Download Immune Response to Self Nuclear Autoantigen Determines the Fate

Altered Expression Level of a Systemic
Nuclear Autoantigen Determines the Fate of
Immune Response to Self
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of June 18, 2017.
Kimito Kawahata, Yoshikata Misaki, Yoshinori Komagata,
Keigo Setoguchi, Shinji Tsunekawa, Yasuji Yoshikawa,
Jun-ichi Miyazaki and Kazuhiko Yamamoto
J Immunol 1999; 162:6482-6491; ;
http://www.jimmunol.org/content/162/11/6482
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Copyright © 1999 by The American Association of
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References
Altered Expression Level of a Systemic Nuclear Autoantigen
Determines the Fate of Immune Response to Self1
Kimito Kawahata,* Yoshikata Misaki,2* Yoshinori Komagata,*† Keigo Setoguchi,*
Shinji Tsunekawa,‡ Yasuji Yoshikawa,§ Jun-ichi Miyazaki,¶ and Kazuhiko Yamamoto*
O
ne of the prominent features of systemic autoimmune
diseases, such as systemic lupus erythematosus (SLE)3
and mixed connective tissue disease (MCTD), is the
presence of autoantibodies. The targets of these autoimmune responses are mostly ubiquitous and organ-nonspecific Ags, including nuclear Ags, such as spliceosome components (U1 small nuclear ribonucleoprotein (snRNP)-A, 70K, C, B/B9, etc.) as well as
nucleosome components (dsDNA, histones) (1). It has been suggested that these autoantibodies are produced by an Ag-driven
mechanism, and that autoantigen-specific T cells are involved in
their production.
Autoreactive T cells are deleted during development in the thymus. Studies on transgenic (Tg) mice that express a TCR specific
for a systemic Ag have confirmed the view that the most efficient
way to achieve self tolerance to a systemic self Ag is central deletion (2). However, in recent studies, the autoreactive T cells responding to systemic autoantigens have been demonstrated to be
*Department of Allergy and Rheumatology, University of Tokyo Graduate School of
Medicine, Tokyo, Japan; †Center for Neurologic Diseases, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA 02215; ‡Medical and Biological
Laboratories, Ina, Japan; §Department of Clinical Laboratory, Medical Institute of
Bioregulation, Kyushu University, Beppu, Japan; and ¶Department of Nutrition and
Physiological Chemistry, Osaka University Medical School, Suita, Japan
Received for publication August 24, 1998. Accepted for publication March 19, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the mixed connective tissue disease research group of the Ministry of Health and Welfare, the Ministry of Education of
Japan, and Schering-Plough.
2
Address correspondence and reprint requests to Dr. Yoshikata Misaki, Department
of Allergy and Rheumatology, University of Tokyo Graduate School of Medicine,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail address: misaki-tky@
umin.ac.jp
3
Abbreviations used in this paper: SLE, systemic lupus erythematosus; MCTD,
mixed connective tissue disease; snRNP, small nuclear ribonucleoprotein; U snRNP,
U-type small nuclear ribonucleoprotein complex; HuA, human U1 snRNP-A protein;
MuA, murine U1 snRNP-A protein; wt, wild type; B6, C57BL/6; m3G, 2,2,7-trimethylguanosine; LAT, local adoptive transfer; DTH, delayed-type hypersensitivity;
Tg, transgenic; LN, lymph node; HEL, hen egg-white lysozyme; MACS, magnetic
cell sorting.
Copyright © 1999 by The American Association of Immunologists
isolated from the peripheral blood not only of patients with autoimmune diseases but also of healthy individuals (3–5). In case of
a microbe infection or chronic inflammation, it is possible that
these autoreactive T cells expand, leading eventually to autoimmune diseases via proposed mechanisms such as molecular mimicry (6, 7) or superantigen. Therefore, peripheral regulation should
be important for the maintenance of tolerance to systemic Ags.
Supporting this idea, it was determined that in MRL-lpr/lpr mice,
which are a prototype model of human SLE, the pathway of peripheral tolerance to eliminate self-reactive lymphocytes is impaired due to the deficient Fas/Fas ligand system (8).
The importance of peripheral tolerance has already been demonstrated using Tg mice in which the extrathymic organs express
neo-self Ags under tissue-specific promoters (9 –14). The Tg Ags
are not expressed in or are not able to gain access to the thymus.
Several mechanisms, including the deletion (15–17) and downregulation of TCRs (18, 19), have been proposed for the establishment and maintenance of peripheral tolerance; it seems that the
nature of Tg Ag, such as antigenicity, expression level, and site of
expression, is important for the mechanism of the tolerance induction. Peripheral tolerance to systemic Ags was also studied using
the adoptive transfer of CD81 T cells from TCR Tg mice into Tg
mice expressing the H-Y Ag (20), Ld (21), and the lymphocytic
choriomeningitis virus GP epitope (22). A transient activation followed by deletion or unresponsiveness was observed. However,
the peripheral regulation of an immune response to intranuclear
autoantigens has not been studied.
To investigate this point, we employed an autoantigen-Tg
mouse system. Because Ag compartmentation and its assembly
with other molecules could significantly influence the Ag-specific
immune response, we generated Tg mice expressing human U1
snRNP-A protein (HuA) under a class I promoter. U1 snRNP is
one of the U-type snRNP complexes (U snRNPs) that constitute a
spliceosome. The constituents (U1A, 70K, and U1C polypeptides)
are recognized by anti-U1RNP Abs that are found in virtually all
patients with MCTD and in ;20 –30% of patients with SLE. The
reasons why we selected HuA as a neo-self Ag are as follows: 1)
0022-1767/99/$02.00
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One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined
the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA)
transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression
levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg
littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower
amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity
spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody
was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type
hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest
that the expression level of systemic autoantigens influences the outcome of the immune response to self. The Journal of Immunology, 1999, 162: 6482– 6491.
The Journal of Immunology
Materials and Methods
Mice
C57BL/6 (B6) mice were obtained from SLC (Shizuoka, Japan); B6-Igha
mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice
were maintained in a temperature- and light-controlled environment with
free access to food and water. Female, age-matched mice were used in all
experiments were 6 –10 wk old at the start of each experiment.
Generation of Tg mice
HuA cDNA was isolated from the HeLa cell l gt11 cDNA library (H.
Miura et al., unpublished data). The coding nucleotide sequence was identical with the published sequence (27). An expression plasmid (pLG-Em)
was constructed by inserting a human Em enhancer into the 59 end of the
Ld class I promoter of pLG-2 plasmid (28). HuA cDNA was fused with an
additional sequence encoding c-myc tag (AEEQKLISEEDL) at the 39 end.
c-myc-tagged HuA cDNA was subcloned into pLG-Em, and the vector
sequence was removed by HindIII-SalI digestion. The HuA transgene construct was microinjected into the pronuclei of fertilized eggs from B6 mice.
Microinjected eggs were transferred into the oviducts of pseudopregnant
females. Mice carrying the transgene were identified by either Southern
blot analysis or PCR analysis of tail DNA.
Examination of intracellular localization
Intracellular localization of the transgene product was studied in the spleen
cells of the Tg mice. Nuclear and cytoplasmic extracts were prepared. Both
samples were separated by 12.5% SDS-PAGE and were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The HuA
expression of each extract was detected by Western blotting with anti-HuA
mAb (a generous gift of Dr. W. van Venrooij) (29) and anti-c-myc mAb
(9E10) (PharMingen, San Diego, CA).
Incorporation of HuA into MuA particles
Incorporation of the HuA transgene product into MuA particles was confirmed by the immunoprecipitation of thymocytes and splenocytes from the
Tg mice using either anti-2,2,7-trimethylguanosine (anti-m3G) mAb (a
generous gift of Dr. R. Lührmann) (30) or Y12 (a generous gift of Dr. Joe
Craft), as described previously (31). Briefly, the nuclear extract of 2 3 107
thymocytes or splenocytes was probed with anti-m3G mAb or Y12 absorbed to protein A/G-Sepharose (Calbiochem, La Jolla, CA) in 500 ml of
IPP150 buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Nonidet
P-40, and 0.1% Tween 20). After 1 h of incubation at room temperature,
the Sepharose particles were collected and washed four times with IPP150
buffer. The washed Sepharose particles were resuspended in 20 ml of SDS-
gel loading buffer and boiled for 5 min. The supernatant was separated by
12.5% SDS-PAGE and transferred to a nitrocellulose membrane. HuA expression was detected by Western blotting as described above.
Preparations of Ags
Escherichia coli BL21 that had been lysogenized with phage lDE3 was
transformed with pET3c (Novagen, Madison, WI) containing HuA cDNA
at NcoI-BamHI sites as described previously (31). After treatment with 0.5
mM isopropylthiogalactose, cells were collected, washed, and disrupted in
50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM PMSF, and 10 mM
MgCl2 by freeze-thawing and sonication. Because our recombinant HuA
protein was insoluble, the insoluble pellets were solubilized in 8 M ureaPBS and were gradually refolded by dialysis against 4 M, 2 M, 1 M ureaPBS sequentially and finally against PBS. The soluble recombinant HuA
was purified by gel filtration on a HiLoad16/60 Superdex 200 column
(Pharmacia, Uppsala, Sweden) and by cation-exchange chromatography on
a MonoS column (Pharmacia) using the fast protein liquid chromatography
system (Pharmacia). The protein concentration was measured by a bicinchoninic acid protein assay (Pierce, Rockford, IL). All samples were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane;
the purity of recombinant HuA was confirmed by staining with Coomassie
brilliant blue. In some experiments, OVA was used as an Ag. Chicken egg
OVA (Sigma, St. Louis, MO) was solved in PBS at various concentrations.
The endotoxin concentration of these samples was measured by Limulus
amebocyte lysate assay (Seikagaku, Tokyo, Japan). All Ags used had ,15
ng endotoxin/mouse at the maximum injected dose.
Immunization
Mice were immunized s.c. in the base of the tail with 50 mg of Ag emulsified 1:1 (v/v) in CFA (Difco, Detroit, MI). In one part of the experiment,
mice were boosted s.c. in the base of the tail 2 wk later with 50 mg of Ag
emulsified 1:1 (v/v) in IFA (Difco).
Adoptive transfer
For adoptive transfer, various numbers of spleen cells or T cell-enriched
fractions in 0.5 ml of PBS were injected i.v. into either naive Tg mice or
their non-Tg littermates. Cell viability was noted to be .97% as determined by trypan blue exclusion.
Preparation of cell populations
Mice were sacrificed at 10 days postimmunization, and spleens or inguinal
and paraaortic lymph nodes (LNs) were taken. Single-cell suspensions of
LN cells or spleen cells were prepared under aseptic conditions by mechanical disaggregation and passed through a sterile nylon mesh, followed
by hypotonic shock to remove contaminated erythrocytes. Purified T cells
were prepared using the method of Julius et al. (32). Briefly, LN cells
(107/ml) suspended in RPMI 1640 complete medium were incubated in a
nylon wool column for 1 h at 37°C. After two washings with medium, T
cells were collected by centrifugation; the enrichment of T cells ($90%)
was examined with flow cytometry. A T cell-rich population was also
prepared by negative selection with magnetic cell sorting (MACS) (Miltenyi Biotech, Bergisch Gladbach, Germany) using anti-B220 mAb (PharMingen) and anti-I-Ab mAb (PharMingen). B cells were prepared by positive selection with MACS using anti-B220 mAb. The enrichment of B
cells ($90%) was examined by flow cytometry. Cell viability was noted to
be .97% as determined by trypan blue exclusion. Depletion of T cells or
B cells was performed by negative selection with MACS using anti-Thy1.2 mAb (PharMingen) or anti-B220 mAb, respectively. The efficiency of
depletion ($96%) was examined by flow cytometry.
ELISA
Anti-HuA Ab production was assayed by ELISA using recombinant protein as described previously (33). Briefly, the microtiter plate (Immulon 4,
Dynatech, Chantilly, VA) was coated with recombinant HuA at 5 mg/ml in
0.03 M carbonate buffer at pH 9.6 by overnight incubation at 4°C. After
blocking with 1% BSA for 2 h at 37°C, the plates were incubated with
mouse serum samples that had been serially diluted in PBS buffer containing 1% BSA and 0.05% Tween 20 for 1 h at 37°C. After washing five times
with 0.05% Tween 20 in PBS, the bound Abs were visualized with goat
anti-mouse IgG mAb coupled to HRP (Zymed, San Francisco, CA), followed by development with 3,5,39,59-tetramethylbenzidine (Kirkegaard
and Perry Laboratories, Gaithersburg, MD). OD was read at 450 nm. All
samples were tested in duplicate. A serum of wt mice immunized with HuA
exhibiting a high level of anti-HuA Abs was selected as a standard serum,
and the level was arbitrarily determined to be 10,000 U/ml. In each experiment, the standard serum was serially diluted and assayed to make a
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In our previous study, the presence of HuA-reactive T cells was
confirmed in patients with SLE or MCTD (3). 2) Although the
amino acid sequence of murine U1 snRNP-A protein (MuA) has a
96% homology to that of HuA (23), it has been demonstrated that
a considerable immune response to HuA can be induced (24). 3) In
murine lupus models, U1A is the immunodominant Ag of U1
snRNP (25), and epitopes spread to other constituents of self U
snRNPs through intermolecular/intrastructural help (26). 4) It was
shown that tolerance to MuA was broken by the coimmunization
of MuA with HuA (26).
We obtained two Tg lines that differ in the expression level of
the transgene. Because it is not feasible to elicit an immunodominant response to systemic autoantigens, we employed an adoptive
transfer strategy: the HuA-specific T cells induced in wild-type
(wt) mice were transferred to HuA Tg mice followed by booster
immunization to enhance the transferred immune response against
HuA. Thus, we were able to observe the fate of the autoimmune
response to a systemic intranuclear autoantigen. The experimental
results were opposite between the two lines. In one line, which
expresses a lower amount of HuA, autoantibody production was
vigorously enhanced and the Tg mice showed severe renal involvement. In the other line, which highly expresses HuA, autoantibody production was markedly suppressed. It was demonstrated that this suppression was Ag-specific and mediated by T
cells. Therefore, we conclude that the expression level of a systemic autoantigen determines the fate of immune response to self.
6483
6484
REGULATION OF AN AUTOIMMUNE RESPONSE TO A SYSTEMIC NUCLEAR Ag
standard curve. The level of anti-HuA Abs in each sample was determined
in comparison with this standard curve and was represented as an arbitrary
unit. Anti-70K Ab production was assayed by ELISA using the recombinant human 70K protein (MBL, Nagoya, Japan). Measurement of allotypespecific anti-HuA IgG2 Abs was performed as described above, except that
IgG2a-specific or IgG2b-specific anti-mouse IgG mAb coupled to HRP
(Zymed) was used as second Ab.
Proliferation assays
Spleen cells and LN cells, obtained as described above, were cultured at
1 3 105 cells/well with various concentrations of HuA in RPMI 1640
medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100
mg/ml streptomycin, 10% heat-inactivated FCS, and 5 3 1025 M 2-ME for
5 days, followed by a final 16 h of culture in the presence of 1 mCi of
[3H]TdR per well. The incorporated radioactivity was counted with a
gamma scintillation counter. The proliferative response was expressed as
Dcpm (mean cpm of test cultures 2 mean cpm of control cultures without
Ag) 6 SD.
Cytokine analyses
Local adoptive transfer (LAT) assays
A LAT assay was employed to detect the regulatory cells that impair the
delayed-type hypersensitivity (DTH) response (36). Spleen cells containing
a population of presumptive regulatory cells (2 3 105 cells) from Tg mice
or their non-Tg littermates (as a control) after adoptive transfer and the
injection of HuA/IFA were mixed with HuA (20 mg/ml) and responder
cells (DTH-mediating effector cells) (2 3 105 cells) obtained from HuAimmunized wt mice. This mixture (10 ml) was injected into the ear pinnae
of wt mice, and ear swelling was measured 24 h and 48 h later. In each
experiment, two non-Tg mice and two Tg mice were examined.
Evaluation of proteinuria
Urine samples were serially collected from Tg mice and non-Tg littermates
to which spleen cells from HuA-immunized wt mice were transferred and
boosted. The presence of proteinuria was assayed by a murine microalbuminuria ELISA kit (Albuwell, Exocell, Philadelphia, PA).
Immunoprecipitations of in vitro-translated protein
mRNA was transcribed from human 70K cDNA subcloned into the pGEM3Zf1 vector (Promega, Madison, WI) using the SP6 RNA polymerase promotor followed by translation in rabbit reticulocyte lysates (Promega) in
the presence of [35S]methionine. The recombinant human 70K protein labeled in vitro with [35S]methionine was immunoprecipitated with sera as
described above and was separated by 7.5% SDS-PAGE.
Results
Two Tg lines differ in HuA expression level
To generate Tg mice with a systemic expression of HuA, we constructed a c-myc-tagged HuA transgene under the control of an Ld
MHC class I promoter and Em enhancer. We obtained two Tg
lines, HAB64 and HAB69. The successful expression of HuA in
Tg mice was confirmed in the total cell extracts of spleen, thymus,
and PBMCs probed by anti-HuA mAb cross-reactive to MuA and
anti-c-myc tag mAb (data not shown).
To investigate the intracellular localization of HuA, we prepared
nuclear and cytoplasmic extracts of spleen cells of Tg mice and
non-Tg littermates in each line and performed Western blotting
using anti-c-myc mAb. The HuA transgene product was found to
be predominantly located in nuclei in both lines, although the ex-
FIGURE 1. HuA protein expression in Tg mice. A, The HuA transgene
product was predominantly expressed in nuclei. Nuclear and cytoplasmic
extracts of splenocytes from wt mice, HAB64 mice, and HAB69 mice were
prepared; samples were separated by 12.5% SDS-PAGE, followed by immunoblotting with anti-c-myc mAb. B, Different amounts of HuA were
incorporated into MuA between two Tg lines, HAB64 and HAB69. Nuclear extracts of splenocytes and thymocytes from HAB64 and HAB69
mice were immunoprecipitated by anti-m3G mAb. Immunoprecipitants
were separated by 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-c-myc mAb.
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The cytokines involved in the inhibitory effect on T cell proliferation were
analyzed. Supernatants were harvested after 40 h of culture and assayed for
IL-4, IL-10, and TGF-b by ELISA. Quantitative determinations of IL-4
and IL-10 were performed by commercial assays (Genzyme, Cambridge,
MA). Active TGF-b (without acid treatment) was determined by a sandwich ELISA using mouse anti-TGF-b mAb (Genzyme), chicken antiTGF-b mAb (R&D Systems, Minneapolis, MN), and recombinant human
TGF-b (R&D Systems) as a standard. In some experiments, to inhibit
cytokines in vitro, neutralizing mAbs to IL-4 (10 mg/ml) (R&D Systems),
IL-10 (10 mg/ml) (R&D Systems), or TGF-b (1 mg/ml) (R&D Systems)
were added in the culture medium at the amounts recommended by the
manufacturer (34, 35).
pression level of HuA was different between the two Tg lines
(Fig. 1A).
In a Xenopus oocyte injection study, it was demonstrated that
HuA injected into the cytoplasm can be transported into nuclei and
that HuA can be incorporated into the U1 snRNP complex (37).
Because HuA has a 96% similarity to MuA regarding the amino
acid sequence, it would be interesting to know whether HuA can
be incorporated into the MuA particle. Therefore, we subsequently
examined the expression level in two Tg mouse lines and the exact
localization of HuA. The nuclear extracts of the same number of
splenocytes and thymocytes of each line were prepared. U snRNP
complexes of nuclear extracts were immunoprecipitated with antim3G mAb (30) and were probed with anti-c-myc tag mAb. Fig. 1B
showed that HuA was incorporated into mouse U snRNP complexes and that the HAB64 line expressed a higher amount of HuA
than did HAB69. We confirmed that the fraction of nuclear extracts that could not be immunoprecipitated by anti-m3G mAb did
not contain HuA by Western blot analysis (data not shown).
To quantify the expression level of HuA, we analyzed these
bands using a densitometer (Table I). The ratio of the expressed
transgene product between HAB64 and HAB69 was 2.2 6 0.5 in
the thymus and 3.7 6 0.8 in the spleen. We also compared the
density of the HuA band with that of the endogenous MuA in the
The Journal of Immunology
6485
Table I. Comparison of the HuA expression level between two Tg lines
by densitometry analysisa
Thymus
Spleen
HAB64/HAB69
n (HAB64/HAB69)
2.2 6 0.5
3.7 6 0.8
12/12
14/14
a
Nuclear extracts of the same number of splenocytes and thymocytes of each line
were prepared. U snRNP complexes of nuclear extracts were immunoprecipitated
with anti-m3G mAb and probed with anti-c-myc tag mAb. The band densities were
analyzed using a densitometer, and the ratio of the expression level was shown.
total cell extract of the same number of B cells from the peripheral
blood of each line using anti-HuA mAb. In one line HAB64, the
amount of HuA was 76% of the endogenous MuA; in the other line
HAB69, the amount of HuA was 35%. Therefore, we could confirm both the different expression level of the HuA transgene and
the incorporation of HuA into mouse U snRNP. These Tg mice
provide us with the advantage of investigating the immune response to an intranuclear autoantigen in physiological conditions.
Because the HuA transgene product is expressed in the thymus, Tg
mice are expected to be unresponsive to HuA. To confirm this
possibility, we immunized Tg mice of both lines and wt mice with
50 mg of HuA emulsified 1:1 (v/v) in CFA in the base of tail. The
sera of these mice were serially collected, and the level of antiHuA IgG Abs was measured by ELISA. The proliferative response
of spleen cells stimulated with HuA was measured by thymidine
incorporation at 10 days postimmunization. Although wt mice responded to HuA, both Tg lines exhibited an unresponsiveness in B
cell response and T cell response (data not shown). To exclude the
possibility that the Tg mice had a defect in their immune response,
we immunized the mice with 50 mg of OVA and examined both T
and B cell responses to the Ag. Both lines of the Tg mice demonstrated a good response to OVA, comparable with that of wt
mice (data not shown), suggesting that the immune responses in
both lines of Tg mice are normal and not significantly different
from each other. Therefore, we concluded that both Tg lines are
unresponsive to HuA and that the insertion effect of the transgene
is negligible.
Autoantibody production can be induced in Tg mice by adoptive
transfer of autoreactive T cells
In systemic autoimmune diseases, expanded autoreactive T cell
clones are thought to drive autoantibody production. To create a
similar situation in Tg mice, we adoptively transferred the autoreactive lymphocytes that are responsive to HuA into HAB64 Tg
mice and observed the immune response to the intranuclear autoantigen. Ten million (1 3 107), 5 3 107, or 2 3 108 viable spleen
cells from wt mice immunized with HuA/CFA were injected i.v.
into naive Tg mice or their non-Tg littermates. After the adoptive
transfer, sera were serially collected and anti-HuA IgG Abs were
measured by ELISA. Both Tg mice and their non-Tg littermates
produced Abs against the HuA protein, and the level thereafter
decreased gradually over time for 6 mo. The level of autoantibodies increased according to the number of transferred cells (Fig.
2A). There were no definite differences in the kinetics and the level
of anti-HuA Abs between HAB64 Tg mice and their non-Tg littermates. In addition, another line, HAB69, also showed similar
results. When a T cell-rich population (.90% purity) was transferred into Tg mice and their non-Tg littermates, anti-HuA IgG Ab
production was also observed (data not shown), indicating that
HuA-specific T cells could provide help for autoantibody production in recipient-derived B cells.
FIGURE 2. Adoptive transfer of HuA-reactive lymphocytes. A, Singlecell suspensions of spleens from wt mice immunized with HuA/CFA were
obtained. For adoptive transfer, 1 3 107, 5 3 107, or 2 3 108 viable spleen
cells in 0.5 ml of PBS were injected i.v. into naive Tg mice and their
littermates. Sera of these mice were serially collected, and anti-HuA IgG
Abs of samples were measured by ELISA. The Ab level of samples was
calculated as the arbitrary unit according to the standard curve and the
mean 6 SD U/ml values are shown. Three mice were tested in each group.
B, HuA-specific T cells could provide help for anti-HuA Ab production in
recipient-derived B cells. A T cell-rich population was obtained from the
spleen cells of HuA-immunized B6-Ighb mice by negative selection with
MACS using anti-B220 mAb and anti-I-Ab mAb; a total of 1 3 107 viable
T cells were adoptively transferred into two B6-Igha mice, and IgG2a and
IgG2b-specific anti-HuA Abs were measured by ELISA. ELISA readings at
a 1/100 dilution of sera from recipients were shown as an OD of 450 nm.
To confirm this observation, we adoptively transferred the T
cell-rich population from HuA-immunized B6-Ighb mice into B6Igha mice and measured allotype-specific anti-HuA IgG Abs by
ELISA. As shown in Fig. 2B, the recipients mainly produced antiHuA IgG2a Abs, indicating that recipient-derived B cells were
involved in anti-HuA IgG Ab production.
Enhancement of autoantibody production in HAB69 resulted in
pathogenic renal manifestations
Although autoantibody production can be successfully induced in
tolerant mice, the autoantibody level was not so high compared
with that seen for MRL/lpr lupus-prone mice. This low autoantibody level seems to be due to a low frequency of autoreactive T
cells. Therefore, we intended to expand autoreactive lymphocytes
by inducing a recall memory response in Tg mice. When 5 3 107
or 1 3 107 viable spleen cells from wt mice immunized with
HuA/CFA were adoptively transferred to the Tg mice of both lines
and their non-Tg littermates, the recipients were boosted with HuA
emulsified in IFA either simultaneously (data not shown) or at
3 mo posttransfer (Figs. 3, A and B). In HAB69, anti-HuA Ab
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Tg mice are unresponsive to HuA
6486
REGULATION OF AN AUTOIMMUNE RESPONSE TO A SYSTEMIC NUCLEAR Ag
FIGURE 3. Enhancement of autoantibody
production in HAB64 and HAB69. A single-cell
suspension of spleens from HuA-immunized wt
mice was prepared. For adoptive transfer, 5 3
107 viable spleen cells were injected i.v. into
three Tg mice and three non-Tg littermates. To
amplify the recall response, these mice were injected with 50 mg of HuA in IFA at 3 mo posttransfer. The sera of these mice were serially collected, and anti-HuA IgG Abs of samples were
measured by ELISA. The Ab level of the samples was calculated as an arbitrary unit according
to the standard curve. The mean unit per milliliter values 6 SD are shown. A, Marked suppression of autoantibody production in HAB64.
B, Enhancement of autoantibody production in
HAB69. The results using 1 3 107 spleen cells
were omitted because they gave similar results
with the exception of the reduced Ab level due to
the reduced number of transferred T cells.
the exaggerated autoimmune response in HAB69 led to an autoaggressive disease phenotype.
Previous reports have described that the coimmunization of self
(murine) snRNPs with HuA could break self tolerance to self
snRNPs and epitope spread to other U snRNP constituents. Based
on this finding, the model of intermolecular/intrastructural help
was proposed to explain autoantibody propagation to the components of an autoantigen complex (26), for example, U1A/70K in
the U1 snRNP complex (26) and La/Ro in the RoRNP complex
(38). Because the HuA transgene product is incorporated into the
MuA complex, HuA Tg mice are a good model to investigate this
mechanism.
U snRNP complexes of nuclear extracts of Ehrlich ascites tumor
cells, a murine lymphoma cell line, were immunoprecipitated with
anti-m3G mAb (30) and probed with sera from Tg mice, which
FIGURE 4. Renal manifestations in HAB69 suffered from autoaggressive immune response. A, Urine albumin concentration of HAB69 mice and their
littermates with an exaggerated HuA Ab response. Urine samples were collected from HAB69 mice and their non-Tg littermates at 5 mo after adoptive
transfer. The results from HAB69 mice and their littermates without any treatment were also included as negative controls. The urine albumin concentration
was measured by ELISA. In each group of mice, 8 or 10 mice were tested. B, A microscopic picture of a kidney from an HAB69 mouse. Marked
proliferation of mesangial cells and matrix was observed (hematoxylin and eosin staining, 3400 magnification). C, Immunofluorescence study of
complement 3. There are granular (mesangium) and linear (tubular and Bowman’s capsular basement membranes) deposits of complement 3 (3400
magnification).
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production was markedly enhanced up to levels that were nearly
comparable with those of the wt controls (Fig. 3B). There were no
differences in the kinetics of autoantibody production as well.
However, HAB69 demonstrated more severe proteinuria than
the control mice at 3 mo after the booster (Fig. 4A). The severity
is significant because 60% of HAB69 mice (n 5 6/10) exceeded
the mean value 1 2 SD of the urine albumin concentration of wt
recipient mice. Light microscopic examinations revealed a marked
proliferation of mesangial cells, and matrix (Fig. 4B) and immunofluorescence microscopy studies showed granular deposits of
complement 3 as well as IgG in the mesangium and in the tubular
and capsular basement membranes (Fig. 4C), indicating an immune complex-type glomerulonephritis. Interestingly, some
HAB69 exhibited more extensive and severe skin manifestations,
including skin ulcers (data not shown). These results suggest that
The Journal of Immunology
produced a high level of anti-HuA autoantibodies. As shown in
Fig. 5A, the Tg mice showing an autoaggressive phenotype developed anti-murine 70K IgG autoantibodies in immunoblots. In
vitro-translated, [35S]methionine-labeled recombinant human 70K
protein could be immunoprecipitated by sera from Tg mice (Fig.
5B), because anti-murine 70K autoantibodies cross-react to human
70K. These results indicate that anti-70K IgG autoantibodies were
produced in HAB69 mice. To compare the level of anti-70K autoantibodies from HuA Tg mice with that of non-Tg mice, sera
from individual mice were examined by ELISA using recombinant
human 70K (Fig. 5C). A larger number of Tg mice showed a
higher level of anti-70K Abs after adoptive transfer of HuA-reactive lymphocytes than did non-Tg mice. These data demonstrate
that once an autoimmune response to HuA occurs, self-reactivity
spreads to another component of the U1 snRNP complex containing HuA via intermolecular/intrastructural help. By these results,
we could again confirm the incorporation of HuA in the mouse U
snRNP particle.
In contrast to the case of HAB69, in HAB64, anti-HuA autoantibody production was markedly suppressed when the recipients
were boosted 3 mo after the successful transfer as shown in Fig.
3A. When we boosted the recipients immediately after the transfer
to exclude the possibility that the donor cells were quickly removed by activation-induced cell death (20 –22), we also observed
a similar suppression (data not shown). Therefore, we postulated
that some regulatory mechanism could appear upon booster
immunization.
To verify this hypothesis, we performed a LAT assay and a
proliferation inhibition assay. The LAT assay has been employed
FIGURE 5. Spreading of self-reactivity to 70K
autoantigen in HAB69. A, Nuclear extracts of Ehrlich ascites cells were immunoprecipitated by antim3G mAb. The immunoprecipitants were separated
by 7.5% SDS-PAGE, transferred to a nitrocellulose
membrane, and probed with a representative serum
from HAB69 exhibiting an enhanced immune response to HuA (lane 3), from an SLE patient who is
known to have a high level of anti-70K autoantibodies as a positive control (lane 2), or from a wt mouse
as a negative control (lane 1). Anti-70K Abs could be
detected by Western blotting as indicated by the arrow. B, Anti-70K Abs were detected by immunoprecipitation reactions using in vitro-translated and
[35S]methionine-labeled human 70K protein. The
70K mRNA was transcribed from the human 70K
cDNA that had been previously subcloned into the
pGEM-3Zf1 vector followed by translation in rabbit
reticulocyte lysates in the presence of [35S]methionine. The human 70K protein labeled in vitro with
[35S]methionine was immunoprecipitated with sera
from HAB69 mice (lane 2) or with sera from wt mice
as a negative control (lane 1). C, The level of anti70K Abs was measured by an ELISA kit using recombinant human 70K. The maximum ELISA readings at a 1/100 dilution of sera from HAB69 Tg mice
and their non-Tg littermates were shown as an OD of
450 nm.
for the detection of regulatory cells that impair the DTH response
(36). Spleen cells from HuA/IFA-immunized HAB64 after adoptive transfer (which presumably contain a population of regulatory
cells) were mixed with HuA and DTH-mediating responder cells
obtained from HuA/CFA-immunized wt mice. This mixture was
injected into the ear pinnae of naive wt mice, and ear swelling was
measured after 24 and 48 h. As a positive control, the spleen cells
from non-Tg littermates immunized with HuA/IFA after adoptive
transfer were employed. The mixture with the spleen cells from
HuA/IFA-injected HAB64 exhibited a lower ear swelling response, indicating the presence of regulatory cells that impair the
expression of DTH (Fig. 6A).
We were also able to confirm the presence of regulatory cells by
an in vitro proliferation inhibition assay. Spleen cells or LN cells
obtained from HuA/IFA-injected HAB64 after adoptive transfer
were mixed with spleen cells or LN cells from HuA/CFA-immunized wt mice at various proportions. Thymidine incorporation of
responder cells was reduced by the addition of splenocytes from
HuA/IFA-treated HAB64. The inhibitory effect on proliferation
was observed when the ratio of regulatory cells to responder cells
was either 3:1 or 1:1, whereas this effect was abrogated when the
ratio was 1:3 (Fig. 6B), suggesting that the regulatory cell frequency might be important for inhibition. This inhibition was also
observed when spleen cells from HAB64 mice, which were only
injected with HuA/IFA without the adoptive transfer, were used as
regulatory cells.
To examine whether this regulation was Ag-specific or not,
spleen cells obtained from HuA/IFA-injected HAB64 after the
adoptive transfer were mixed with the spleen cells obtained from
OVA/CFA-immunized wt mice and were cultured in the presence
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Regulatory mechanism of autoreactivity in HAB64
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REGULATION OF AN AUTOIMMUNE RESPONSE TO A SYSTEMIC NUCLEAR Ag
of OVA and HuA. In these experiments, The proliferation response to OVA was not inhibited (data not shown). These data
suggest that the regulatory cells in HuA/IFA-injected HAB64 mice
are autoantigen specific.
Interestingly, when we compared the inhibitory activity between
HAB64 and HAB69 using spleen cells from HuA/IFA-injected
mice after the adoptive transfer, we found that there was a clear
difference between both lines (Fig. 6C). Spleen cells from HAB69,
the autoaggressive phenotype line, did not exhibit an impairment
of proliferation to HuA, suggesting that the suppression of the
immune response to HuA in HAB64 can be attributed to the induction of regulatory cells by HuA/IFA immunization.
We subsequently examined which cell population is responsible
for this inhibition. T cells or B cells were depleted from the spleen
cells obtained from either Tg mice or non-Tg littermates after the
injection of HuA/IFA by MACS using either anti-Thy-1.2 mAb or
anti-B220 mAb. T cell- or B cell-depleted spleen cells were mixed
with spleen cells as responder cells obtained from HuA/CFA-immunized wt mice and cultured in the presence of HuA (Fig. 6D).
Anti-Thy-1.2 mAb treatment abrogated the inhibitory effect for
HuA-reactive T cell proliferation, suggesting that regulatory T
cells mediated the inhibition.
Several studies have reported that IFA induced Ag-specific tolerance or unresponsiveness. In most of them, the regulatory mechanism is attributed to the induction of Th2 cells by i.p. Ag/IFA
injection as demonstrated in a beef insulin Tg model (39) and in
neonatal tolerance models (40). Another candidate for the mechanism is Th3 cells secreting TGF-b, which has been shown to play
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FIGURE 6. Inhibition of the DTH response and the proliferative response in HAB64. A, The DTH response was suppressed by spleen cells from Tg
mice injected with HuA/IFA. Spleen cells containing a population of presumptive regulatory cells (2 3 105 cells) obtained from HAB64 mice or their
non-Tg littermates (as a control) injected with HuA/IFA after adoptive transfer were mixed with HuA (20 mg/ml) and responder cells (DTH-mediating
effector cells) (2 3 105 cells) obtained from HuA/CFA-immunized wt mice. This mixture (10 ml) was injected into the ear pinnae of the wt mice, and ear
swelling was measured after 24 h (open bars) and 48 h (striped bars). These data are representative of two separate experiments. In each experiment, two
non-Tg mice and two Tg mice were tested. B, Impairment of a proliferative response by the lymphocytes from HAB64. Spleen cells or LN cells were
obtained from HAB64 mice or their non-Tg littermates (as a control) that had been injected with HuA/IFA after the adoptive transfer as regulator cells.
As responder cells, spleen cells or LN cells were obtained from HuA/CFA-immunized non-Tg mice. Regulatory cells and responder cells were mixed at
3:1, 1:1, and 1:3 and cultured with HuA (50 mg/ml) in RPMI 1640 for 5 days. Proliferation was measured by thymidine incorporation. Results are
representative of three independent experiments. C, Proliferative responses of HuA-reactive lymphocytes in the presence of lymphocytes from HuA/IFAinjected HAB64 and HAB69. Proliferation inhibition assays, as described above, were performed. Presumptive regulator cells (105 cells/well) and responder
cells (105 cells/well) were mixed and cultured. The percent inhibition was calculated in comparison with the proliferation of responder cells in the absence
of regulator cells. Results are representative of three independent experiments. D, Inhibition of the proliferative response in HAB64 was mediated by
regulatory T cells. T cells or B cells were depleted from the spleen cells obtained from Tg mice and non-Tg littermates after the injection of HuA/IFA by
MACS using anti-Thy-1.2 mAb or anti-B220 mAb, respectively. T cell- or B cell-depleted spleen cells were used as presumptive regulator cells; proliferation inhibition assays, as described above, were performed. The percent inhibition was calculated in comparison with the proliferation of responder cells
in the absence of regulator cells. Results are representative of three independent experiments. E, Inhibitory cytokines did not abrogate the proliferative
response in HAB64. Proliferation inhibition assays were performed in the presence of neutralizing mAb to IL-4, IL-10, or TGF-b. Proliferation was
measured by thymidine incorporation. Results are representative of two independent experiments.
The Journal of Immunology
Discussion
A few studies have examined Ag compartmentation and immunological tolerance (44, 45). Previous studies have revealed that the
availability of self Ag depends upon processing and Ag compartmentation (e.g., mitochondria membrane, plasma membrane, and
nucleus) and that it could influence the emergence of autoreactive
T cells (45). These studies demonstrated that CD4 T cell tolerance
via negative selection, a pivotal mechanism for self tolerance, can
be achieved by MHC class II Ag presentation of endogenous proteins on thymic epithelial cells, although the contribution of autophagy cannot be excluded. Our Tg model, expressing the neoself
Ag in thymocytes, is tolerant of intranuclear neo-autoantigen, and
they did not exhibit spontaneous autoimmunity. Although we were
not able to define the precise expression site of HuA within the
thymus and the amount of circulating Ag, it would be interesting
to examine whether bone marrow-derived dendritic cells or thymic
epithelial cells contribute to the induction of tolerance. Therefore,
the next question would be how the immunological tolerance is
induced to intranuclear Ag under physiological conditions, as in
the case of our Tg mice, and whether autoantigen-reactive T cells
are deleted or anergic in the thymus.
By the adoptive transfer of autoreactive (HuA-reactive) T cells
from non-Tg mice immunized with HuA/CFA into naive Tg mice
or their non-Tg littermates, anti-HuA Abs were successfully induced in both. Therefore, it seems that the autoimmune response is
acceptable to some extent in the periphery. Recently, using adoptive transfer of naive autoreactive T cells, it was demonstrated that
autoreactive T cells led to an induction of tolerance after a period
of transient activation by encountering an autoantigen (22, 46).
Because we transferred the autoreactive T cells from immunized
mice instead of naive T cells, it is possible that the activated T cells
in the transferred T cell population induced Ab production efficiently before being tolerant. However, because the autoantibody
level increased until 1 wk after the transfer and there is no difference concerning both the level and the kinetics between Tg and
non-Tg mice, it is rather likely that the tolerance induction to the
transferred autoreactive T cells in HuA Tg mice might not be so
effective as the reported cases. It would be interesting to know
whether this inefficiency is related to the accessibility and availability of the autoantigen.
HAB69, a low expresser, exhibited enhanced production of antiHuA and anti-70K autoantibody, resulting in glomerulonephritis.
However, because anti-HuA Ab and proteinuria could be also
induced in wt mice to some extent, glomerulonephritis might
be induced by the deposition of immune complexes. Nevertheless,
the significant difference in the severity of proteinuria suggests
that the HuA transgene product plays some role in the pathogenesis of the kidney in these mice. In SLE patients, proteinuria was
usually not associated with anti-U1RNP autoantibody but with antidsDNA autoantibody. Ab to native DNA in sera from SLE patients
and BWF1 lupus-prone mice has been shown to have cross-reactivity with U1A and Sm D proteins frequently, and this crossreactivity was suggested to be part of the original immunogenic
drive in the production of anti-native DNA autoantibody (47). We
could also detect anti-dsDNA reactivity in HAB69 (our unpublished observations). This implies the possibility that enhanced
immune responses to HuA led to the production of nephritogenic
Abs such as anti-dsDNA autoantibody and that these nephritogenic
Abs were involved in the severity of glomerulonephritis in
HAB69.
HAB64, a high expresser, demonstrated a regulatory phenotype.
This suppression was Ag-specific. Thus far, a few reports have
described the relationship between the amount of tolerogen and T
cell tolerance (19, 48). It has been shown that peripheral tolerance
has multiple levels that are significantly influenced by the amount
of tolerogens in organ-specific autoantigens (19). This view has
also been proposed in studies that investigated the immune response to a systemic Ag (20, 21). According to the previously
reported two Tg lines producing different amounts of secreted hen
egg-white lysozyme (HEL), T cells specific for the minimal immunodominant epitope of HEL were deleted or inactivated in both
lines, whereas T cell clones of lower affinity reacting with epitopes
on longer peptides persist only in the line that produced a lower
amount of HEL (48). Therefore, we can postulate that in our Tg
mice, the different expression levels of the nuclear autoantigen led
to the difference of either the amount or affinity of T cell clones
that were deleted or inactivated, and that the tolerized T cells compete for IL-2 (49), resulting in the inhibitory effect on Ag-specific
T cell proliferation. Because our system does not use TCR Tg
mice, and we could not examine the phenotype of single T cell
clones (e.g., TCR down-regulation), we can not completely exclude the possibility that the tolerance level of a single T cell clone
is more profound in HAB64. However, this mechanism is less
likely, because an addition of IL-2 could not restore the inhibitory
effect (our unpublished observations).
We prefer to postulate the presence of regulatory T cells, which
appear after HuA/IFA immunization. It has been proposed that
CD41 T cells are the executors of Ag-specific surveillance of autoantibody production (50). This mechanism depends upon the
Fas/Fas ligand system (51, 52). Based on this idea, an alternative
explanation might be possible. That is, the memory HuA-specific
T cells transferred and activated by HuA/IFA immunization could
exhibit cytolytic activity against the Tg mice B cells, leading to a
loss of APC. However, when we introduced the HuA transgene
into B6/lpr/lpr to investigate the involvement of the Fas/Fas ligand
system, a suppression of autoantibody production was observed.
Because the spleen cells with the suppressive activity did not show
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an important role in oral tolerance (41). Therefore, we examined
the possibility that these humoral factors are involved in the inhibition. At first, we collected culture supernatants at 48 h in the
proliferation inhibition assays and analyzed the cytokines. We
were unable to detect any significant increase in Th2-type cytokines (IL-4 and IL-10) (42, 43) and TGF-b in the supernatant of
responder cells mixed with the regulatory spleen cells from
HAB64 or in the supernatant of HAB64 spleen cells stimulated
with the Ag. We could observe only lower amounts of IL-2 in the
supernatant of responder cells mixed with spleen cells from
HAB64 mice (131.3 6 1.1 pg/ml) compared with that seen for wt
mice (177.7 6 2.1 pg/ml).
Because it is possible that the frequency of the Ag-specific T
cells was not sufficient to be distinguished by their lymphokine
secretion, we employed neutralizing Abs to the inhibitory cytokines. However, the addition of Abs to IL-4, IL-10, and TGF-b did
not abrogate the inhibition, suggesting that the inhibitory effect is
not mediated by these cytokines (Fig. 6E).
Taken together, the two HuA autoantigen Tg lines, HAB64 and
HAB69, which show different expression levels of the autoantigen,
demonstrated different outcomes when autoreactive lymphocytes
were expanded. Pathogenic lesions (manifestations of autoimmune
diseases) appeared in one line, whereas a regulatory mechanism
was seen in the other. Therefore, it seems that a peripheral immunoregulatory mechanism might exist that depends upon the expression level of an autoantigen; our data suggest that the regulatory T cells are involved in this phenomenon.
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Acknowledgments
We thank Drs. Walther van Venrooij, Reinherz Lührmann, Joe Craft, and
Yoshiyuki Kanai for providing the necessary reagents. We are also grateful
to Dr. Nobukata Shinohara for valuable discussions and to Dr. Kiyoshi
Kitamura for encouragement.
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any killing activity against Tg spleen cells as target cells, the involvement of CD81 CTLs is also unlikely (our unpublished observations). Interestingly, Marusic and Tonegawa reported that a
mechanism other than Th2 deviation or activation-induced cell
death can be induced by peritoneal IFA injection; they observed an
unresponsive induction using either Fas-deficient or IL-4 deficient
anti-myelin basic protein TCR Tg mice (53).
In conclusion, these data suggest that the immune system to
intranuclear autoantigens has a regulatory mechanism that appears
according to the intensity and the context of the immune response
to self. This peripheral regulation might be a fail-safe mechanism
against the disturbance of self tolerance by several mechanisms
including cryptic epitope (54), molecular mimicry (6, 7) and bystander activation (55). Our Tg mouse models can be expected to
present the key for the understanding and treatment of systemic
autoimmune diseases such as SLE and MCTD, because the location and assembly with other molecules of the introduced Ag are
physiological rather than artificial. Further studies, including those
using TCR Tg mice to dissect the fate of autoreactive T cells at a
single cell level, are underway to understand how this autoimmune
response is regulated.
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