the incidence of non - hodgkin lymphoma ( nhl ) is rising worldwide and is higher in developed countries than in africa and asia .
b cell non - hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis c virus ( hcv ) infection . the prevalence of hcv infection in patients with b cell non - hodgkin lymphoma is approximately 15% .
the hcv envelope protein e2 binds human cd81 , a tetraspanin expressed on various types of cells , including lymphocytes , and activates b cell proliferation .
infection and replication of hcv were observed in b cells [ 6 , 7 ] although the direct effects , particularly in vivo , have not been clarified . to determine the direct effect of hcv infection on b cells in vivo , we crossed transgenic mice with an integrated full - length hcv genome ( rz ) under the conditional cre / loxp expression system , with mice expressing the cre enzyme   under transcriptional control of the b lineage - restricted gene cd19 .  
to investigate the mechanism of development of lymphoproliferation or b cell non - hodgkin lymphoma in hcv patients , we also developed a transgenic mouse model that conditionally expressed hcv cdna ( nucleotides 2943435 ) , including the viral genes that encode the core , e1 , e2 , and ns2 proteins , by using the cre / loxp system ( in core - ns2 [ cn2 ] mice ) [ 10 , 11 ] .
conditional transgene activation of the hcv cdna ( core , e1 , e2 , and ns2 ) protects mice from fas - mediated lethal acute liver failure , by inhibiting cytochrome c release from mitochondria .
persistent hcv protein expression is established by targeted disruption of interferon regulatory factor-1 ( irf-1 ) , and high incidences of lymphoproliferative disorders are noted in irf-1 cn2 mice .
previously , transgenic mice that expressed the hcv core protein were established using a promoter derived from hepatitis b virus , whereas mice that expressed structural or complete viral proteins were established using promoters derived from the albumin gene .
these mice were immunotolerant to the transgene and did not develop hepatic inflammation . however , they developed age - related hepatic steatosis and hepatocellular carcinomas .
in contrast , the cn2 mice used in the present study were not immunotolerant to the hcv gene and developed hepatitis after the onset of hcv gene expression .
however , the expression of hcv in these mice was usually lost after 21 days .
therefore , an animal model of persistent hcv protein expression is required for examining the effects of chronic hcv infection in vivo .
ifn signaling mediates tumor - suppressor effects and antiviral responses and is regulated by key transcription factors of the interferon - regulatory factor ( irf ) protein family , including irf1 , irf2 , irf3 , irf7 , and irf9 .
targeted disruption of irf-1 results in aberrant lymphocyte development and a marked reduction in the number of cd8 t cells in the peripheral blood , spleen , and lymph nodes .
the mechanisms by which hcv infection induces ifn resistance and influences the development of lymphomas are poorly understood .
therefore , we established an irf-1 cn2 mouse model of persistent hcv expression , which allowed us to investigate the effects of hcv on lymphatic tissue tumor development .
the full - genome hcv expression was induced by the cre / loxp system with cd19cre ( figure 1(a ) ) .
the incidence of b cell lymphomas in rzcd19cre mice was 25.0% ( 22.2% in male and 29.6% in female mice ) and was significantly higher than the incidence in the hcv - negative groups .
most were cd45r positive and located in the mesenteric lymph nodes ( figure 1(c ) ) .
indeed , the expression of the hcv or hcv proteins induces the spontaneous development of b cell lymphomas , irrespective of the integrated site in the mouse genome .
serum concentrations of il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-9 , il-10 , il-12(p40 ) , il-12(p70 ) , il-13 , il-17 , eotaxin , g - csf , gm - csf , ifn- , kc , mcp-1 , mip-1 , mip-1 , rantes , tnf- , il-15 , fgf - basic , lif , m - csf , mig , mip-2 , pdgf , vegf , alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) were not significantly different in the presence or absence of b cell lymphomas .
interestingly , the average sil-2r level was significantly higher in the sera from rzcd19cre mice with b cell lymphomas ( 830.3  533.0 
pg / ml ) than in that from tumor - free control groups , including the rzcd19cre , rz , cd19cre , and wild - type ( wt ) mice ( 499.9  110.2  pg / ml ; p < 0.0057 ) ( figure 1(d ) ) .
the difference in the average sil-2r levels between the sera from groups with tumors other than b cell lymphomas ( 430.46  141.15 
pg / ml ) and that from tumor - free control groups was insignificant ( p > 0.05 ) .
moreover , all rzcd19cre mice with a relatively high level of sil-2r ( > 1000  pg / ml ) presented with b cell lymphomas .
a significant increase in sil-2r was also observed in mxcre / cn2 - 29 mice that expressed the hcv cn2 gene   and had b cell lymphomas , compared with tumor - free control ( cn2 - 29 ) mice . to examine
whether sil-2r was derived from lymphoma tissues , we quantified il-2r concentrations in splenocytes , peripheral blood lymphocytes ( pbls ) , and b cell lymphoma tissues .
the concentration of il-2r was significantly higher in splenocytes from rzcd19cre mice than in splenocytes from cd19cre mice .
moreover , the concentration of il-2r in b cell lymphoma tissues from rzcd19cre mice was higher than that in splenocytes .
these results strongly suggest that b cell lymphomas directly contribute to the elevated serum concentrations of sil-2r in rzcd19cre mice .
they also strongly support the possibility that persistent expression of hcv could directly induce transformation of b cells .
rzcd19cre mice are immunotolerant to hcv , because hcv is expressed in b cells before birth .
in contrast , cn2 mice express hcv after they are administered the recombinant adenovirus that expresses the cre enzyme ( figures 2(a ) and 2(b ) ) .
the expression of hcv in these mice is usually lost after 21 days ( figure 2(b ) ) through removal of hcv - expressing hepatocytes by the immune response . to establish persistent hcv expression , we disrupted irf-1 by crossing of rzcd19cre mice with irf-1 mice .
irf-1 plays a significant role in the th1-type immune response and its absence is expected to decrease the elimination of hcv - expressing cells .
as expected , hcv expression in irf-1 cn2 mice persisted for more than 500 days ( figure 2(c ) ) .
a significant percentage of the mice that expressed the hcv core protein ( irf-1 cn2 mice ) showed polyclonal lymphoid growth disturbances , including splenomegaly , expanded lymph nodes , adenocarcinoma in the abdomen or leg , and lymphoma of the liver or peyer 's patches ( figures 2(d ) and 2(e ) ) .
in contrast , hepatocytes with abundant expression of hcv proteins rarely developed into hepatocellular carcinomas .
hematoxylin and eosin ( h&e ) staining of splenomegalic tissue showed extensive hyperplasia of the white pulp zones , in which the cortical zones contained lymphoid follicles and scattered germinal centers although mitotic figures were rarely observed .
these results indicate that persistent expression of hcv proteins frequently induces lymphoproliferative disorders in addition to liver hyperplasia , which is consistent with the phenotype of patients with hepatocellular carcinoma .
the average ratio of t cells to b cells in the lymph nodes and spleens of cn2 mice was significantly higher than that in wt mice .
the majority of cd3 lymphocytes and a few cd8 lymphocytes expressed cd4 on their surfaces .
these results confirm that hcv protein expression induces lymphoproliferative disorders that involve excessive expansion of both t cells and b cells .
the cell population that showed negative results for t cell receptor (  ,  ,  , and  isoforms ) staining was smaller in irf-1 cn2 mice than that in the other mice .
the disruption of irf-1 inhibited fas - induced apoptosis , presumably by decreasing the levels of caspase-6 and caspase-7 messenger rna .
these results suggest that the reduced expression of effector caspases delays fas - mediated apoptosis in irf-1 mice and prevents the elimination of hcv - expressing cells in vivo .
the cn2 mice showed significantly increased levels of serum il-2 , il-10 , and il-12 ( figure 3(a ) ) .
notably , the cn2 mice with proliferative disturbances in the lymph nodes and spleen had dramatically elevated levels of these cytokines , suggesting that altered cytokine production is involved in aberrant lymphocyte proliferation or differentiation .
in contrast , the irf-1 cn2 mice did not show elevated levels of serum il-12 , but had significantly higher levels of serum il-2 and il-10 than did irf-1 mice ( figure 3(b ) ) .
thus , the disruption of irf-1 negated the increase in the il-12 level , but augmented the increases in the levels of il-2 and il-10 in cn2 mice .
these results indicate that il-2 and il-10 play key roles in the induction of the lymphoproliferative phenotype in irf-1 cn2 mice .
a significantly positive correlation was found between the cytokine levels and spleen weights of cn2 gene - expressing mice with the irf-1 background ( r = 0.43 , p < 0.05 , and r = 0.53 , p < 0.05 , resp . ) .
these results indicate that il-2 and il-10 are involved in lymphoproliferation in viral protein - expressing mice .
bcl-2 is an integral inner mitochondrial membrane protein , overexpression of which blocks the apoptotic death of pro - b - lymphocyte death .
bcl-2 transgene expression increases the oncogenic potential   and is linked with b cell neoplasm and t(14;18 ) translocation .
we therefore examined the level of bcl-2 protein and found that it was upregulated in the lymph nodes of irf-1 cn2 mice after 400 days ( figure 3(c ) ) .
il-10 treatment in the presence of il-2 greatly inhibited fas - induced apoptosis in irf-1 cn2 mice compared with other groups ( table 1 ) .
furthermore , irf-1 disruption accelerated the resistance of splenocytes to fas - induced apoptosis in the presence of il-2 , il-10 , and/or il-12 .
in particular , il-2 plus il-10 treatment produced the strongest upregulation of the bcl-2 mrna levels in splenocytes of irf-1 cn2 mice .
this indicates that il-2 , il-10 , and/or il-12 contribute to upregulation of bcl-2 expression , which subsequently inhibits fas - induced apoptosis .
these results indicate that aberrant cytokine expression and disruption of ifn signaling affect bcl-2 expression synergizing with hcv proteins , which is associated with the inhibition of caspase expression .
these results indicate that the hcv core and e2 proteins are responsible for il-2 , il-10 , and il-12 expression
. core protein expression and il-10 stimulation most strongly induced bcl-2 expression ( table 2 ) . from these results
, core protein contributes significantly to the induction of bcl-2 in the presence of cytokines .
our results show that the conditional expression of hcv proteins induces inflammation and lymphoproliferative disorders .
furthermore , established animal models will probably provide critical information for the elucidation of the molecular mechanism(s ) underlying the spontaneous development of b cell non - hodgkin lymphoma after hcv infection .
therefore , irf-1-inducible genes probably play essential roles in suppressing hcv - induced lymphoma and in eliminating hcv protein - expressing cells .
the overexpression of apoptosis - related proteins ( including bcl-2 ) and/or aberrant cytokine production are the primary events in hcv - induced lymphoproliferation .
the hcv gene has the potential to induce b cell lymphomas in rzcd19cre mice , without inducing host immune responses against hcv gene product .
this is in agreement with the results of a previous study , which indicate that viral elimination reduces the incidence of malignant lymphoma in patients infected with hcv .
the incidence of b cell lymphoma in the hcv transgenic mouse strain ( mxcre / cn2 - 29 ) is high , and this strongly suggests that development of b cell lymphomas occurs via expression of the hcv transgene .
the level of il-2r was higher in splenocytes of rzcd19cre mice than in those of cd19cre mice ; however , the differences in the serum concentrations of sil-2r between rzcd19cre mice without b cell lymphomas and other control groups ( rz , cd19cre , and wt ) were insignificant .
these results indicate that hcv increases il-2r expression in b cells ; proteolytic cleavage of il-2r increased after b cell lymphoma development in the rzcd19cre mice . the detailed mechanism by which hcv expression induces il-2r remains unclear , but hcv core protein induces il-10 expression in mouse splenocytes .
il-10 upregulates the expression of il-2r ( tac / cd25 ) in normal and leukemic b lymphocytes .
therefore , through il-10 , the hcv core protein might induce il-2r in b cells of the rzcd19cre mouse .
this leads to lymphoproliferative diseases resulting from reduced apoptosis ( i.e. , lower levels of caspase-1 , caspase-6 , and caspase-7 expression ) .
hcv cn2 transgenic ( tg+ ) mice are resistant to fas - induced apoptosis because of the inhibition of cytochrome c release from mitochondria
. mice with disruption of irf-1 have several defects in their innate and adaptive immunities , including lineage - specific defects in thymocyte development , and the development of immature t cells into mature cd4 cells but not cd8 t cells [ 16 , 23 ] .
irf-1 controls the positive and negative selection of cd8 thymocytes   and is required for the development of the th1-type immune response .
the absence of irf-1 induces th2-type immune response [ 16 , 25 ] . the number of natural killer cells is dramatically reduced in irf-1 mice .
this defect may markedly increase viral protein expression and inhibit tumor surveillance mechanisms , leading to the development of non - hodgkin lymphoma .
hypermutation of the immunoglobulin genes in b cells induced by hcv infection is the cause of the lymphomagenesis observed in hcv infection [ 16 , 26 ] .
this finding may provide a more direct insight into lymphoma production , because hcv - induced hypermutation causes genetic instability and chromosomal aberrations , possibly resulting in neoplastic transformation .
in addition , the antiapoptotic phenotype resulting from sustained viral protein expression may enhance the survival of lymphocytes and inhibit activation - induced cell death to turn off the activated lymphocytes .
the dysregulated cytokine profiles and sustained lymphocyte survival may alter the fates of regulatory t cells and dendritic cells . in summary , the mouse model of b cell lymphoma and lymphoproliferative disorder represents a powerful tool to address the molecular mechanism of lymphoma development by hcv .
 b cell non - hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis c virus ( hcv ) infection . 
 the mechanism by which hcv infection leads to lymphoproliferative disorder remains unclear . 
 our group established hcv transgenic mice that expressed the full hcv genome in b cells ( rzcd19cre mice ) . we observed a 25.0% incidence of diffuse large b cell non - hodgkin lymphomas ( 22.2% in male and 29.6% in female mice ) within 600 days of birth . 
 interestingly , rzcd19cre mice with substantially elevated serum - soluble interleukin-2 receptor -subunit ( sil-2r ) levels ( > 1000  pg / ml ) developed b cell lymphomas . 
 another mouse model of lymphoproliferative disorder was established by persistent expression of hcv structural proteins through disruption of interferon regulatory factor-1 ( irf-1_/_/cn2 mice ) . 
 irf-1_/_/cn2 mice showed extremely high incidences of lymphomas and lymphoproliferative disorders . 
 moreover , these mice showed increased levels of interleukin ( il)-2 , il-10 , and bcl-2 as well as increased bcl-2 expression , which promoted oncogenic transformation of lymphocytes . 
1. Introduction
2. Spontaneous Development of B Cell Lymphomas in the RzCD19Cre Mouse
3. Persistent HCV Expression and Lymphoproliferative Disorder
4. Conclusion
 Conflict of Interests