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Transcript
Page 1 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
Combined action of anti-CD4 autoantibodies and rheumatoid
factor in the development of CD4 lymphocytopenia in rats
immunized with HIV-1 gp120
Liubov Beduleva, Tatyana Khramova, Igor Menshikov, Elena
Stolyarova, Svetlana Pavlova
Udmurt State University, Department of Immunology and Cell
Biology,
1
Universitetskaya
St.,
Izhevsk,
426011,
Russian
Federation. Tel.: +73412916426.
Correspondence to Liubov Beduleva; E-mail: [email protected]
Running Head: RF is a cofactor of anti-CD4 autoantibodies
Page 2 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2
Abstract
The development of immunodeficiency in HIV-infected patients is
known to result from CD4+ lymphocyte depletion. Most CD4+
lymphocyte cells destined to die are not infected. The mechanism of
HIV-uninfected cell death has not yet been fully elucidated. The aim
of this study is to examine the role of anti-CD4 autoantibodies and
physiological rheumatoid factor (RF) in the development of CD4+
lymphocytopenia. Immunization of Wistar rats with gp120 HIV-1
induces
chronic
production
of
anti-CD4
autoantibodies
and
decreases CD4+ lymphocytes in the blood. However, the anti-CD4
autoantibodies produced as part of the immune response to gp120
do not kill CD4+ cells directly. In rats producing anti-CD4
autoantibodies, a low level of peripheral CD4 lymphocytes is
associated with high blood RF levels. The sera containing RF killed
lymphocytes when the lymphocytes were pre-treated with sera
containing anti-CD4 autoantibodies. Thus, the death of CD4+
lymphocytes in rats immunized with gp120 is a result of the
combined action of anti-CD4 autoantibodies and rheumatoid factor,
and the action of these factors can be separated in time. The fact
that two signals are needed for CD4+ lymphocyte death in HIV
gp120-immunized rats does not contradict the hypothesis of the
Page 3 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3
activation-induced death of uninfected CD4+ cells in HIV-infected
humans.
Keywords:
gp120
HIV-I,
CD4+
lymphocytopenia,
anti-CD4
autoantibodies, rheumatoid factor, idiotypic regulation
Introduction
The development of opportunistic infections and progression to
AIDS in HIV-infected patients is known to result from CD4 T
lymphocyte depletion. Although a direct cytopathic effect of HIV-1 on
CD4 T cells contributes to the depletion, most cells destined to die
are not infected with HIV. Understanding how uninfected CD4 Т
cells are eliminated is critical to the development of effective
therapies. The mechanism of death of HIV-uninfected cells that
results in immune deficiency has not yet been fully elucidated.
Several hypotheses were offered. The role of apoptogenic HIV
proteins and antibodies to gp120 that induce cross-linking of gp120
on CD4+ lymphocyte membrane has been considered.1-3 The
counter-argument to hypotheses that consider viral proteins,
independently or together with anti-gp120 antibodies, to be the
cause of CD4 T lymphocyte death is that such theories do not
explain the lack of correlation between viral load and blood levels of
CD4 T cells in HIV-infected patients nor the AIDS-like symptoms
Page 4 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4
without HIV infection that are observed, for instance, in idiopathic
CD4 T lymphocytopenia and mixed connective tissue disease.4-8
The hypothesis of activation-induced cell death of uninfected CD4 Т
lymphocytes appears less contradictory.9 Under this hypothesis, in
HIV infection, uninfected CD4 Т lymphocytes undergo a generalized
activation
that
sensitizes
the
CD4
T
cells
to
apoptosis.
Subsequently, when the activated cells encounter an apoptosis
inducer, they undergo apoptosis. However, neither the factor that
induces
this
generalized
activation
of
uninfected
CD4
Т
lymphocytes, nor the apoptosis inducer, i.e. the factor that kills the
activated CD4 T lymphocytes, is yet known.
Anti-CD4 autoantibodies can be the factor that causes the chronic
polyclonal activation of uninfected CD4 cells in HIV infection and
sensitizes them to apoptosis.10 The hypothesis that the autoimmune
response to CD4 plays an important role in AIDS pathogenesis is
not a new idea.11 Depletion of CD4+ T cells in HIV- or SIV-infected
individuals was associated with plasma antibodies autoreactive with
CD4+ T cells.12-15 Furthermore, Coore et al. demonstrated the
presence of idiotype-anti-idiotype interactions between anti-gp120
antibodies and autoantibodies to CD4 in HIV-infected people, and
the mechanism by which the autoantibodies to CD4 form during HIV
Page 5 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5
infection is clear.16 However, the autoimmune hypothesis of AIDS is
not generally accepted. Perhaps the reason is that none of the
studies conducted have clearly identified the mechanism by which
anti-CD4 autoantibodies could mediate CD4+ T cell depletion.
The aim of this study is to examine the role of anti-CD4
autoantibodies induced by gp120 HIV-1 in the development of CD4+
lymphocytopenia. The model chosen for study was immunization
with purified gp120 protein from HIV, since under the autoimmune
theory of AIDS the immune response to gp120 serves as the cause
of anti-CD4 autoantibody induction.16 This model precludes a
contribution by the virus or its apoptogenic proteins to the depletion
of CD4 T lymphocytes. Humanized mice are the most appropriate
for inducing an autoimmune response to CD4. However, in the pilot
version we tried using Wistar rats. The basis for this attempt was the
homology between rat CD4 and human CD4 in the area responsible
for binding with HIV-1 gp120.17 The homology suggests that HIV-1
gp120 may be complementary to rat CD4, and consequently that the
anti-CD4 lymphocytes that may be activated by anti-gp120
lymphocytes through idiotype-anti-idiotype interactions will be
specific to rat CD4.
Page 6 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6
Previously we showed that resistance to experimental autoimmune
diseases was associated with the production of rheumatoid factor
(RF), which was detected by the method of agglutination of tanned
erythrocytes loaded with homologous IgG.18 The role of RF in the
formation of resistance to developing an autoimmune response to
CD4 or regulating the immune response to gp120 has not been
studied. Therefore, one of the aims was to investigate the role of RF
in regulating the immune response to HIV gp120.
Materials and methods
Rats
Wistar rats (8–10 weeks old) were used. They were brought from
the Stolbovaya breeding facility (Russia) and housed under standard
laboratory conditions with food and water at a constant temperature
of 20±5 °C. Animal experiments were performed in accordance with
European Commission Directive 86/609/EEC (European Convention
for the Protection of Vertebrate Animals used for Experimental and
Other Scientific Purposes) and approved by the local animal care
commission of Udmurt State University.
Gp120 HIV-1 immunization and blood collection
Recombinant
GP120
HIV-1
glycoprotein
(ACROBiosystems,
Newark, USA) was emulsified with the same volume of incomplete
Page 7 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7
Freund’s adjuvant (IFA) (Sigma, St Louis, MO, USA). Rats (n=7)
were injected at the tail base with gp120/adjuvant emulsion (20 μg
of gp120 per rat). No booster immunization was performed. Control
rats (n=8) received an injection of an emulsion containing saline
solution and IFA. Blood sampling for anti-gp120 antibody, anti-CD4
autoantibody, and RF detection was carried out every week. From
time to time blood samples were used to determine CD4+ cell
counts. The number of exposed rats and days of CD4+ cell count
determinations are presented in Fig. 2a.
Determination of anti-gp120 antibodies by ELISA
Plates (Corning-Costar, Acton, MA, USA) were coated overnight at 4
°C with 50 µl of gp120 HIV-1 (ACROBiosystems, Newark, USA) (10
µg/ml in 0.15 M PBS). Plates were blocked with 150 µl 5% milk
powder in PBS. Serum samples were added in a 1:200 dilution with
PBS/Tween-20 and incubated for 1 h at RT. The plates were then
incubated for 1 h at RT with 100 µl of goat anti-rat Ig (IgG, IgM, IgA)
conjugated to horseradish peroxidase (IMTEC, Moscow, Russia).
Then the substrate mixture (5 ml of citrate buffered solution [pH
5.0]/3 mg ortho-phenylenediamine/15 ml 3% H2O2) was added.
Absorbances were read after 15 min at 492 nm.
Determination of anti-CD4 antibodies by ELISA
Page 8 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8
Plates (Corning-Costar, Acton, MA, USA) were coated overnight at 4
°C with 50 µl of recombinant rat CD4 (R&DSystems, Minneapolis,
USA) (10 µg /ml in 0.15 M PBS). Plates were blocked with 150 µl
5% milk powder in PBS. Serum samples were added in a 1:100
dilution with PBS/Tween-20 and incubated for 1 h at RT. The plates
were then incubated for 1 h at RT with 100 µl of goat anti-rat Ig (IgG,
IgM, IgA) conjugated to horseradish peroxidase (IMTEC, Moscow,
Russia). Then the substrate mixture (5 ml of citrate buffered solution
(pH 5.0)/3 mg ortho-phenylenediamine/15 ml 3% H2O2) was added.
Absorbances were read after 15 min at 492 nm.
Measurement of RF
The RF titer was determined in an agglutination test using rat-IgGloaded tanned erythrocytes. Group 0 human erythrocytes were fixed
with 1% glutaric dialdehyde. The washed-off fixed erythrocytes (10%
in PBS [pH 7.2]) were incubated for 10 min with the same amount of
tannin solution in PBS at room temperature. The erythrocytes were
then washed off. For sensitization, 4 ml of PBS (pH 6.4), 1 ml of a
solution of 0.5 mg/ml normal IgG from rat serum (Equitech-bio,
Kerrville, USA) in 0.9% NaCl, and 70 µl of the pelleted tanned
erythrocytes were mixed. Incubation lasted for 20 min at room
temperature; erythrocytes were washed with 0.9% PBS containing
Page 9 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
9
0.2% bovine serum albumin. Twofold serial dilutions of serum were
prepared and put into wells in the amount of 50 µl. The same
amount of rat-IgG-loaded 1.5% erythrocyte suspension was then
added. Agglutination results were read in 3 h.
Determination of CD4+ cell counts
The lymphocytes were isolated in ficoll-urografin gradient (ρ=1.08
g/cm3). Then aliquots of cells washed in PBS were stained for 1 h at
37 °C with FITC-conjugated anti-rat-CD4 (eBioscience, San Diego,
USA), 0.25 mg antibody per 106 cells in a final volume of 100 µL.
The lymphocytes were then washed off. After that CD4+ lymphocyte
counts were determined using laser-based flow cytometry (BD
FACSCanto II Flow Cytometer).
Viability
of
lymphocytes
treated
with
anti-rat
CD4
autoantibodies and rheumatoid factor in vitro
The lymphocytes were isolated in ficoll-urografin gradient (ρ=1.08
g/cm3) from intact rats. The lymphocytes were washed off in RPMI1640. After that 100 µl of serum from gp120-immunized rats
containing anti-rat CD4 antibody or 100 µl of serum from IFAinjected rats containing no anti-rat CD4 antibody was added to 106
lymphocytes in RPMI-1640 in a final volume of 1 ml. The rheumatoid
factor titer of the sera that were added was ≤1:4. The lymphocytes
Page 10 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
10
were incubated for 24 hours in a 37 °C, 5% CO2 incubator. The
lymphocytes were then washed off in RPMI-1640 and were
suspended in a volume of 100 µl. Then 100 µl of serum containing
rheumatoid factor (RF titer ≥1:32) and obtained from intact rats or
100 µl of serum containing no rheumatoid factor (RF titer ≤1:4) and
obtained from intact rats was added. After incubation for 3 hours in a
37 °C, 5% CO2 incubator the number of cells was counted using
trypan blue.
Statistical analysis of the data
The significance of differences was assessed by the t-test.
Results
Production
of
anti-CD4
autoantibodies
and
CD4+
lymphocytopenia in rats immunized with HIV 1 gp120.
Immunization of Wistar rats (n=7) with HIV-1 gp120 induced the
production not only of antibodies to HIV-1 gp120, but of
autoantibodies to rat CD4 (Fig. 1). Anti-CD4 autoantibody production
was revealed in all of the gp120-immunized rats. In rats receiving
IFA (n=8), no anti-CD4 autoantibodies were found (Fig. 1). Fig. 1
shows that the production of anti-gp120 antibodies induced by a
single injection of HIV-1 gp120 did not attenuate over the 4 months
of observation. The production of anti-CD4 autoantibodies also was
Page 11 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
11
chronic in nature; the level of anti-CD4 autoantibodies in the blood of
rats immunized with gp120 remained significantly higher than that of
IFA-injected rats even 4 months post immunization. In summary,
immunization
with
HIV-1
gp120
induces
chronic
anti-CD4
autoantibody production in Wistar rats.
The count of CD4+ cells in the blood of the gp120-immunized rats
and the IFA-injected rats is shown in figure 2a. In rats that have
developed an autoimmune response to CD4, the average number of
CD4+ cells was significantly lower than the average number of
CD4+ cells in the peripheral blood of IFA-injected rats (Fig. 2b). The
lowest number of CD4+ cells induced in rats immunized with gp120
that developed an autoimmune response to CD4 was 293 cells per
µl of blood (Fig. 2c, left picture). Representative flow cytometry of
CD4+ cells in IFA-injected rats is shown in Fig. 2c, right picture. The
decrease in CD4+ lymphocytes in rats immunized with gp120
protein was transitory in nature; a drop was followed by a
compensatory rebound in CD4 lymphocyte levels.
Contrary to expectations, no correlation was found between the level
of anti-CD4 autoantibodies and the number of CD4+ lymphocytes in
the blood (r=0.24, p=0.27). The drop in the number of CD4+ cells in
the blood can be observed against a background of stable, relatively
Page 12 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
12
low anti-CD4 antibodies in the blood, and vice versa - when the level
of anti-CD4 antibodies is increasing rapidly, CD4 numbers can
remain stable as compared to the cell numbers in IFA-injected rats.
In sum, immunization of Wistar rats with HIV-1 gp120 induces
chronic production of anti-CD4 autoantibodies and decreases CD4
lymphocytes in the blood. However, no correlation was found
between antibodies to CD4 lymphocytes and the number of CD4
lymphocytes in the blood.
Role of RF in the development of CD4+ lymphocytopenia in rats
immunized with HIV-1 gp120
The time course of RF in response to HIV-1 gp120 immunization
and in IFA-injected rats is shown in figure 3. RF levels were found
not to increase in response to gp120 immunizations. The surges in
RF production observed at 5, 9, 12, 15, and 17 weeks after gp120
immunization were delayed relative to immunization with the gp120
protein of HIV, while the kinetic curves of RF in gp120-immunized
and IFA-injected rats almost entirely coincide (Fig. 3). Therefore, the
observed changes in RF level in the blood of gp120-immunized rats
are not the result of the immune response to gp120 nor the
administration of animal IFA, but are spontaneous in nature and are
natural variations in RF level. In sum, rheumatoid factor is not
Page 13 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
13
involved in downregulating the immune response to gp120 HIV in
rats.
In an individual analysis of the parameters studied, we noticed that
in the rats immunized with HIV gp120, the minimum of the blood
CD4 lymphocytes corresponded with the maximum of the blood RF
levels (Fig. 4a). No such association was found in the rats that had
received IFA in place of gp120.
We compared the level of CD4 cells at a relatively high RF level
(titer >1/16) and a relatively low RF level (titer ≤1/16) in the blood of
rats immunized with gp120. The same test was performed in IFAinjected rats. In the gp120-immunized rats, when RF blood levels
were relatively high, the number of CD4 lymphocytes was
significantly lower than in the rats with a relatively low RF level (Fig.
4b). In the IFA-injected rats, whether the blood RF was relatively
high or relatively low, the blood CD4 cell levels were the same (Fig.
4b). In sum, in rats immunized with gp120 and producing anti-CD4
autoantibodies, a low level of peripheral CD4 lymphocytes is
associated with high blood RF levels. In the IFA-injected rats, which
have no anti-CD4 autoantibodies, the number of CD4 cells does not
depend on blood RF levels. This information prompts us to suggest
that the death of CD4 lymphocytes in rats immunized with gp120 is
Page 14 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
14
a result of the combined action of CD4 autoantibodies and
rheumatoid factor, and the action of the factors can be separated in
time.
Effect of serum containing anti-CD4 antibodies and serum
containing RF on lymphocyte viability in vitro
To test the assumption that CD4+ lymphocyte death in rats
immunized with gp120 results from the combined action of anti-CD4
autoantibodies and rheumatoid factor, we performed additional in
vitro studies. Lymphocytes from intact rats (n=11) were incubated
with sera containing anti-CD4 antibodies and obtained from gp120immunized rats (n=11). Next, the lymphocytes were treated with
serum from intact rats (n=11) that contains RF.
We found that the lymphocytes pretreated with serum containing
anti-CD4 antibodies died in response to the presentation of serum
containing RF (Fig. 5). Lymphocytes did not die if serum without RF
was added to lymphocytes incubated with serum containing antiCD4 (Fig. 5). The lymphocytes also stayed alive when the
lymphocytes were incubated in serum without anti-CD4 antibodies
and then presented with RF-containing serum. Thus, anti-CD4
autoantibodies and rheumatoid factor do not induce lymphocyte
death on their own; the death of CD4 lymphocytes results from the
Page 15 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
15
combined action of rheumatoid factor and anti-CD4 antibodies
obtained from rats immunized with HIV-1 gp120.
Discussion
The aim of this study was to test the hypothesis that the cause of
CD4 lymphocytopenia in HIV infection is an autoimmune response
to CD4 induced by HIV-1 gp120. It is known that in HIV infection
lymphocytes specific to CD4 are activated by lymphocytes against
HIV-1 gp120 through idiotype-anti-idiotype interactions. The ability of
lymphocytes against HIV gp120 to enter into idiotype-anti-idiotype
interactions with lymphocytes against human CD4 is prompted by
the complementarity of the antigen-recognizing receptors of these
lymphocytes, which itself is a consequence of the complementarity
between the gp120 CD4-binding sequence and the first domain of
human CD4. In choosing rats as the subjects for testing the
hypothesis, we assumed that rat lymphocytes, which can be
activated by anti-gp120 lymphocytes through idiotype-anti-idiotype
interactions in response to HIV-1 gp120 immunization, would not be
specific to rat CD4. This doubt was supported by data on the
homology of about 50% between the first domain of human CD4,
which is responsible for HIV-1 gp120 binding, and the analogous
section of the rat CD4 molecule. The fact that antibodies to rat CD4
Page 16 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
16
appeared in the rats in response to gp120 immunization indicated
that the rat lymphocytes specific to HIV gp120 enter into idiotypeanti-idiotype interactions with autoreactive lymphocytes against rat
CD4 and that immunization of rats with HIV-1 gp120 can be used to
test the hypothesis.
To measure anti-CD4 autoantibodies, recombinant rat CD4 was
used. Some researchers are skeptical of the use of recombinant
CD4 (rCD4) to measure anti-CD4 lymphocyte antibodies, because
they
believe
that
rCD4
differs
from
the
membrane-bound
molecule.19, 21 However, rCD4-specific antibodies can appear in rats
in response to gp120 immunization only if the lymphocytes that
produce antibodies specific to rCD4 enter into idiotype-anti-idiotype
interactions with lymphocytes against gp120, and subsequently
carry the internal image of gp120. Considering that gp120 interacts
with membrane-bound lymphocyte CD4, we can assert that the
rCD4 preparation we used also bears sections responsible for
gp120 binding, and consequently in this area it is the same as
membrane-bound CD4, and the auto-antibodies revealed by
recombinant CD4 are specific to the available determinants on
membrane-bound CD4. This assertion does not contradict the result
Page 17 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
17
obtained by Corre et al. that anti-idiotypic antibodies to human antigp120 antibodies bind recombinant and cellular human CD4.16
According to the autoimmune hypothesis of AIDS, the rat anti-CD4
autoantibodies should induce death of rat CD4 T lymphocytes. In
rats immunized with gp120 and producing anti-CD4 antibodies, the
average levels of CD4 lymphocytes were significantly lower for the
experiment than in the IFA-injected rats. However, the anti-CD4
autoantibodies produced as part of the immune response to gp120
do not kill cells directly. The lack of a correlation between anti-CD4
autoantibodies and the number of CD4 lymphocytes in the blood of
gp120-immunized rats, as well as the results of in vitro experiments
in which we tried to induce lymphocyte death in the presence of
serum that contained CD4 autoantibodies obtained from gp120immunized mice, serve as evidence of this. The death of
lymphocytes exposed to anti-CD4 autoantibodies induced by gp120
immunization commences in the presence of rheumatoid factor, both
in vivo and in vitro. The fact that two signals, from anti-CD4
autoantibodies and rheumatoid factor, are needed for CD4
lymphocyte death in HIV gp120-immunized rats does not contradict
the hypothesis of the activation-induced death of uninfected CD4+
cells in HIV-infected humans. This hypothesis also indicates that two
Page 18 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
18
factors are needed: a factor that activates the cells and a factor that
kills the activated cells, and the action of the factors can be
separated in time. Furthermore, our data coincide with results
obtained by Susal et al., who found the association between a high
anti-Fab/anti-rCD4 ratio and a low level of CD4+ helper T cells in
AIDS patients. Anti-Fab antibodies and rheumatoid factor have
similar characteristics. Rheumatoid factor and anti-Fab antibodies
both possess anti-Fab activity and downregulate the immune
response.18,24
At the same time, our data raise new questions, particularly
concerning the mechanism of action of rheumatoid factor that results
in CD4 lymphocyte death.
We previously conducted detailed studies of the rheumatoid factor
population, which is associated with the drop in blood CD4
lymphocytes in HIV gp120-immunized rats. This RF population is
found in intact animals; therefore it can be considered physiological.
We showed that an increase in RF production in response to
immunization with antigen-inducers of experimental autoimmune
diseases prevents the development of those diseases and is also
associated with termination of the normal immune response.18 A
study of RF specificity found that every RF molecule has two kinds
Page 19 of 32
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
19
of paratopes: a unique individual paratope and a shared paratope;
the specificity of the shared paratope is the same across the
different RF molecules. The individual paratopes of RFs represent
internal images of the foreign antigens; therefore, the RFs are antiidiotypic antibodies, which recognize the antigen-binding sites of
antibodies to foreign antigens. So the RF naturally occurring in the
blood is heterogeneous in its specificity population of anti-idiotypic
antibodies.18 The shared paratope recognizes the recurrent
sequence that exists on antibodies of various specificities and is
located in the Fab region but outside the antigen binding site.18
Furthermore, we showed that the antigenic determinants for the
shared RF paratope can be created in the Fc hinge region of IgG
fragments through papain cleavage of IgG.18 The specificity of the
shared paratope of RF to the hinge region of IgG Fc fragments
distinguishes the given RF from other known RF populations, which
have determinants localized between the CH2 and CH3 domains.25
If RF is classified in accordance with the list of types of anti-idiotypic
antibodies proposed by Parsons et al.,26 the properties of the
individual paratope of the RF molecule enable the RF to be classed
as Ab2 Beta (Ab recognizes the antigen binding site), while the
properties of the shared paratope of RF resemble the properties of
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Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
20
the Ab2 Delta class (Ab recognizes non-binding site, non-disease
specific, and outbred shared idiotope). Thus, RF is simultaneously
both Ab2 Beta and Ab2 Delta.
Based on this information, we proposed a hypothesis explaining how
RF prevents autoimmune response.21 We think that RF normally
acts as a factor depleting activated antigen-specific CD4 cells. In the
course of an immune response to a foreign antigen (including
antigens that induce experimental autoimmune diseases), RF is
formed as anti-idiotypic antibodies; therefore, its target is only
antigen-specific
lymphocytes.
RF
does
not
recognize
CD4
lymphocytes with other specificities. Transmission of the death
signal to the activated antigen-specific CD4 lymphocytes may occur
by virtue of the shared paratope on RF. The ligand for the shared
paratope on the CD4 lymphocyte membrane may be an inducible
molecule that is structurally similar to the Fc fragments of IgG and
appears only in activated lymphocytes.
Anti-CD4
autoantibodies
produced
in
response
to
gp120
immunization can induce polyclonal activation of peripheral CD4
lymphocytes and the appearance thereon of the ligand for the
shared paratope of RF (Fig. 6). Therefore, the RF normally in the
blood gets the opportunity to bind with CD4 lymphocytes of any
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Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
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21
specificity and exercise its ability to kill the lymphocytes (Fig. 6). In
this way, we suggest that anti-CD4 autoantibodies turn CD4
lymphocytes into a target for rheumatoid factor and involve RF in a
pathological process that results in immunodeficiency.
Acknowledgements
This study was supported by the Ministry of Education and Science
of the Russian Federation, grant number 14.124.13.1159-MD. We
are grateful to Jennifer Guernsey for editorial assistance. We thank
Tatiana Tolstolutskaya at the Izhevsk State Medical Academy for
technical assistance.
Conflict of interest statement
The authors declare no conflicts of interest.
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AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Correspondence to Liubov Beduleva; E-mail: [email protected]
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Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
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Page 27 of 32
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Figure 1. Anti-CD4 autoantibodies production and time course of anti-gp120 antibodies
in rats immunized with gp120 HIV-1. Each point is represented as mean±SD of 7 (gp120
immunized rats) and 8 (IFA injected rats).
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Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
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28
Figure 2. CD4+ cell counts in the blood of gp120 HIV-1 immunized and IFA injected
rats. (a) CD4+cell counts monitoring in the course of immune response to gp120.
Mean, highest and minimum CD4+cell number in each experimental point. Numbers
show how many rats were studied in each point. (b) Average number of CD4+
lymphocytes observed in blood samples obtained from gp120 immunized and IFA
injected rats throughout the entire experiment (mean±SD). (с) Lowest number of
CD4+cells in rats immunized with gp120 (left picture). Representative flow cytometry
of CD4+cells in IFA injected rats (right picture).
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
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Page 29 of 32
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Figure 3. The time course of rheumatoid factor in rats in response to HIV-1 gp120
immunization.
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30
Figure 4. CD4+lymphocyte numbers in the blood of rats with high and low titer of
rheumatoid factor. (a) Individual drop in CD4+ cell count coincident with high level of
rheumatoid factor in gp120 immunized rats. Anti-CD4 autoantibodies level is
presented as ODA of serum, predeluted 100-fold with PBS. (b) Average number of
CD4+lymphocytes observed in blood samples of gp120 immunized rats and IFA
injected rats with high and low RF titer (means±SD). Blood samples were separated
into groups according to their RF titer. n- number of blood samples.
AIDS Research and Human Retroviruses
Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
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Figure 5. Death of lymphocytes after incubation with or without sera containing antiCD4 autoantibodies and treatment with RF-containing sera (means±SD).
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Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358)
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32
Figure 6. A hypothetical scheme of death of HIV-uninfected CD4 cells in the course of
HIV infection. Anti-CD4 autoantibodies produced in response to gp120 HIV-1 induce
polyclonal activation of peripheral CD4 lymphocytes and the appearance thereon of
the ligand (IgG Fc fragment-like molecule) for the shared paratope of rheumatoid
factor. The rheumatoid factor normally in the blood gets the opportunity to bind with
CD4+ lymphocytes and exercise its ability to kill the activated lymphocytes.