During the last years, our group has been interested to
understand how some viruses could modulate immune responses concomitant
to infection, and what could be the results of this immunomodulation on
diseases not directly related to the infection. Other projects involved
the analysis of expression of the cellular receptor for mouse hepatitis
virus (MHV), in relationship with the pathogenicity of this virus;
study on polioencephalomyelitis induced by lactate
dehydrogenase-elevating virus (LDV); and analysis of the mode of action
of total immunoglobulins as a treatment for autoimmune diseases.
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1. Immunomodulation
triggered by virus infection
We had observed previously that some mouse viruses could induce in vivo
a polyclonal activation of B lymphocytes, characterized by a
T-dependent IgG2a-restricted hypergammaglobulinemia and an
immunostimulant effect on immunization with non-viral protein antigens
concomitant with the infection. Currently, we examine more closely the
effect of viruses on B lymphocytes, T helper lymphocytes, NK cells and
macrophages that could lead to this virally-induced immunomodulation.
We are also interested by the possible consequences of this
immunomodulation on some autoimmune diseases. To find out more
information about platelets and
thrombocytopenia click
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1.1 B lymphocytes
We have analyzed B lymphocyte activation after infection with LDV,
adenovirus and MHV. Infection triggers production by B lymphocytes of
large amounts of IgG2a, most without viral specificity. This polyclonal
immunoglobulin production is dependent on the presence of functional T
helper cells, although B cell proliferation is T-independent. A
differential regulation of antiviral responses and polyclonal IgG2a
production was observed: specific IgG2a antibody production requires
gamma-interferon (IFN-g), but not IL-6, whereas total IgG2a production
depends on IL-6, but not on IFN-g.
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1.2 T helper lymphocytes
The effect of viral infections on T helper lymphocyte differentiation
was analyzed at the level of cytokine expression . IL-4 and IFN-g gene
expression by lymph node cells from mice immunized with keyhole limpet
haemocyanin was analyzed. The expression of IL-4 message observed in
normal mice was drastically decreased in animals infected with LDV.
These results were completed by analysis of IL-9, another Th2 cytokine.
In vivo, IL-9 mRNA was detected in lymph nodes after immunization of
normal mice with soluble antigens. IL-9 expression preceeded that of
IL-4 and was not affected in IL-4 knockout mice. Treatment with
anti-CD4 antibody and analysis of purified CD4 cells confirmed that
IL-9 was produced by T helper lymphocytes. Moreover, similarly to what
was shown for IL-4, IL-9 message induction was strongly decreased by
infection with LDV. Taken together, our results indicate that viruses
can in some circumstances modulate the differentiation of T helper
lymphocytes, by suppressing Th2 responses.
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1.3 Macrophages
Our results indicated that the message for the p40 component of IL-12
was transiently increased shortly after infection with LDV, MHV and
adenovirus. IL-12 was mainly expressed by macrophages. Therefore,
production of IL-12 might constitute the initial event that would
determine the subsequent characteristics of the immune response
elicited by viral infections.
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1.4 Consequences
of virally-induced immunomodulation on concomitant diseases
The effect of LDV and MHV infection on autoimmune responses was
examined in mice immunized with rat red blood cells, that made
antibodies reactive with both rat and mouse erythrocytes. Whereas
uninfected animals developed a progressively increasing autoantibody
titer, infected mice quickly attained high anti-erythrocyte
autoantibody titers that remained rather constant. Virus infection
enhanced all the IgG subclass responses, with the exception of IgG1, to
both rat and mouse erythrocytes.
In collaboration with C.J. Pfau (Troy, USA), we analyzed
also a model of hemolytic anemia induced in C3H mice by lymphocytic
choriomeningitis virus (LCMV). This model is characterized by the
production of anti-erythrocyte autoantibodies. Treatment with the T
helper-depleting GK1.5 monoclonal antibody indicated the central role
of the immune system in this anemia by showing that depletion of CD4+
cells largely abrogated this anti-erythrocyte autoimmune reaction. In
contrast to LCMV, LDV had no apparent effect on erythrocytes, even
though this virus also induced a sharp increase in plasma IgG levels.
Therefore, B cell polyclonal activation is not sufficient to explain
this autoimmune anemia.
We also found that the pathogenicity of autoantibodies
may be dramatically modified by the immune environment, and more
precisely that viruses can trigger the onset of autoimmune disease by
enhancing the phagocytic activity of macrophages for
autoantibody-coated target cells . Indeed, a strong enhancement in the
pathogenicity of an IgG2a anti-erythrocyte monoclonal autoantibody, was
observed after infection of mice with LDV or MHV. While injection of
the anti-red cell antibody alone induced only a moderate anemia, the
concomitant infection with LDV, which is harmless in most normal mice,
led to a dramatic drop in the hematocrit and to the death of infected
animals. In vitro and in vivo analysis showed a dramatic increase in
the ability of macrophages from LDV-infected mice to phagocytose
antibody-coated erythrocytes, when compared to cells from normal
animals.
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2. MHV receptor
In collaboration with K.V. Holmes (Denver, USA), expression of Bgp1a, a
glycoprotein that serves as receptor for MHV-A59 has been analyzed in
various mouse tissues and correlated with the pathogenicity that this
virus induces in the corresponding organs. In contrast to some cell
types, like hepatocytes that express Bgp1a and may be infected and
destroyed in vivo by MHV, other cell types that express the viral
receptor are not infected after inoculation of this virus. For
instance, BALB/c mice develop a neurologic demyelinating disease after
inoculation of MHV-A59, by the intracranial, but not by the
intraperitoneal route. Bgp1a is strongly expressed on the endoluminal
pole of these cells and MHV is able to bind endothelial cells via this
receptor. Despite this expression of a functional viral receptor, in
normal mice infected with MHV by the intra-peritoneal route, no in vivo
viral replication could be detected in endothelial cells from the
brain, contrasting with the equivalent cells from the liver. However,
shortly after administration of sodium dodecylsulfate, virus infection
of some cerebral endothelial cells was detected and MHV was able to
cross the blood brain barrier. These results suggest that the
protective role of the blood-brain barrier against spreading of MHV
into the central nervous system is determined by a specific restriction
of viral entry into the endothelial cells of cerebral origin.
The expression of Bgp1a was also analyzed in cells from
the immune system. This molecule was highly expressed in B lymphocytes,
including cells of the B-1a (CD5+) lineage, and in macrophages, but was
not detectable in resting T lymphocytes. Bgp1a is also expressed in
endothelial and thymic epithelial cells. Thus, some alterations of
immune responses induced by MHV might be explained by the expression of
the viral receptor on immune cells. For instance, MHV-A59 infection of
adult BALB/c mice induces a severe, transient atrophy of the thymus.
This thymus atrophy is not mediated by glucocorticoids, as it was also
found in adrenalectomized, infected mice. In infected thymus, immature
CD4+ CD8+ lymphocytes are selectively depleted, and apoptosis of
lymphocytes is increased. In a small number of stromal epithelial
cells, but in very few lymphocytes, the viral genome was detectable by
in situ hybridization. These observations suggested that, rather than a
generalized lytic infection of T lymphocytes, MHV-A59-induced thymic
atrophy results from apoptosis of immature double-positive T cells that
might be caused by infection of a small proportion of thymus epithelial
cells.
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3. LDV
infection and polioencephalomyelitis
Mouse infection with LDV leads to lifelong viraemia, despite the
production of neutralizing antiviral antibodies. To test whether viral
persistance correlates with the development of resistance to
antibodies, we compared the neutralization of viral particles derived
from acutely and chronically infected animals, using polyclonal and
monoclonal anti-LDV antibodies. Whereas virus isolated during acute
infection was efficiently neutralized, titres of LDV from chronically
infected mice were only slightly reduced by antiviral antibodies. In
addition, LDV from animals acutely infected with such poorly
neutralizable virus from chronically infected mice were resistant to
anti-LDV antibodies like their parental viral particles. These results
suggest that LDV variants capable of escaping neutralization by
antiviral antibodies can emerge in chronically infected animals.
Immunosuppression, occurring naturally with aging, or
experimentally after cyclophosphamide treatment or irradiation, is
required for the development in C58 mice infected with LDV of a severe
polioencephalomyelitis that is caused by viral destruction of anterior
horn neurons. We have shown that depletion of T helper lymphocytes by
administration of an anti-CD4 antibody is followed by a progressive
paralysis typical of polioencephalomyelitis in C58/J mice inoculated
with a neurovirulent strain of LDV. Although it was clear that other
cell subsets are also required to assure complete protection of
genetically-susceptible mice, our results show that T helper
lymphocytes play a major role in the prevention of LDV-induced
polioencephalomyelitis. In addition, the genetic background requested
for the development of this disease was analyzed. The Fv1 gene
determines the susceptibility to retrovirus replication. We sequenced
the open reading frame of the Fv1nr allele of resistant 129/Sv mice. It
differs by only one nucleotide, modifying one amino acid in the encoded
protein, from the Fv1n allele of susceptible AKR and C58 animals. We
excluded that the resistance of 129/Sv mice to LDV-induced
polioencephalomyelitis resulted from the absence of endogenous N-tropic
retrovirus, by infecting (129/Sv x C58/J) F1 animals. Therefore it is
possible that the amino acid that defines the Fv1nr allele is
responsible for resistance of 129/Sv mice to N-tropic MuLV expression
and to LDV-induced polioencephalomyelitis.
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4. Mode
of action of total immunoglobulins as a treatment for autoimmune
diseases
In order to gain insight into the mechanisms by which the infusion of
IgG can improve some autoimmune diseases, we induced hemolytic anemia
in mice by the injection of anti-erythrocyte monoclonal antibodies.
Treatment of mice with pools of either human or mouse IgG clearly
attenuated the anemia induced by an IgG2a autoantibody.Similar
protection was obtained with human monoclonal IgGs from myeloma
patients. Prior absorption by mouse red blood cells did not affect the
efficacy of the injected IgG. Treatment with Fc fragments also reduced
the anemia. In vitro phagocytosis of autoantibody-coated red cells by
murine macrophages was completely inhibited by the addition of
polyclonal or myeloma IgG or of human Fc fragments. These results
indicate that, in this model of autoimmune pathology, the protective
effect of IgG is mediated by its interaction with the macrophage Fc
receptors.
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5. Immunology of Platelets
Our Lab is also dealing with various mouse models of immune
thrombocytopenia. To find out more information about platelets and
thrombocytopenia please click
here
