Hepatitis C virus-mixed cryoglobulinemia-lymphoma relationship.
ABSTRACT HCV (hepatitis C virus) chronic hepatitis has become one the most expensive diseases for public health systems all over the world in the past 10-20 years, a real epidemic, the second most frequent, after hepatitis B virus infection. Due to the complex manifestations, one may consider HCV infection as a "systemic" disease. Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of HCV infection, but cryoglobulinemic vasculitis (CV) is considered to be relatively sparse although prevalence studies are needed. Presence of serum cryoglobulins is essential for MC diagnosis, but serum levels do not correlate with the disease activity or prognosis. MC can be defined as a B lymphocyte proliferation disease being characterized by polyclonal activation and antibody synthesis. Evolution to lymphoma should be considered continuous but also other infectious, environmental or genetic factors could be involved. The t (14.18) translocation and Bcl-2 activation in B lymphocytes, B cell-activating factor (BAFF), E2-CD81 interaction, immunoregulatory T CD4+CD25(high) + lymphocytes and type III IFNs might play an important role in MC and lymphoma evolution in HCV patients.
Hepatitis C Virus – Mixed Cryoglobulinemia – Lymphoma Relationship
ADRIANA NICOLAU1, R. TĂNĂSESCU²,3, EUGENIA BĂLĂNESCU1,
P. BĂLĂNESCU1, RUXANDRA PĂTRAŞCU1,3, C. TĂNĂSESCU1,3
1 Internal Medicine, “Colentina” Clinical Hospital, Bucharest, RO
2Neurology, “Colentina” Clinical Hospital, Bucharest, RO
3University of Medicine and Pharmacy “Carol Davila”, Bucharest, RO
HCV (hepatitis C virus) chronic hepatitis has become one the most expensive diseases for
public health systems all over the world in the past 10–20 years, a real epidemic, the second most frequent,
after hepatitis B virus infection. Due to the complex manifestations, one may consider HCV infection as a
“systemic” disease. Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of HCV
infection, but cryoglobulinemic vasculitis (CV) is considered to be relatively sparse although prevalence
studies are needed. Presence of serum cryoglobulins is essential for MC diagnosis, but serum levels do not
correlate with the disease activity or prognosis. MC can be defined as a B lymphocyte proliferation disease
being characterized by polyclonal activation and antibody synthesis. Evolution to lymphoma should be
considered continuous but also other infectious, environmental or genetic factors could be involved. The t
(14.18) translocation and Bcl-2 activation in B lymphocytes, B cell-activating factor (BAFF), E2-CD81
interaction, immunoregulatory T CD4+CD25high+ lymphocytes and type III IFNs might play an important
role in MC and lymphoma evolution in HCV patients.
Key words: hepatitis C virus, mixed cryoglobulins, lymphoma, t (14.18), Bcl-2, BAFF, E2-
CD8, Tregs, type III IFNs.
HCV (hepatitis C virus) chronic hepatitis has
become one of the most expensive diseases for public
health systems all over the world, in the past 10–
20 years. We can say there is a real epidemic, with
a prevalence of over 170 million, an incidence of
3.3% per year and responsible for over 12000 deaths/
year. Nearly 170 million people are already infected,
meaning about 3% of world population, a number
5 times higher compared to HIV . It was the
major cause of posttransfusional hepatitis after 1990.
When serological screening was introduced the
incidence decreased significantly but still remains a
problem for intravenous drug users.
HCV infection is associated with autoimmune
extrahepatic manifestations such as cryoglobulinemic
vasculitis, nephritis, arthritis due to immunological
imbalances that HCV can induce. A study group
reported that 74% of patients with chronic hepatitis
C had at least one extrahepatic manifestation, the
most common being mixed cryoglobulinemia (MC–
40%) followed by arthritis or arthralgia (23%),
paresthesia (17%), myalgia (15 %), pruritus (15%)
and sicca syndrome (11%). Due to the complex
manifestations, one may consider HCV infection as
a “systemic” disease .
MC is the most common extrahepatic manifes-
tation of HCV. Recently it has been shown that
most of the MC patients had also HCV infection.
This correlation was found between 30 and 96% of
cases, depending on the geographic area .
Some authors consider today that HCV infection
can be excluded in MC patients only after the virus
is not detected in the cryoprecipitate and/or tissue
samples (skin, peripheral nerves, kidneys, lympho-
cytes). Its heterogenic composition can be assessed
by cryoprecipitate analysis, which includes immuno-
electrophoresis or immunofixation, or even more
sensitive methods such as immunoblotting and two-
dimensional polyacrylamide gel electrophoresis.
Molecular biology techniques (including VHC-
RNA via in-situ hybridization) and immunohisto-
chemistry showed that HCV antigens are directly
involved in cryoglobulinemic vasculitis (mediated
by immune complexes) and its manifestations [6–9].
Thus, anti HCV antibodies and HCV-RNA are
frequently detected in cryoprecipitate having concen-
trations of 30 to 1000 times higher than the serum
levels, where cryoglobulins may remain undetected.
So “essential cryoglobulinemia”term is used to a
lesser extent and only after absence of immune and
ROM. J. INTERN. MED., 2011, 49, 1, 3–10
4 Adriana Nicolau et al. 2
HCV-RNA detection in the cryoprecipitate .
Also, the fact that viral particles were detected by
in-situ hybridization from nerve and muscle samples
from HCV infected patients with peripheral
neuropathy but without MC, raised the question if
the virus has a direct cytopathic action .
Fig. 1. Etiopathogenesis of HCV syndrome, adapted with permission after Ferri et al., B-cells and mixed cryoglobulinemia
Autoimmunity Reviews 7 (2007) 114–120.
HCV AND CRYOGLOBULINS
Cryoglobulins are serum immunoglobulins
that precipitate at temperatures below 37ºC and
redissolve when the temperature rises. Cryoglobulins
can be associated with hematological, infectious or
rheumatologic diseases. The immunochemical
composition divides cryoglobulins in three groups,
all of them could be HCV associated:
• type I (25%) monoclonal immunoglobulins
(IgM more frequently) mainly associated
with hematological malignancies (lympho-
proliferative disorders, plasmocytoma,
Waldenstrom macroglobulinemia, multiple
myeloma, monoclonal gammopathy with
undetermined significance). They are
frequently asymptomatic or associated with
clinical symptoms derived from rheological
changes (acrocyanosis, Raynaud syndrome,
• type II (25%) and III (50%) mixed cryo-
globulinemia (MC)-usually polyclonal IgG
with monoclonal IgM (type II) or poly-
clonal IgG with polyclonal IgM (type III).
Monoclonal IgM has rheumatoid factor
activity interacting with both F(ab2)2 IgG
fragment or with Fc IgG fragment thus
forming immune complexes.
• type II/III (unknown prevalence) mono-
clonal or microheterogeneous IgM, identified
by more specific and sensitive methods
(immunoblotting, two-dimensional poly-
acrylamide gel electrophoresis).
3 Hepatitis C virus – mixed cryoglobulinemia – lymphoma relationship 5
• some authors claim a continuous transition
from pure polyclonal forms to some partial
monoclonal forms due to a continuous
clonal selection [13–15].
Cryoglobulins can change their immuno-
chemical profile during chronic HCV infection.
Type II MC is more stable in comparison to type
III or type II/III.
In some cases cryoglobulinemia can be
asymptomatic or may form immune complexes that
are deposited and can activate the complement
cascade (pathogenic mechanism for cryoglo-
bulinemic vasculitis-CV). According to Chapel Hill
Consensus Conference classification CV affects
small and medium size vessels by deposition of
immune complexes with complement activation
Another argument for a causal relationship
between HCV-MC is the fact that HCV-MC
prevalence depends on the dominant genotype and
HCV incidence in different populations, and has
the same evolutionary trends in a specific
population. For example the prevalence of HCV-
CM increases in southern Europe, Africa and Asia,
where the circulating genotypes and the HCV
prevalence is growing but decreases in Italy and
some Mediterranean countries, where significant
reduction of HCV pool in the population was
achieved by active detection and preventive measures
. Cryoglobulins are detected in 29–45% in HCV
patients, type II being most frequent. There are
studies showing higher prevalence in patients of
certain ethnic group or from a particular geographic
area (for example higher prevalence in southern
Europe compared with northern Europe or North
America [18–23]. HCV-CM prevalence is higher
with a particular genotype for example, Japanese
genotype 1b when compared to Egyptian’s genotype 4
. HCV treatment improves CV, suggesting a
cause-effect relationship between HCV and MC
CV represents the most frequent extrahepatic
manifestation. It is characterized by the difficulty
of the therapeutic approach coming from the absence
of a standardized protocol that is implemented for
this specific group of patients. The disease is
considered to be relatively sparse, but the prevalence
studies are not confident due to the clinical
polymorphism and the dispersed addressability to
different medical specialties .
Increase of HCV prevalence in the world will
determine an increase in the prevalence of secondary
MC, particularly in undeveloped countries with
high HCV prevalence . Serum cryoglobulins are
essential for MC diagnosis but serum levels do not
correlate with the disease activity or prognosis .
Sansonno et al. determined intralymphocytic viral
load in patients with HCV infection with or without
MC. Intracellular viral load was higher in patients
with MC. These data explain the associated low
viral loads in CV . An abruptly cryoglobulin
level decline and their peripheral disappearance
(sometimes associated with higher C4 serum levels)
may be associated with incipient signs proliferation
malignant B .
HCV, MC AND B CELLS
There is still debate whether MC is the
consequence of the concomitant intervention of
viral, genetic and environmental factors. HCV is
hepatotropic and also lymphotropic, suggested by
the active viral replication in marrow and
peripheral lymphocytes .
Moreover, t (14.18) translocation and Bcl-2
activation (anti apoptotic molecule) in B lympho-
cytes are observed in HCV infected patients (for
instance t (14.18) approximately 37% –particularly
in MC type II patients). They seem to be responsible
for CD5+ B cell clonal expansion in the blood,
liver, blood and marrow, for the increased
polyclonal IgM production (with rheumatoid factor
activity), for autoantibodies synthesis – including
here cryoglobulins – and for lymphoproliferation
. CD5+ B cells oligoclonal prolix-
feration seems to be the most important charac-
teristic of MC. Actually MC can be defined as a B
lymphocyte proliferation disease being charac-
terized by polyclonal activation and antibody
synthesis (Fig. 2).
B cell-activating factor (BAFF) is a recently
discovered TNF- α member playing a key role in
B lymphocyte survival . BAFF changes have
been associated with autoimmune diseases such as
Sjogren syndrome, systemic lupus erythematosus
and rheumatoid arthritis. Recent studies showed
higher BAFF serum levels in patients with chronic
VHC infection with or without MC [36–38]. Dif-
ferent BAFF mutations were described (– 871C/T )
and are associated with monocyte BAFF mRNA
increase in rheumatoid arthritis and lymphopro-
liferative diseases . Giani et al. demonstrated
that this BAFF promoter mutation could play a
pathogenic role in MC by activating the immune
system and promoting lymphoproliferation. In this
case, along with other gene mutations, one may speak
about a specific cryoglobulinemic genotype .
6 Adriana Nicolau et al. 4
Fig. 2. B cell lymphoma evolution during HCV chronic infection, adapted after Ferri C. et al., B-cells and mixed cryoglobulinemia.
Autoimmunity Reviews 7 (2007) 114–120 (abbreviations: MLDUS – Monoclonal Lymphoproliferative Disorders of Undetermined
Significance, B – B lymphocyte).
E2 antigen binding on CD81 expressed by B
lymphocytes could represent the chronic stimulus
for the continuous, polyclonal activation thus
increasing CD5+ lymphocyte number and peripheral
autoantibody production in HCV infection. Evolution
to lymphoma should be considered continuous but
also other infectious, environmental or genetic
factors could be implicated. For instance, Charles
et al. described that MC patients with HCV
infection also had CD27+ CD21lowk-IgM+IgDlow
lymphocyte population expansion . Vallat et al.
also identified clone B lymphocyte expansion in
chronic HCV patients blood and liver, positively
correlated with the age of patients, duration of the
HCV infection, serum cryoglobulins levels, symptoms
and clinical severity. A significant percentage
(25%) developed neoplasia . Kronenberger et
al. studied CD81 expression variations during
HCV infection treatment. Flow-cytometry analysis
of CD81 and CD8+, CD4+, CD19+and CD56+
during interferon and ribavirine therapy revealed T
CD8+ lymphocytes downregulation , important NK
CD56+ cells downregulation, transient B CD5+
lymphocyte upregulation and weak and delayed T
CD4+ lymphocyte downregulation. No correlation
between viral load and CD81 expression was
determined. After the treatment, CD81+CD8+T
lymphocytes remained decreased but in non-
responders the number started to increase along
with CD56+ NK cells. It was suggested that CD81
variations during antiviral treatment are interferon
related and do not correlate with HCV RNA.The
mechanism is still unclear, and variations in
lymphocyte subclasses are described. CD81 decrease
after interferon treatment could induce a decrease
in the lymphocyte HCV replication and thus of the
immune associated phenomena .
IL-6 and IL-8. HCV B cell activation can also
be achieved by means of other than E2-CD81
interaction. For example, HCV core protein can
stimulate IL-6 and IL-8 synthesis by interaction of
a specific NS3 region with TLR2. Serum IL-6
levels were higher in chronic HCV infected patients
compared to healthy control, became normal after
infection eradication by antiviral treatment.
Antonelli et al. compared IL-6 and TNF-α levels in
patients with and without MC. IL-6 and TNF-α
serum levels were higher in MC patients when
compared with patients without MC and negative
controls . These data suggest that IL6 reflects
the HCV immune stimulation. IL-6 is a strong
stimulus for B lymphocyte maturation and develop-
ment. Significantly elevated IL-6 levels were found
in patients with HCV and MC when compared with
the patients without MC stressing the importance
of TLR2 in the HCV related lymphoproliferative
5 Hepatitis C virus – mixed cryoglobulinemia – lymphoma relationship 7
manifestations. TLR2 implication in other lympho-
proliferative disorders has already been studied, for
example in MALT after chronic Helicobacter
pylori infection . These data may reveal new
therapeutic targets in MC and nonHodgkin lym-
phoma in HCV patients.
T regulatory lymphocytes(Treg). MC develop-
ment in chronic HCV infected patients seems to be
due to some host particularity rather than aminoacid
changes in viral particles (like the first hyper-
variable sequence (HVR1)) . The fact that T
lymphocytes are present in the inflammatory
infiltrate, associated with antibody and autoantibody
synthesis and some HLA specific genotypes in CV
asociated with CVH, suggest that autoimmune
phenomena are implicated in pathogenesis of
CV[47, 48]. Immunoregulatory T CD4+CD25high+
lymphocytes play a key role in the physiologic
control over autoimmune phenomena. Treg’s deficient
mice infused with T lymphocytes developed auto-
immunity , and Treg infusion in these mice
determined inhibition of these phenomena .
Some FoxP3 (specific marker of natural and adaptive/
induced Treg) mutations could induce severe and
fatal autoimmune disease due to its associated Treg
deficiency . Researchers focused upon their
implication in diabetes mellitus, systemic lupus
erythematosus, rheumatoid arthritis, asthma, inflam-
matory bowel disease, multiple sclerosis, hoping
that cellular therapies using Foxp3 positive cells
may, one day, help overcome these diseases [52–56].
Programmed Death 1, or PD-1, is a Type I
membrane protein, a member of the extended
CD28/CTLA-4 family of T cell regulators .
Many studies indicate that PD-1 and its ligands
negatively regulate immune responses. First, PD-1
knockout mice develop lupus-like glomerulo-
nephritis and dilated cardiomyopathy on the C57BL/6
and BALB/c backgrounds, respectively . In
vitro, treatment of anti-CD3 stimulated T cells with
PD-L1-Ig results in reduced T cell proliferation and
IFN-γ secretion . Reduced T cell proliferation
correlated with attenuated IL-2 secretion, which
can be rescued by addition of cross-linking anti-
CD28 antibodies or exogenous IL-2. Together,
these data suggest that PD-1 negatively regulates
T cell responses. Blocking PD-1 in HCV lymphoma
patients determine a decrease in the inhibitory Treg
activity, suppressing T cell responses, thus PD-1
seems to play an important role in Treg function.
Future studies are necessary to evaluate its im-
plication in HCV lymphoma . Treg depletion in
symptomatic CV patients seems to play an important
role in CV HCV associated pathogenesis. The
number of Treg cells is severely decreased before
the interferon therapy. Interestingly there was no
difference in the peripheral Treg number in
asymptomatic MC patients when compared to
healthy controls . Responder patients have a
significant increase in peripheral Treg number
compared to the non-responders where there were
no changes during interferon therapy. This correlation
suggests an important role of Treg depletion in CV
pathogenesis. Tregs play a dual role, on the one
hand, they limit autoimmune phenomena and, on
the other hand, they decrease viral clearance
. There is also the possibility that Treg
cells are recruited at the inflammation sites deter-
mining peripheral depletion but a recent study in
rheumatoid arthritis showed that Treg accumulation
in the synovial fluid does not associate with
peripheral depletion. Future long prospective
studies should focus upon the triggers of Treg
deficiency in CV. Treg depletion can promote
T CD4+ stimulatory function on autoantibody
synthesis. In vitro studies showed that Th2
lymphocytes from MC patients are oligoclonally
expansioned upon IL-2 stimulation, thus suggesting
a larger antigen activated Th2 pool in these patients
. Additionally, hypergammaglobulinemia and
autoantibody secretion in mice is the result of virus
specific CD4+ T cells and HCV infected B
lymphocyte interaction .
IL-29 (IFN-λ1), Il28A (IFN-λ2) si IL28B
(IFN-λ3), type III IFN or IFN lambda-members of
a newly discovered class II cytokine family,
induced by class I IFN and by viral infection, have
a demonstrated antiviral action, similar with type
I IFN family [64–67]. Between IL-28A and IL-29
there is a 81% structural similarity. IL-28 and IL-
29 receptors have 2 polypeptide chains- one
common chain present in most class II cytokines
receptors (IL-10Rβ) and one specific orphan chain,
IL-28Rα . Although this receptor is widely
expressed (hepatocytes, pneumocytes, myocardial
cells) it is less expressed in hematopoietic cells
thus future IFNλ therapies would have less
hematologic side effects. Type II IFN receptor is
mainly expressed by B lymphocytes. T lymphocytes,
NK cells and other myeloid derived cells (mono-
cytes, neutrophils) express only IFNAR2 receptor
for type I IFN. Therefore, IL-29 induces STAT-1
phosphorylation in B lymphocytes but not in T and
NK cells hence IFNλ therapy would have less
systemic immune effects compared to type I IFN
therapy . The serological marker for IL-29
activation seems to be represented by β2-micro-
globulin (B2M), an extracellular component of
8 Adriana Nicolau et al. 6
class I major histocompatibility complex (MHC I).
MHC I presents intracellular antigens to T CD8+
lymphocytes, its expression being enhanced by
type I IFN with serum β2M levels increase. Serum
β 2M levels increase also via IL-29 stimulation in a
dose-dependent manner .Recent studies focused
upon a point IL-28 mutation – rs minor 8099917
allele – which is associated with non-responsive
therapeutic behavior especially in genotype 1 or 4
HCV chronic infection. Future studies should focus
upon type III IFN and autoimmune phenomena in
HCV chronic infection [65, 67], and whether their
mutations might contribute to the continuum CVH-
Hepatita cronică C a devenit în ultimii 10–20 de ani o problemă de sănătate
publică în toată lumea evoluând ca o reală epidemie, a doua ca frecvenţă după cea
cu virus hepatitic B. Datorită manifestărilor clince complexe infecţia cronică cu
virus hepatic C poate fi considerată o boală sistemică. Crioglobulinemia mixtă
(CM) este una din cele mai frecvente manifestări extrahepatice ale hepatitei cu
virus C, dar vasculita crioglobulinemică are o prevalenţă relativ scăzută deşi sunt
încă necesare studii epidemiologice. Prezenţa crioglobulinelor serice este
obligatorie pentru diagnosticul CM, dar criocritul nu se corelează cu manifestările
clinice şi nici cu prognosticul bolii. CM poate fi definită ca un status de proliferare
limfocitară B, caracterizată prin activare policlonală şi sinteză de anticorpi.
Evoluţia spre limfom poate fi considerată astfel un continuum, dar alţi factori
infecţioşi, de mediu sau genetici ar putea fi implicaţi. Translocaţia t(14;18) şi
activarea bcl-2 în limfocitele B, BAFF,interacţiunea dintre E2 şi CD81, limfocitele
T, reglatorii CD4+CD25+ şi interferonii de tip III ar putea juca un rol important
în evoluţia CM către limfom.
Chronic HCV infection should be considered
as a continuous evolution from asymptomatic to
lymphoma. Any MC stage (from asymptomatic to
CV) should be considered as a prelymphomatous
disorder. In this review we presented only a few
recent discoveries that try to decipher the
mechanisms of transition that predispose or induce
autoimmunity or advanced malignancy in chronic
HCV infected patients. Nevertheless, current
technological progress will give us, in a not too
distant future, the opportunity to precociously
select individuals at high risk.
Corresponding author: Adriana Nicolau MD
19–21, Ştefan cel Mare Street, Colentina Clinical Hospital,
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Received February 17, 2011