3. Schematic representation of mixed cryoglobulinemia (MC)spectrum. MC may be classified according to different clinico-virol-ogical subsets: 1) the ‘essential’ mixed cryoglobulinemia (EMC);2) MC associated with autoimmune–lymphoproliferative disorders(ALD); 3) HCV-positive MC in the setting of well-known ALD;4) HCV-positive MC syndrome; and 5) MC associated with variousinfectious agents other than HCV.

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predisposing factors, such as a peculiar susceptibility ofRF producing B-cells to be activated, as well as thepresence of HCV epitopes, which selectively activatethese cells, may induce an elevated production of RFIgM. Moreover, the inhibition of B-cell apoptosis byBcl-2 overexpressed protein, may prolong the survival ofactivated B-cells favoring the development of MC syn-drome (Figs. 1 and 2). Similarly, the prolonged B-cellsurvival may represent a predisposing condition forfurther genetic aberrations, which may lead to frank B-cell malignancy [23,24].

A B-NHL may develop in patients with type II MC,usually after a long-term follow-up [1,4,6,11,25]. It canvary from diffuse large B-cell lymphoma (observed in40–50% of cases) to marginal-zone lymphoma (extra-nodal, nodal or splenic) or, more rarely, B-cell chroniclymphocytic leukemia (B-CLL) and lymphoplasmacyticlymphoma/immunocytoma (LPL/Ic) [4,26]. The malig-nancy may be related to peripheral B-cell expansion[6,23] and to lymphoid infiltrates observed in the liverand bone marrow of MC patients [25]. These infiltrateshave been regarded as “early lymphomas”, since they aresustained by lymphoid components indistinguishablefrom those of B-CLL and LPL/Ic [25]. However, un-like frank malignant lymphomas, they tend to remainunmodified for years or even decades and are fol-lowed by overt lymphoid tumors in about 10% of cases[4,25,26]. These characteristics justify the proposed termof “monotypic lymphoproliferative disorder of undeter-mined significance (MLDUS)” [4,25,26]. Of interest,type II MC-related MLDUS has its highest incidence inthe same geographic areas where about 30% of ‘idio-pathic’ B-CLL patients also display HCV-positivity, andwhere an increased prevalence of HCV genotype 2a/chas been observed in both MC and B-CLL [4,25–27].

4. B-cell and HCV syndrome

A small but significant percentage of HCV-infectedindividuals may develop a variable combination of bothhepatic and extra-hepatic complications, usually as latemanifestation. We proposed the term ‘HCV syndrome’to fully describe this complex etiopathogenetic and clin-ical spectrum [8]. The common immunological alter-ation of this syndrome is the HCV-driven B-lymphocyteproliferation responsible for a variety of autoimmunephenomena [1,4,6–9].

Fig. 2 shows the natural history of HCV syndromethat is the consequence of a multifactorial and multistepprocess; usually the syndrome develops from mild,often isolated symptoms (thyroiditis, diabetes, glomer-ulonephritis, arthritis, etc.) to systemic manifestations

(cryoglobulinemic vasculitis), in some cases complicat-ed by malignancy [4,6–9].

Among immune-rheumatic complications, MCrepresents a crossing-road between classical Sjogren'ssyndrome and/or polyarthritis, and other autoimmune(organ-specific or systemic) and/or lymphoproliferativedisorders ([4,6–9,28–33], Fig. 2).

5. HCV-negative cryoglobulinemia

In the classical description by Meltzer et al. [3] theterm ‘essential’ MC syndrome was referred to a distinctdisease if present the typical clinical triad — purpura,weakness, arthralgias-, serum mixed cryoglobulins,hypocomplementemia, and multiple organ involvement,in the absence of well-known infectious, autoimmune,hematological, and neoplastic diseases. After thedemonstration of the association with HCV in 1991[4,6–9], MC syndromemay be classified as ‘essential’ inonly a percentage of cases; the prevalence of ‘essential’MC syndrome largely vary among patients’ series fromdifferent countries [4,6–9]. On the other hand, focusingon the presence of circulating mixed cryoglobulins perse, regardless the etiology and possible clinical associa-tions, a higher prevalence of HCV-negative MC is found[9]. In particular, patients with HCV-negative MC maybe classified in the setting of various autoimmune–lymphoproliferative disorders (ALD) or as ‘essential’MC. Fig. 3 shows the main subsets of patients withserum mixed cryoglobulins according to both involvedetiological factors and clinical characteristics. Thespectrum includes: MC without any apparent causativeagent, namely ‘essential’MC and patients with differentALD; the large group of HCV-positive MC, more oftenas classical clinical syndrome or in association with otherwell-known ALD; and MC correlated to other infectious

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agents such as hepatitis B virus. MC syndrome, with orwithout HCV infection, in association with differentALD can be classified as ‘overlapping disorder’; anexample of this condition is the MC/Sjogren's over-lapping syndrome [8]. The above MC subsetting cannotquantify the actual dimension of different MC subgroupsin referring centers from different geographical areas; butit can be usefully employed in the clinical practice aswell as in the pathogenetic studies.

6. Therapeutic strategies

A correct therapeutic strategy to HCV-related MCmust deal with concomitant, conflicting conditions:HCV infection, autoimmune, and lymphoproliferativealterations. Following the pathogenetic process leadingfrom HCV infection to B-cell expansion, and finally toovert MC syndrome (Fig. 2), we can treat the disease atthree different levels by means of etiological, pathoge-netic, and/or symptomatic therapies [4,6–9,34–39]. Animportant therapeutic approach is the use of immuno-suppressors, chiefly the anti-CD20 treatment (Ritux-imab), able to reduce the B-lymphocyte proliferationthat represents the main pathogenetic step of the dis-ease. When possible, sequential or combined — anti-viral/immunosuppressive-therapy can represent a morecorrect strategy [39]. During the asymptomatic phasesof the disease patients usually do not need any treat-ment, even in the presence of high levels of cryocrit. Onthe whole, the treatment of MC should be tailored forthe single patient, according to the severity of clinicalsymptoms [39].

7. Concluding remarks

Although an increasing number of new insights in theetiopathogenesis of MC have been achieved during thelast 16 years, the disease presents some challenging openquestions. HCV infection is the main causative agent inover 3/4 of patients, but in a significant percentageof cases the etiology of MC remains still obscure. HCV-negative MC is clinically comparable to the HCV-asso-ciated disease, even if it may present a worse clinicalcourse in term of increased risk of developing B-NHL[40]. HCV-associated cryoglobulinemic vasculitis is oneof the most investigated models of virus-driven autoim-munity and cancer; however, the involved pathogeneticmechanism(s) are still controversial with regards to theactual role of HCV and possible co-factors. In addition,the appearance of B-NHL in the setting of HCV-positivepatients is particularly intriguing; future investigationsshould clarify whether it is the possible evolution of

virus-driven lymphomagenesis or it may appear asex novo event. Finally, the introduction of anti-CD20monoclonal antibody significantly improved the thera-peutic options, even if in some cases the treatment isparticularly difficult. Future clinical and laboratory stud-ies might be crucial for a better understanding of MCetiopathogenesis and not secondarily for their therapeu-tic implications.

Take-home messages

• B-cell expansion is responsible for bothMC and otheroverlapping disorders; this is a benign lymphoproli-feration that may evolve to frank lymphoma through amultifactorial and multistep pathogenetic process.

• HCV infection is the triggering factor in the majorityof MC patients; the prevalence of HCV-related MClargely varies among patients' populations from dif-ferent geographical areas. The same virus is respon-sible for various autoimmune-lymphoproliferativemanifestations; the term ‘HCV syndrome’ can bereferred to the wide spectrum of virus-related hepaticand extrahepatic diseases.

• B-cell proliferation in the setting of HCV-infectedpatients represents an important model of virus-driven autoimmune/neoplastic disorder.

• Cryoglobulinemic vasculitis can be treated by meansof etiological, pathogenetic, and/or symptomatictherapies. Sequential or combined-antiviral/immuno-suppressive-treatments should be tailored for thesingle patient, according to the severity of clinicalsymptoms.

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