Hepatitis C Virus Infection, Mixed Cryoglobulinemia ...

Leukemia and Lymphoma, 1998, Vol. 31(5-6),pp. 463-476

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Hepatitis C Virus Infection, Mixed Cryoglobulinemia, and Non-Hodgkin’s Lymphoma: an Emerging Picture FRANC0 DAMMACCO*, PIETRO GATTI and DOMENICO SANSONNO Department of Biomedical Sciences and Human Oncology Section of Internal Medicine and Clinical Oncology University of Bari Medical School Bari, Italy

(Received 20 February, 1998)

Hepatitis C virus (HCV) is a single-stranded RNA agent which expresses its genetic informations in the form of a single, large polyprotein encoded by an open reading frame (ORF) that extends through most of its genomic RNA. Proteolytic cleavage of the ORF product is essential for the virogenesis and the production of viral progeny. HCV is responsible for chronic liver disease, cirrhosis and possibly hepatocellular carcinoma. Viral persistence is considered the greatest problem in the management of HCV infection. It may result from several mechanisms, two of which are established. In the first, the high rate of genetic variations during viral replication results in the production of mutants capable of escaping the immune attack. In the second, the virus infects cells of the immune system itself, which represent a privileged site that cannot be reached by virus-specific T cell response. Involvement of lymphoid cells in the early stages of HCV infection may provide insight into the pathobiologic patterns of extrahepatic dissemination (lymph nodes, major salivary glands, kidneys, blood vessels). Dissemination of HCV-infected lymphoid cells throughout the organism is likely to maintain a mobile and extensive reservoir of the virus. In this respect, extrahepatic sites may act as a source of continuous reinfection of hepatocytes. Studies of intrahepatic B lymphocytes indicate that they are infected with HCV, clonally expanded and activated to secrete IgM molecules with rheumatoid factor activity. This strongly suggests that HCV directly stimulates B cell expansion, which may result in an indolent stage of lymphoproliferation (i.e., mixed cryoglobulinemia) or in frank B cell non-Hodgkin’s lymphoma (NHL). The frequency of NHL, however, is much lower than that of HCV infection, suggesting that HCV alone is not able to induce tumors and that cellular events, in addition to the presence of virus and virus-encoded products, are necessary in order to obtain a malignant B cell phenotype. The demonstration of HCV productive infection in bone marrow-recruited and circulating pluripotent hematopoietic CD34’ stem cells indicates that HCV replication occurs in the early differentiation stages of hematopoietic progenitors. These are stable cell populations and are likely to represent the initial site of infection and a continuous source of virus production.

Keywords: hepatitis C Virus infection, HCV, mixed cryoglobulinemia, non-Hodgkin’s lymphoma

* Correspondence to Franco Dammacco, M.D., Department of Internal Medicine and Clinical Oncology, Policlinico - 11, Piazza G. Cesare - 70124 Bari Italy - Phone: (39) 80 547.88.62 -Fax (39) 80 547.88.20. 463

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INTRODUCTION Hepatitis C virus (HCV) is a major human pathogen that causes acute and chronic infections (1). Although acute infection rarely results in severe illness, more than 80 % of the infected patients become chronic HCV carriers and may progress to a broad spectrum of liver disease, ranging from chronic hepatitis to cirrhosis and hepatocellular carcinoma (2), possibly as the consequence of complex interactions between viral and host factors (3). The occurrence of HCV infection and replication in sites other than the liver is a prominent biologic feature of its variable clinical outcome (4). The peculiar tropism of HCV for immunologically privileged tissues helps to explain its persistence and the development of immunologic abnormalities (5). The presence of HCV in lymphocytes and lymphoid organs, indeed, may result in a high mutation rate of HCV genome and the production of variant strains that escape the immune response. In addition, involvement of different cells (i.e., B and T lymphocytes, monocytes, pluripotent hematopoietic stem cells) may help to elucidate the mechanisms that seem to link HCV infection with autoimmunity, B cell dyscrasia and lymphomagenesis (6). Here, we will review recent evidence which correlates HCV infection with benign and malignant lymphoproliferative disorders. The provisional associations with chronic HCV infection will only be briefly reviewed, mainly to emphasize their expanding spectrum.

HEPATITIS C VIRUS GENOME, GENE-PRODUCTS AND PUTATIVE FUNCTIONS HCV is a lipid-enveloped, single-stranded, positive-sense RNA virus (7). It shows similarities with the family of Flaviviridae and has now been classified with these groups as a separate genus. Based on phylogenetic analysis of the core, E l and NS5 regions, six major genotypes (1 to 6) with more closely related variants within these groups (subtypes) were defined

(8). HCV genome of approximately 9,600 nucleotides in length contains highly-conserved noncoding regions (NCR) at both the 5’ and 3’ termini, which flank a large open reading frame (ORF) coding for a polyprotein of - 3,000 amino acids (Fig. 1). This represents a precursor that is subsequently cleaved into functional proteins by host and virus-encoded proteases. Core, E l and E2 are structural proteins and are located in the N-terminal quarter of the polyprotein. Downstream from the structural coding regions there are a series of nonstructural (NS) genes that code for proteins with enzymatic functions including protease, helicase, and polymerase. The complete 5’NCR consists of 341 nucleotides. Four stem-loops with the initiator AUG codon for translation of the polyprotein have been indicated in its secondary structure (9). This is the most conserved portion of the HCV genome and peculiar nucleotide variations characteristic of different genotypes do not modify its basic secondary and tertiary structure. Increasing evidence suggests that 5’NCR acts as internal ribosome entry site that directs translations of the RNA genome through an uncapped message (10). In the opposite position, the 3’NCR is a further highly-conserved portion of HCV genome, suggesting that it exerts a major regulatory role in the replication andor stabilization of viral RNA. It contains 30 nucleotides downstream the stop codon of the ORF with consistent variability between genotypes followed by a poly(U) tract of variable length, a polypyrimidine c(U)n stretch and a highly-conserved end of 98 base sequence (11). Viral proteins are generated by co- and post-translational cleavage of the precursor polyprotein, while host peptidases localized in the endoplasmic reticulum catalyze the cleavage of structural proteins. Cleavage between NS2 and NS3 occurs autoproteolytically via a viral protease encoded by NS2 and the N-terminal portion of NS3. Further distinct viral proteases act to produce NS3, NS4 and NS5 proteins (12). HCV core, a 21 kD protein (p21) of 191 amino acids in length, is a highly basic molecule with a hydrophobic region at the C terminus that likely acts as a signal for E l translocation to ER (13). A 19 kD (p19) product, which corresponds to a C-terminally


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FIGURE 1 Structure and genetic organization of the HCV genome. Processing products of the viral coding regions are represented as precursors (empty boxes) and mature (full boxes) proteins generated by sequentially proteolytic events. Inserted box shows hydrophobicity plots of HCV structural proteins. Envelope proteins contain hydrophobic sequences in their C-termini, supporting their role for the membrane anchoring. Hphob: hydrophobicity; Hphil: hydrophilicity

truncated form of p21, has been identified in vitro (14). The basic nature of the N-terminus portion of the core protein contains a DNA binding motif that may result in its nuclear translocation (15). However, there are no evidences of nuclear localization of the core protein in naturally-infected human livers (16). The core protein has also been shown to have RNA binding activity localized in the N-terminus of the molecule (14), whose significance remains to be established. Glycoproteins E l (gp31) and E2 (gp70) are the putative viral envelope proteins (17). It has been shown that El and E2 interact to form a complex. The resulting heterodimers remain totally membrane-associated (18). The administration of purified E1E2 proteins is capable of eliciting some degree of protection in immunized chimpanzee (19). The N-terminus of the E2 protein is a hypervariable segment that accounts for - 50 % of the nucleotide changes

and 60 % of the amino acid substitution of the entire region (20). The variability occumng in this region, referred to as highly variable region 1 (HVRl), possibly represents the molecular basis to generate efficient and rapid mechanisms for the virus to escape the host immune response. However, there are some evidences suggesting the presence of neutralizing epitopes within E2 protein, that likely are not confined to this specific region only (21, 22). NS2 protein forms part of an HCV-encoded protease which overlaps the NS3 serine protease domain. NS2-3 protein mediates autoproteolytic cleavage of 2/3 site of polyprotein (23). Protease activities mediating the cleavage of downstream NS proteins are contained in the first 181 amino acids of NS3 protein. It has been shown that NS4A protein acts as a cofactor in NS3 activity and seems to be essential for the NS3/4A and NS4B/5A cleavage. A trypsin-like molecule has been suggested by crystallizing studies of


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NS3 protease (24). The C terminal portion of NS3 protein seems to contain NTPase and RNA helicase activities (25). It is likely that RNA helicase is involved in the strand separation during viral replication and transcription. HCV helicase has 3’to 5‘ direction and intrinsically contains NTPase activity (26). The NS4 region encodes two viral proteins designated NS4A and NS4B. NS4A protein acts as a cofactor in NS3 protease activity (27). It contains a highly hydrophobic portion which may direct NS3 to the membrane (28). The function of NS4B is unknown. NS5 region codifies for two further proteins referred to as NS5A and NSSB that seem to associate important enzymatic activities. Aminoacid sequence variability identified in the C-terminus of NS5A appears to regulate interferon sensitivity (29). NSSB possesses, as predicted by comparative studies with other viruses, an RNA-dependent RNA polymerase (RpRd) activity (30). However, in vitro experiments demonstrate that replication directed by NS5B-associated RpRd is not only limited to those HCV template containing complete 3’NCR, suggesting that other viral and/or host components contribute to the specificity of RdRp activity in vivo.

REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN THE DETECTION OF GENOMIC STRANDS Since an effective cell culture system for the virus is still lacking, almost all data regarding HCV gene and gene-product functions are based on experiments established in mammalian cell lines capable of supporting the growth of HCV. Furthermore, all these systems rely on the use of reverse transcriptase-polymerase chain reaction (RT-PCR) for evaluating viral genomic strands. Particularly, data regarding negative strand RNA intermediate likely reflecting active replication in infected cells are based on the demonstration of negative sense molecules (2). The presence of HCV replicative intermediates has been questioned and relative results taken with extreme caution because of the poor specificity of the current techniques. False priming of the incorrect

strand, self priming due to complex S’NCR secondary structure, or random priming by small cellular nucleic acids are the possible events occumng during cDNA synthesis (31). To overcome falsely primed cDNA products, “tagged” PCR was recently developed. The primer used in the cDNA synthesis procedure contains additional nucleotides at the Send without homology to the HCV sequence. Primers specific for tug sequence on the one hand, and for HCV on the other, are used in the PCR development generating strands with much greater specificity, However, nonspecific detection of negative strands is not completely abolished, even with tagged primers; otherwise, the use of primers located outside SNCR seems to result in more specific products (32).

ZN SZTU DETECTION OF HCV Significant advances have been made in the localization of HCV in the tissues (33-39). Analysis of distribution of either antigens or genomic HCV RNA sequences in tissues preserving morphological details is crucial to understand virus-induced damage (40). In addition to hepatocytes, HCV has been found in epithelial cells of parotid glands (41) and in endothelial cells in cryoglobulinemic vasculitis (42). A peculiar tropism of HCV infection for immunologically privileged tissues has been emphasized, and it has been convincingly shown that HCV RNA can be detected in both peripheral blood and bone marrow mononuclear cells (4345). HCV antigens were demonstrated by immunofluorescence as diffuse and homogeneous deposits involving cytoplasmic rims and as distinct brilliant granules localized in submembrane areas or along the cytoplasmic membrane of both bone marrow and peripheral blood monocytes/macrophages, as well as B and T lymphocytes of chronically HCV-infected patients. Cell membrane signal accumulation was shown by immunodetection in unfixed cell suspensions, suggesting that sample processing and preparation are crucial steps in the demonstration of cell membrane expression of HCV proteins whose antigenicity is likely reduced or destroyed by fixative agents (46).



Immunofluorescence studies on the distribution of HCV-related proteins within subcellular compartments are consistent with a process of complete virus formation in hematopoietic cells. Cytoplasmic, submembrane or membrane staining may reflect intracellular HCV accumulation, a pre-secretory process and integration of the virus with cell membrane components, respectively. Furthermore, the demonstration of HCV RNA genomic strands by in situ hybridization in lymphocytes strongly suggests that these cells actually contain the virus (47). It has been shown that monocytes are the primary cells implicated in the pathogenesis of Flavivirus infection (48). Monocytes bearing receptors for the Fc portion of immunoglobulins bind IgG. Complexes of virus and antibody bind to the Fc receptor. If the antibody is non-neutralizing, the virus infects the normally poorly permissive Fc-bearing cells. This mechanism is likely to be operative in HCV infection, since the virus is usually complexed with non-neutralizing IgG antibodies (49). Granular distribution of HCV-reactive deposits is particularly evident in the macrophage cytoplasm with appearance of inclusion bodies. These undefined subcellular structures have been defined, by confocal microscopy in combination with membrane staining, as HCV immunoreactants distributed on the surface of lipid droplets in liver cells (16). Since this demonstration is lacking in monocytes, it can be inferred that “granules” are indeed phagosomal structures in that HCV is assembled and partially processed. Infection of immunocytes may be the underlying mechanism which accounts for the striking tendency of HCV infection to become chronic in a very high proportion of patients. It has been calculated that 70 to 80 % of infected patients become chronic carriers of the virus (2). This persistence seems primarily attributable to HCV’s ability to mutate rapidly and exist simultaneously as a series of related but immunologically distinct variants (quasispecies nature). Coexistence of multiple mutants provides a very efficient and rapid mechanism for the virus to escape host immunological pressure (50). Infection of blood mononuclear cells seems to be strictly correlated with progression of HCV carriage state. HCV was not

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detected in the peripheral blood mononuclear cells during the incubation period of acute-phase hepatitis C (51). The susceptibility of B, T and monocyte/macrophage cell lines to HCV infection has been demonstrated by the observation of PCR-driven HCV-specific sequences. These findings have been corroborated by in situ hybridization techniques in circulating and/or bone marrow-recruited mononuclear cells (43, 47). The multilineage character of HCV infection supports the contention that other cells in the hematopoietic microenvironment may serve as a potential virus reservoir. Recently, we have demonstrated that pluripotent hematopoietic CD34’ stem cells support productive HCV infection in chronically-infected patients (52). It was shown that HCV-harboring CD34+ cells are a consistent biologic feature, since in more than 80 % of HCV chronic carriers viral RNA was amplified both on extracted nucleic acids and in situ on intact CD34’ purified samples. Morphological evidence for the cellular accumulation of HCV proteins was provided by immunohistochemistry. In immunoreactive cells viral antigens were displayed as diffuse and homogeneous immune reactants within cytoplasmic compartments. Submembrane immune deposits were demonstrated for the E2 antigen, whereas core antigen was preferentially located around nuclear membrane. Flow cytometry analysis, furthermore, showed that both these proteins were consistently detected either at the surface or in the cytoplasm of the CD34’ cells, thus confirming that these cells do contain the virus which is not just adsorbed to their surface. An apparently complete extent for the viral cycle is likely to occur in CD34’ cells under conditions of spontaneous cultures, in that significant progressive increases of viral RNA concentrations were shown over time. Because the CD34’ hematopoietic progenitor cells are a stable population, they possibly represent the initial site of HCV infection and serve as unceasing source of the virus and its dissemination. Indeed, CD34 reactivity is maintained in the liver (53). This organ may retain a hematopoietic environment from the fetal period, and CD34’ cells present in


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the liver are likely independent of those from the bone marrow. Alternatively, the liver simply acts as a reservoir for circulating CD34’ cells because of the favourable conditions related to the hematopoietic microenvironment still remaining in adult organ (53). The same considerations possibly apply to lymph nodes, in which CD34+-reactive cells showed the same morphological features as those expressing HCV reactivities. However, CD34 antigen is expressed on endothelial cells and may be involved in leukocyte adhesion and “homing” during inflammatory processes, as well as in the localization of progenitor cells in bone marrow (54). The demonstration of CD34-positive cells in the liver may lead to hypothesize that this organ is indeed a possible primary target of HCV infection, and that infected CD34’cells leave the liver and migrate to the bone marrow, where they establish a life-long infected cell population. On the other hand, antigen-presenting stromal cells derived from hematopoietic stem cells migrate to the thymus and present viral peptides on its MHC molecules to remove self-reactive T cells. This attractive hypothesis may explain the mechanisms underlying the persistence of HCV infection. Whether HCV infection of CD34’ cells is related to lymphomagenesis, is an intriguing possibility. A pathogenetic role of HCV has been hypothesized for a subset of B-cell non-Hodgkin’s lymphomas and, in particular, for those non-Hodgkin’s lymphomas developing in patients with mixed cryoglobulinemia, a chronic immune-complex mediated disease sustained by B-cell clonal proliferation (55).

LIVER AS THE MAJOR SITE OF B-CELL LYMPHOPROLIFERATION HCV may sustain indolent stages of B cell lymphoproliferation. In the course of B cell clonal proliferation, somatic mutations arising in V region genes of immunoglobulins (IgV) generate different types of mutants (56). PCR has been succesfully employed to detect B cell clonality in lymphoid proliferation (57). PCR directed against the variable-diversity-joining

(V-D-J) region of Ig genes defines the unique combination of N-regions along with variations in the DH and JH regions and can be used as a clonal marker of the cell progeny. Applying this technique to characterize B lymphocytes isolated from liver tissue samples of HCV-infected cryoglobulinemic patients, we have demonstrated that B cell clonal expansion occurs in the liver of almost 90 % of these patients. In addition, intrahepatic B lymphocytes were infected with HCV and capable of spontaneous production of rheumatoid factor (RF) displaying the WA cross-reactive idiotype (Xld) in cultures. Therefore, liver may be considered as a major site of lymphocyte infection by HCV that likely stimulates B cells to produce 17.109 IgM molecules with RF activity. These proteins, thought to be germline gene products of WA group ( 5 8 ) , have been recently shown to be a constant component of soluble non-precipitating immune complexes in patients with acute and chronic HCV infection (49). WA Xld-positive immunoglobulins are molecules without RF activity and are thought to play the role of “natural” antibodies to common pathogens (59). Results from our laboratory indicate a close association between WA Xld IgM with RF activity and V-D-J Ig gene rearrangements, suggesting that RF activity is clonally-related and derived from somatically mutated molecules. Indeed, differences in Xld and fine specificities between RFs of different origin have been confirmed by the analysis of RF-encoding Ig V-genes; in addition to a broader use of different unmutated germ-line heavy and light chain variable region genes, somatically mutated V genes suggestive of an antigen-driven response was found (60). The accumulation of somatic mutations in Ig V-genes forms the molecular basis for the production of antibodies with high affinity. Somatic mutations are T cell-dependent, take place in the germinal centers of secondary lymphoid organs and characterize B cells (61). Since germinal center-like aggregates of lymphocytes are a consistent feature in the liver of patients with HCV-induced chronic disease, analysis of Ig V-genes amplified directly from lymphoid aggregates becomes especially important. These observations may lead to conclude that, like synovial membrane in patients with rheumatoid arthritis (62),


HCV. CRYOGLOBULINEMIA& NHL

the liver generates a microenvironment outside lymphoid tissue, in which a germinal center-like reaction is built-up. Analysis of V-D-J PCR products includes olygoclonal to monoclonal patterns, indicating that B cell expansion in the liver originates from very few or single cells. Each focus may derive from a different B cell of the polyclonal repertoire, with the result that different foci contain unrelated B cell clones. Whether these cells are primarily generated in the liver or whether they infiltrate this organ after pre-selection in germinal centers of lymph nodes, remains to be clarified.

MIXED CRYOGLOBULINEMIA AS AN HCV-ASSOCIATED B CELL LYMPHOPROLIFERATIVE PROCESS The demonstration of HCV in MC has led to hypothesize that mixed cryoglobulins (MCs) result from chronic stimulation of a population of WA Xld-positive B cells. Immunochemically MCs are classified as type I1 or type 111 on the basis of the presence of monoclonal or polyclonal IgM with RF activity, respectively. MCs account for almost 70 % of cases of cryoglobulinemia and may be secondary to lymphoid malignancies, infections and autoimmune disorders. However, in a major proportion of cases no primary cause is detected, and MCs are then designated as “essential” (EMC). EMC is definitely associated with chronic liver disease. Impaired liver function has indeed been reported in approximately two thirds of these patients (63). Whether cryoglobulins are promoted by the liver damage itself owing to reticuloendothelial system impairment in the blood clearance of circulating immune complexes, is still a matter for speculation. Many investigators have reported a 30-100 % prevalence of HCV antibodies in the sera of MC patients ( 64-66). In assessing the prevalence of anti-HCV antibodies in EMC, the results offered by the first-generation test, which included c 10&3 antigen as the target protein in the enzyme immunoassay (EIA), were accepted with caution, especially when sticky serum samples were considered. Subsequent

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introduction of the second-generation HCV EIA (c200k22-3) improved sensitivity in individuals at high risk for HCV infection and assay specificity when screening low-risk populations. These results were also corroborated by an independent supportive assay, namely a second-generation immunoblotting assay (4-RIBA) which, with the additional incorporation of c33c and c22-3 to the first-generation RIBA, significantly increased both sensitivity and specificity, so that this assay was proposed for the discrimination of infectious from noninfectious EIA-positive samples (67). Even though cryoglobulinemic sera are sticky samples, the second-generation EIA has been found to be a reliable test for anti-HCV detection. Cryocrit levels and RF titers are not disturbing factors, and consistent results are obtained when whole sera and clear supernatants are separately studied. It is assumed that the molar ratio between IgG and IgM in the cryoprecipitates is approximately 1.5:1, and that almost half of IgG possess anti-HCV activities other than anti-hepatitis B core, anti-Epstein-Barr virus, and anti-cytomegalovirus (68). The most definitive test for diagnosing active HCV infection is the RT-PCR procedure, which measures the presence of viral RNA. HCV RNA occurs in more than 80 % of MC patients, and a higher concentration is always found in the cryoprecipitates than in the supernatants (69, 70). We have shown that HCV RNA is entirely confined to the cryoprecipitates in 18 of 26 (69.2 %) patients, and is 103-105times more concentrated in the remaining 8 cases (31.8 %). Analysis of purified components of cryoprecipitates after chromatographic separation at 37°C shows that cryoglobulins dissociate almost completely to hepatitis C virions, monomeric IgG, and monomeric monoclonal RF (mRF) (71). WA is the major Xld among mRF in EMC (72). It is almost always associated with the light-chain Xld 17-109 and the heavy-chain Xld G6, which are postulated as the products of the restricted expression of germline genes with few or no somatic mutations (73). It had been inferred that VkIIIb light-chain detected by 17109 monoclonal antibody (Mab) and encoded by the HumKv 325 gene was the structural basis for the mRF WA Xld and was unique to IgM with RF activity


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(74). However, WA Xld was subsequently found in polyclonal IgM with and without RF activity (58). Our recent data (49) on the composition of soluble immune complexes ( 1 0 ) in acute and chronic HCV infection without cryoglobulinemia show that hepatitis C virions are bound to IgG molecules with specific anti-HCV activities, which in turn are linked to IgM bearing 17-109 Xld. The 17-109 Xld was detected on IgM in sera without cryoglobulins, even at low concentrations, consistent with the range indicated for natural antibodies and with the amount reported for polyclonal RFs occurring in patients with rheumatoid arthritis (75). The observation that these ICs are uniquely associated with HCV infection supports the view that, in this situation, WA Xld RF derives from a specific antigen-driven response. The demonstration of WA Xld-positive, RF-negative cells in normal individuals seems compatible with the suggestion that their physiological role is the secretion of natural antibodies to common pathogens, and that RF activity is a cross-reaction that develops with somatic mutations accompanying their proliferation (76). The factors controlling the expression and levels of particular Xld in a normal immune response still remain a subject of speculation. Our results suggest that the immune response to HCV infection basically includes an IgM bearing 17109 Xld in addition to IgG with anti-HCV activity. Despite several efforts, demonstration of anti-HCV activity of IgM 17-109 Xld was consistently met with failure. A 17-109 Mab-based test was used to study IgG anti-HCVAgM RF 17-109 Xld (IgG-IgM) ICs in whole cryoprecipitates and in purified components of cryoproteins (49). Since it was assumed that ICs could be detected by 17-109 Mab once they were combined with HCV-related antigens fixed onto a solid phase, these results confirmed that ICs in HCV infection comprise IgG with anti-HCV activity linked by IgM 17-109 Xld, which does not directly bind to HCV antigens. It is not known, however, whether IgM can be cross-reactive with an HCV-induced self antigen. Several IgM mRF encoded by germline genes have been found to cross-react with non-lgG antigens (77). Furthermore, the concentration of

IgG-IgM ICs increases in supernatants in relation to decrements of cryocrit and RF concentration. This suggests that IC cold-dependent insolubility is the result of a critical antigen-to-antibody (1gG:IgM) ratio, in that an excess of antibody component appears to promote the cryoprecipitating mechanism. Suggestive evidence for the direct role of HCV in the pathogenesis of cryoglobulinemic vasculitis is the demonstration of HCV RNA or HCV-related proteins in the vessel walls of HCV-infected MC patients (42). Active cutaneous vasculitis was strikingly associated with the presence of HCV in the blood vessels. We have recently found (unpublished results) HCV RNA sequences in situ in 94.7 % of patients with MC cutaneous vasculitis, and in 18.2 % of patients with noninflammatory changes (p < 0.001). In the adjacent sections, viral proteins were found in 78.9 % of patients with vasculitis and in 27.3 % of those without (p c 0.005). Although the demonstration of HCV antigens in the vessel walls of MC patients suggests a pathogenetic role for this virus in the induction of vasculitis, the lack of a direct correlation between vascular HCV antigen reactivity and inflammation indicates that further mechanisms are likely to be involved in the pathogenesis of vasculitis, and that HCV antigen deposition precedes and possibly triggers tissue damage. Evidence for the role of cryoproteins is the demonstration of IgM, IgG, and complement fractions at the sites of vascular damage. However, the failure to demonstrate a direct relationship between the amount of circulating cryoproteins and vascular damage argues against their direct role in the production of vasculitis, and suggests that peculiar characteristics of ICs and/or local environment are crucial factors in the endothelial damage. The extent of cryoglobulin deposition may be the result of a critical antigen:antibody ratio in circulating ICs, in that high-molecular weight ICs are rapidly cleared from the circulation, whereas low-molecular weight material containing dissociated IgG and IgM is trapped within tissues (78). This strongly suggests that tissue-specific ICs are formed in situ through the binding of HCV proteins expressed on endothelial cells to serum immunoglobulins.



There is also evidence that HCV is an important cause of cryoglobulinemic nephropathy (79, 80). Glomerulonephritis in patients with MC is classified as membranoproliferative (MPGN) with subendothelial deposits. Johnson et a1 (81) have reported a strict association between MPGN and HCV infection. They studied kidney biopsy material from eight patients with subendothelial immunodeposits displaying the classic cryoglobulin-like substructure. Even though all patients had both HCV RNA and antibodies to HCV in their serum and circulating cryoglobulins, HCV RNA or HCV-related proteins could not be demonstrated in their tissue. Renal damage could thus be the result of an immune-mediated reaction in that a tissue antigen released by the virus or a neoantigen induced by viral infection may act as the main target in the cytopathogenetic mechanism(s). This hypothesis, moreover, is in keeping with many examples of animal and human models of IC disease, in which glomerular damage has been strictly associated with viral infection despite the lack of a direct evidence of viral antigens in tissue lesions (82). We used a panel of Mabs directed against structural and non structural proteins to demonstrate HCV-specific immunoreactive deposits in kidney biopsy specimens from HCV-chronically infected patients with MPGN and MC (71). Two main immunohistochemical patterns were established as regards glomerular deposition of HCV antigens: 1) diffuse granular deposits in the mesangial and paramesangial cells; and 2) heavy, diffuse, and homogeneous deposits along the glomerular capillary walls. Furthermore, fine as well as coarse HCV immunoreactive deposits were found within tubulointerstitial arterioles, sometimes completely filling the microvascular spaces. HCV antigen reactivity confined to arteriolar vessels was also detected in apparently normal vascular structures and in the absence of inflammatory infiltrates. HCV antigen-positive signals were shown within and between endothelial cells up to the layers of basal lamina. Scanty HCV antigen deposits were found in glomerular and tubular epithelium. Immunodetection of HCV antigens in the kidney was compared with reactivity for Ig and complement fractions. While IgM and IgG deposits were shown in

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88.8 % and 44.4 % of the cases, faint IgA deposits were found in only 11.1 %. In addition, complement fraction 3 (C3) was almost invariably detected, but C4 deposits were never found. Immunodeposits of Ig and C3 resembled the glomerular pictures of HCV antigen deposits. The two HCV-related immunohistochemical patterns were found simultaneously in a small proportion of patients, suggesting that they may represent different stages of disease or different phases of HCV infection. Indeed, as compared with patients with homogeneous glomerular HCV deposition, those with a granular pattern had more pronounced renal damage, whereas no significant differences were found in terms of other clinical parameters, viremic state, and cryocrit levels. Major questions to be answered are why only a small proportion (10 to 15 %) of patients with HCV develop MC while other do not, and why a consistent RF production occurs in cryoglobulinemic patients only. Possibly, affinity maturation through hypermutation can take place specifically in lymphocytic infiltrates of a subgroup of HCV-infected patients, in which there is a failure of the mechanism(s) for the silencing of higher affinity, potentially pathologic RF-expressing B cells, as recently indicated (83). This is a T cell-dependent mechanism, whereby a complete B cell activation involving the formation of germinal centers and sustained high levels of RF secretion occurs only if T cell help is provided. These data support a primary pathogenetic role of HCV in indolent stages of B cell lymphoproliferation which underlines chronic production of cryoglobulins, and emphasize that MC is a condition with high potential risk of developing a B cell lymphoma.

NON-HODGKIN’S LYMPHOMAS (NHLS) AND CHRONIC HCV INFECTION Data from Italy (84, 85) and USA (86) indicate that the prevalence of HCV infection in B cell NHL ranges from 9 % to 32 %, and these values are significantly higher than those found in other hematologic disorders. A recent study (87) based on stringent cri-


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teria to characterize B-cell NHL in HCV-infected patients revealed a frequent extranodal localization, with peculiar target organs (i.e., the liver and the major salivary glands), a diffuse large cell histotype without history of low-grade B-cell malignancy or bone marrow involvement and, surprisingly, a weak association with full-blown predisposing autoimmune diseases, including cryoglobulin production. These features strongly indicate that HCV-related B cell NHLs are distinct from immunocytomas frequently diagnosed in HCV-positive patients with mixed cryoglobulinemia. As stated in this series, extranodal sites were primarily involved at the onset of NHL. In particular, the liver and the major salivary glands were significantly overrepresented. Both these organs are targets of active HCV infection; since nonlymphoid cells are typically infected by HCV, a local role of HCV as an exogenous trigger of B-cell proliferation should be considered in NHL lesions. HCV localization in lymph nodes has been recently described (88). Infected cells are mainly detected in extrafollicular areas. Rare but distinct HCV-harboring cells were detected in germinal centers, indicating their participation in physiologic germinal center reactions which ensure their long survival. HCV-infected mononuclear cells were found in the hilar or capsular vessels of lymph nodes, suggesting a highly dynamic traffic between lymph nodes and infected compartments. Thus, it may be assumed that cells in transit through the germinal centers would be exposed continually to a reservoir of readily available virus. The efficiency of infection may depend on many variables and is probably determined by infectious and replication-competent virus particles and the rate of cell passage through lymphoid tissue. The events occurring in lymph nodes in HCV-infected individuals without peripheral lymphadenopathy are unknown, since tissue from such individuals rarely becomes available for examination. The above described pictures of distribution of HCV-infected mononuclear cells seen in hyperplastic lymphadenopathy is completely lost in frank nodal NHL. HCV-infected cells are generally confined to limited areas of the cortex or randomly distributed in

the neoplastic tissue. HCV-specific signal, furthermore, is confined to small cells and is associated with low-grade disease, suggesting that the expression of HCV gene products is closely related to certain stages of cell differentiation. This is similar to the situation found in hepatocellular carcinoma, in that many more HCV-infected cells are found in non-neoplastic areas as compared with their neoplastic counterparts (40). Cell de-differentiation may change the affinity of neoplastic cells for HCV and indicates that transformed cells are no longer permissive to HCV replication. This implies that HCV is not a proliferating stimulus in the late stage of the neoplastic process and that other factors contribute to ongoing disease. A distinct feature of HCV-specific signal in lymph nodes is its detection in single, elongated elements reminiscent of interdigitating reticulum cells. This point, in our opinion, deserves further comments. It appears that the follicular dendritic cell network in lymphoid tissue serves as a filter that retains the virus, enclosed in immune complexes, on the surface of the follicular dendritic processes. The virus-antibody complex would thus be available to any susceptible cell that might migrate through the germinal centers. It is reasonable to assume that cells in transit would be exposed continually to a reservoir of readily available infectious virus. Otherwise, depopulation of HCV-infected cells in late, progressive NHL may be related to disorganization of the follicular dendritic cell network with subsequent decreased trapping and release of virus-antibody complexes from lymph nodes.

WHAT IS THE ROLE OF HCV IN THE PATHOGENESIS OF NHL? It can be postulated that HCV is the stimulus not only for the apparently benign lymphoproliferative process underlying MC, but also for progression to frank malignancy in a subgroup of patients. Somatic mutations are introduced into Ig V-region genes in a stepwise fashion during the course of clonal proliferation leading to high-, low-, and auto-affinity mutants. Survival of these cells seems to be dependent on their



binding to the low levels of antigen-antibody immune complexes that are exposed on the surface of follicular dendritic cells. It is clear from our studies that the number of HCV-infected cells in neoplastic lymph nodes is lower than that found in hyperplastic lesions. This supports the contention that HCV infection precedes the expansion of the tumor clone, possibly contributing to tumor development. In addition, down-regulation of HCV-carrying cells in frank lymphoma indicates that it does not directly participate in the pathogenesis of the advanced stages of the tumor. The potential role of viral antigens in the growth stimulation andor clonal selection of NHL has been strengthened (89). Indeed, comparative analysis of the sequence of the Ig heavy chain variable region expressed by a lymphoma subgroup and its germline counterpart during the course of the disease revealed that these regions carry somatic mutations analogous for type and distribution to those that normally are selected by antigens. The emerging data imply a causative role for HCV in both benign and malignant lymphoproliferation. Experiments addressed to characterizing the acquisition of transforming mutations which may lead to lymphoma will possibly allow to redefine NHL classification and to design more appropriate therapies.

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