вход по аккаунту


Tissue targeting of anti-RNP autoimmunityEffects of T cells and myeloid dendritic cells in a murine model.

код для вставкиСкачать
Vol. 60, No. 2, February 2009, pp 534–542
DOI 10.1002/art.24256
© 2009, American College of Rheumatology
Tissue Targeting of Anti-RNP Autoimmunity
Effects of T Cells and Myeloid Dendritic Cells in a Murine Model
Eric L. Greidinger,1 YunJuan Zang,2 Irina Fernandez,3 Mariana Berho,4 Mehdi Nassiri,2
Laisel Martinez,2 and Robert W. Hoffman1
Objective. To explore the role of immune cells in
anti-RNP autoimmunity in a murine model of pneumonitis or glomerulonephritis, using adoptive transfer
Methods. Donor mice were immunized with 50 ␮g
of U1–70-kd small nuclear RNP fusion protein and
50 ␮g of U1 RNA adjuvant. Whole splenocytes as well as
CD4ⴙ cell and dendritic cell (DC) subsets from the
immunized mice were infused into naive syngeneic recipients. Anti-RNP and T cell responses were assessed
by immunoblotting, enzyme-linked immunosorbent assay, and flow cytometry. Development of renal or lung
disease was assessed by histology and urinalysis.
Results. Unfractionated splenocytes from donor
mice without proteinuria induced predominantly lung
disease in recipients (8 [57%] of 14 versus 2 [14%] of
14 developing renal disease; P ⴝ 0.046). However,
infusion of CD4ⴙ cells from donors without proteinuria
induced renal disease more frequently than lung disease
(7 [70%] of 10 versus 2 [20%] of 10; P ⴝ 0.01); adoptive
transfer of RNPⴙCD4ⴙ T cells from short-term culture
yielded similar results (renal disease in 8 [73%] of 11
recipients versus lung disease in 3 [27%] of 11). Cotransfer of splenic myeloid DCs and CD4ⴙ T cells from
immunized donors prevented induction of renal disease
in all 5 recipients (P ⴝ 0.026 versus recipients of fresh
CD4ⴙ cells alone), although lung disease was still
observed in 1 of 5 mice. Transfer of myeloid DCs alone
from immunized donors induced lung disease in 3 (60%)
of 5 recipients, without evidence of nephritis. Cotransfer
of splenocytes from mice with and those without nephritis led to renal disease in 4 of 5 recipients, without
evidence of lung disease.
Conclusion. These findings indicate that RNPⴙ
CD4ⴙ T cells are sufficient to induce anti-RNP autoimmunity, tissue targeting in anti-RNP autoimmunity can
be deviated to either a renal or pulmonary phenotype
depending on the presence of accessory cells such as
myeloid DCs, and DC subsets can play a role in both
propagation of autoimmunity and end-organ targeting.
Autoimmunity to RNP autoantigens is frequently
seen in systemic autoimmune diseases, including lupus
and mixed connective tissue disease (MCTD) (1). The
induction of anti-RNP autoantibodies is closely linked,
in terms of time to onset, with the initial presentation of
clinical manifestations of autoimmune disease in patients, suggesting that anti-RNP responses may have
direct pathogenic roles in autoimmune diseases (2,3).
We recently demonstrated that in mice that were not
otherwise disease-prone, a single direct immunization
with an RNP peptide plus adjuvant can induce a persistent autoimmune response, including development of
autoantibodies and end-organ injury, both of which are
consistent with the characteristics of human anti-RNP
syndromes (4). The findings from this model also suggest
that the pattern of tissue injury that emerges could
Dr. Greidinger’s work was supported by the US Department
of Veterans Affairs (Merit Review grant), the NIH (grant AI-1842),
and the Lupus Research Institute. Dr. Hoffman’s work was supported
by the US Department of Veterans Affairs and the NIH (grants
AR-43308 and AR-48805).
Eric L. Greidinger, MD, Robert W. Hoffman, DO: Miami
VA Medical Center, and University of Miami Miller School of
Medicine, Miami, Florida; 2YunJuan Zang, MD, Mehdi Nassiri, MD,
Laisel Martinez, MS: University of Miami Miller School of Medicine,
Miami, Florida; 3Irina Fernandez, MS: Miami VA Foundation for
Medical Research, Miami, Florida; 4Mariana Berho, MD: Cleveland
Clinic Foundation, Weston, Florida.
Dr. Nassiri holds a patent for a molecular fixative, for which
he receives royalties through Sakura FineTek USA.
Address correspondence and reprint requests to Eric L.
Greidinger, MD, Division of Rheumatology and Immunology, University of Miami, 1400 NW 10th Avenue, Suite 602, Miami, FL 33136.
Submitted for publication July 1, 2008; accepted in revised
form October 17, 2008.
develop into either MCTD-like lung disease or lupuslike nephritis, depending on the immune context present.
We have since found that serum transfer from
immunized mice could exacerbate lung injury in recipients in the setting of acute inflammation but causes
minimal tissue injury in normal physiologic conditions
(5). Prior studies have indicated that the functions of
T cells may be sufficient to convey anti-RNP autoimmunity (6). This led us to investigate the role of cellular
immunity in mediating anti-RNP autoimmunity. Thus,
in the present study, we examined the ability of immune
cells from anti-RNP–immunized mice to induce disease
after adoptive transfer into naive syngeneic animals.
Our results show that transfer of whole splenocytes conveys anti-RNP autoimmunity and lung disease
from donors to recipients, whereas transfer of only
CD4⫹ splenocytes conveys anti-RNP autoimmunity but
changes the clinical phenotype from lung disease to
nephritis in the recipients. We were able to identify a
population of nonplasmacytoid splenic dendritic cells
(DCs), referred to as myeloid DCs, that can prevent the
induction of nephritis, when cotransferred with CD4⫹
cells, or can induce anti-RNP immunity with lung disease, when transferred alone. However, splenocytes
from mice without nephritis, when transferred along
with splenocytes from mice with established nephritis,
failed to prevent nephritis, and a small number of
plasmacytoid DCs from the immunized mice with renal
disease was sufficient to convey nephritis to naive recipients. These results suggest that adaptive and innate
immune cells collaboratively shape the tissue expression
of systemic autoimmune disease. Moreover, we are able
to identify distinct steps in the development of the
autoimmune responses and the differentiation of autoimmune tissue targeting.
Mice. Experiments were performed using C57BL/
6NTac H2-Abl–knockout DR4-transgenic mice (Taconic, Germantown, NY). These C57BL/6 mice (herein referred to as
DR4 mice) are transgenic for the expression of a chimeric
human/mouse class II major histocompatibility complex in
which the extracellular antigen-presentation domains of HLA–
DR4 have replaced the native murine class II regions, with the
remainder of the native murine molecule kept intact, as we
have previously reported (4). Earlier studies used the same
mouse strain successfully in adoptive transfer experiments
involving immunization with type II collagen (CII), in which
transfer of anti-CII immunity and induction of arthritis were
successfully achieved in transfer recipients, without indications
of lupus or MCTD-like immunity (7). All experiments were
conducted in accordance with Institutional Animal Care and
Use Committee–approved protocols and were carried out in
facilities certified by the Association for Assessment and
Accreditation of Laboratory Animal Care.
DR4 mice, at ages 8–10 weeks, were subcutaneously
immunized once in the flank with 50 ␮g of U1–70-kd small
nuclear RNP fusion protein (70K) and 50 ␮g of U1 RNA
adjuvant. As previously reported, a high proportion of the
directly immunized mice developed anti-RNP autoantibodies
(4), RNP-specific T cells (8), and interstitial pneumonitis that
persisted for months after the single immunization, and this
was associated with anti-70K T cell epitope spreading (4). In
order for a mouse to be qualified as immunized for use in the
current study, anti-RNP antibodies were required to be demonstrated by enzyme-linked immunosorbent assay (ELISA)
and/or immunoblotting.
Adoptive transfer. To assess the contribution of immune cells to this process, we harvested splenocytes from
anti-RNP–immunized mice 2 months after immunization;
splenocytes from naive syngeneic mice were used as controls.
Whole spleens were mechanically disrupted and passed
through a fine wire mesh in cold phosphate buffered saline
(PBS) to create a single-cell suspension. The cells were treated
with red blood cell (RBC) lysis buffer, washed, and resuspended in sterile PBS for infusion or further cell separation.
We infused cells (10 ⫻ 106 whole splenocytes, 2 ⫻ 106 CD4⫹
cells, and 0.1 ⫻ 106 myeloid DCs or 0.1 ⫻ 106 plasmacytoid
DCs, in a final volume of 50 ␮l of sterile PBS) into 8–10-weekold naive syngeneic (nonirradiated) recipients via the tail vein;
thereafter, the recipient mice were followed up for 2 months.
As we have previously reported, recipient mice in the whole
splenocyte transfer experiments developed anti-RNP antibodies that were typically of lower titer than that found in
donor mice (4,8). Moreover, the anti-RNP T cells from recipients had T cell fine specificity and also had T cell receptor
(TCR) V-gene usage similar to that of the anti-RNP–specific
T cells derived from donor mice (8).
Cell separation. CD4⫹ cells were obtained from RBCdepleted splenocytes by positive selection with anti-CD4 microbeads, using AutoMACS (Miltenyi, Auburn, CA). Myeloid
DCs were obtained from RBC-depleted splenocytes, using
OptiPrep density centrifugation in accordance with the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO) to
isolate the DC layer, followed by negative selection with
anti-murine plasmacytoid dendritic cell antigen 1 (PDCA-1)
microbeads to deplete plasmacytoid DCs, and positive selection with anti-CD11c microbeads to select myeloid DCs (AutoMACS; Miltenyi). Plasmacytoid DCs were obtained using
OptiPrep density centrifugation as described above, followed
by use of the Miltenyi murine Plasmacytoid Dendritic Cell
Negative Selection Kit, then positive selection with antimurine PDCA-1 microbeads (Miltenyi).
Cell culture. Murine T cell lines were generated and
characterized using an approach similar to one that has been
used extensively by our group for generating human
autoantigen-specific T cell lines and one used more recently to
generate murine autoantigen-specific T cell lines (8–12).
Briefly, spleen and lymph node cells were obtained at necropsy, in sterile conditions, and then mechanically disrupted,
filtered through a sterile 100-␮m nylon mesh filter, and
subjected to density-gradient centrifugation using Histopaque
(Sigma-Aldrich). Cells obtained from immunized mice were
used immediately for the generation of T cell lines. Cells
obtained from naive mice were irradiated with 30 Gy for use as
antigen-presenting cells (APCs) to stimulate T cells and for use
in proliferation assays.
Approximately 5 ⫻ 106 cells were cultured in Dulbecco’s modified Eagle’s medium with 2 mM L-glutamine (complete medium) supplemented with 20 ␮g/ml gentamicin/15%
fetal calf serum and containing fusion protein at a final
concentration of 50 ␮g/ml. The 70-kd fusion protein was used
as antigen, as described previously (8–12). Cells in a final
volume of 5 ml were placed in a 25-cm2 flask and incubated in
5% carbon dioxide at 37°C. Cells were restimulated with 5 ⫻
106 murine APCs, which had been irradiated with 30 Gy,
together with antigen in fresh medium on days 7–10. Following
1 or 2 cycles of stimulation, cells were harvested for adoptive
transfer, in vitro proliferation assays, and/or TCR analyses. For
TCR analyses, cells were stimulated with plate-bound antiCD3 in the absence of APCs, and at 48 hours, RNA was extracted from the cells and analyzed using RNeasy (Invitrogen,
Carlsbad, CA).
Disease assessment. The presence of active urinary
sediment was determined by urinalysis on urine specimens
obtained at the time the mice were killed. The presence of
histologic abnormalities was determined in a blinded manner
by assessing UM-Fix–prepared, hematoxylin and eosin–stained
tissue, as previously reported (4,13). Additional sections of the
kidney, obtained after the mice were killed, were embedded in
OCT compound and snap-frozen for immunofluorescence
assays with fluorescein isothiocyanate–conjugated anti-mouse
C3 and anti-mouse IgG antibodies. Serum C3 levels were
measured in mouse serum samples, using a commercial ELISA
(Immunology Consultants, Newberg, OR).
Flow cytometry. Fluorochrome-conjugated antibodies
to CD3, CD4, CD8, CD11c, and HLA–DR4 were purchased
from Becton Dickinson (Franklin Lakes, NJ), 5,6carboxyfluorescein succinimidyl ester (CFSE) was obtained
from Molecular Probes (Eugene, OR), and fluorochromeconjugated anti-murine PDCA-1 was from Miltenyi. For flow
cytometric analyses, purified myeloid DCs were labeled with
CFSE in a 10-minute incubation, in which the cells were
incubated in complete medium containing a final concentration of 2.5 ␮M CFSE. Cells were washed and resuspended in
cold sterile PBS prior to infusion into the tail vein. Cells were
quantitated on a BD LSRII instrument, and data were analyzed using BD FACSDiva software (Becton Dickinson).
Anti-RNP assays. Anti-RNP autoantibodies were detected by immunoblotting against intact and apoptotic Jurkat
cell lysates and by an ELISA against purified 70K fusion
protein as antigen, both of which have been previously described in detail (14). Anti-RNP T cells were detected using a
previously described method (8,12), in which we noted elevations in the stimulation indices in response to known DR4restricted 70K T cell epitopes that were presented by the
irradiated APCs from syngeneic naive mice. T cell lines
proliferating in response to RNP peptides were generated as
described above. The V␤ regions were sequenced using primers for the TCR heavy-chain V␤ region, as previously described
(8), to isolate the recombined area of TCRs from RNPresponsive cells.
Statistical analysis. Categorical variables were compared using Fisher’s exact test. Results were calculated using
the Prism program, version 3.0 (GraphPad, San Diego, CA).
Disease induction with RNPⴙ splenocyte adoptive transfer. Direct immunization of DR4 mice (n ⫽ 80)
with a single subcutaneous dose of 50 ␮g of 70K peptide
along with 50 ␮g of U1 RNA adjuvant induced anti-70K
immune responses in the mice, as confirmed by ELISA
and/or immunoblotting. Of these mice, 47 (59%) were
determined, by urinalysis, to be without proteinuria,
while 33 (41%) had at least trace proteinuria. Among
the 47 mice without proteinuria, 18 (38%) developed
histologic manifestations of interstitial lung disease,
whereas none of these mice developed histologically
evident renal disease. In contrast, among the 33 mice
with proteinuria, 5 (15%) developed histologic manifestations of lung disease (P ⫽ 0.027 versus mice without
proteinuria, by Fisher’s exact test) and 9 (27%) developed histologic manifestations of renal disease (P ⫽
0.0002 versus mice without proteinuria, by Fisher’s exact
test). We therefore hypothesized that the presence or
absence or proteinuria may be a marker of immunologic
conditions that could favor the induction of nephritis
over the induction of lung disease.
We performed adoptive transfer studies using
serum transfer of splenocytes from immunized DR4
mice into naive syngeneic recipients. Two months after
transfer of 10 million RBC-depleted whole splenocytes
from mice without proteinuria, 8 (57%) of 14 recipients developed interstitial lung disease, while only 2
(14%) of 14 mice showed evidence of renal disease
without lung disease (Fisher’s exact P ⫽ 0.046) (Table 1
and Figure 1). Examination of the joints, heart, liver,
spleen, skin, esophagus, salivary glands, and intestines
revealed no gross or histologic differences between the
directly immunized mice and the recipients of the
splenocyte adoptive transfer.
In contrast, 2 months after transfer of 10 million
splenocytes from mice with proteinuria to naive syngeneic recipients, none of the 5 recipient mice developed
lung disease, and 3 (60%) of 5 developed renal disease.
Similar transfer of 10 million splenocytes from naive
syngeneic donors led to no identifiable lung or renal
disease in any of the 5 recipients. We could identify
anti-RNP antibodies and anti-RNP T cells in the recipients of cells from immunized mice but not in the
recipients of cells from naive mice.
Table 1. Protective effect of myeloid DCs on CD4⫹ cell transfer–
induced nephritis*
Transfer experiment
Whole splenocytes
Fresh CD4⫹ cells alone
Cultured CD4⫹ cells
Fresh CD4⫹ cells plus
myeloid DCs
Myeloid DCs alone
No. of
No. (%)
No. (%)
8 (57)
2 (20)
3 (27)
1 (20)
2 (14)
7 (70)†
8 (73)‡
0 (0)§
3 (60)
0 (0)
* The development of lung disease and renal disease was assessed in
naive syngeneic female mice 2 months after adoptive transfer of 10 ⫻
106 red blood cell–depleted whole splenocytes, 2 ⫻ 106 purified splenic
CD4⫹ cells that were either immediately transferred after purification
(fresh) or cultured for 2 weeks in vitro with irradiated naive syngeneic
antigen-presenting cells and U1–70-kd small nuclear RNP fusion
protein (70K) (cultured), and/or 0.1 ⫻ 106 myeloid dendritic cells
(DCs). All donor mice were syngeneic animals killed 2 months after
anti-70K immunization, and were confirmed to have anti-70K antibodies by enzyme-linked immunosorbent assay and to have consistently normal results on urinalyses (i.e., no proteinuria).
† P ⫽ 0.01 versus renal disease after whole splenocyte transfer, by
Fisher’s exact test.
‡ P ⫽ 0.005 versus renal disease after whole splenocyte transfer, by
Fisher’s exact test.
§ P ⫽ 0.026 versus renal disease after fresh CD4⫹ cell transfer alone,
by Fisher’s exact test.
Renal disease after adoptive transfer of RNPⴙ
CD4ⴙ splenocytes from donors without proteinuria.
After harvesting cells from the spleens of immunized
RNP⫹ DR4 mice without proteinuria, we separated the
CD4⫹ cells from the spleens by magnetic bead positive
selection. We then adoptively transferred 2 million
CD4⫹ cells, approximating the number of CD4⫹ cells
recoverable from a single immunized donor mouse
Figure 1. Development of lung disease after adoptive transfer of
splenocytes from immunized DR4 mice into naive syngeneic recipients. Representative hematoxylin and eosin–stained sections of the
lung and kidney from a naive syngeneic recipient are shown. Transfer
of whole splenocytes from a U1–70-kd small nuclear RNP fusion
protein plus U1 RNA adjuvant–immunized HLA–DR4⫹ donor into
the recipient resulted in a mixed connective tissue disease–like lung
disease (A), but no evidence of renal disease (B). Results from
urinalysis (not shown) were also normal. (Original magnification ⫻ 10
in A; ⫻ 40 in B.)
Figure 2. Induction of nephritis, rather than lung disease, after adoptive transfer of CD4⫹ splenocytes from non-nephritic donors. Representative hematoxylin and eosin–stained sections of the lung and
kidney from naive syngeneic recipients are shown. CD4⫹ cells were
directly isolated from a U1–70-kd small nuclear RNP fusion protein
(70K) plus U1 RNA adjuvant–immunized naive syngeneic donor and
transferred into the recipient (A and B), or CD4⫹ cells were harvested
from a 70K-immunized syngeneic donor and grown in vitro for 2 weeks
with 70K-loaded, irradiated antigen-presenting cells from a naive
syngeneic donor before being transferred into the recipient (C and D).
In both cases, minimal interstitial pulmonary infiltrates were evident,
but renal lesions consistent with nephritis were present. Results from
urinalysis (not shown) also revealed active urinary sediment in both
cases. (Original magnification ⫻ 10 in A and C; ⫻ 40 in B and D.)
spleen, to naive syngeneic recipients. In contrast to our
findings in recipients of whole splenocytes, renal disease
was common in recipients of CD4⫹ cells from donors
without proteinuria, since it was identified in 7 (70%) of
10 recipient mice (P ⫽ 0.01 versus whole splenocyte
transfer recipients with renal disease, by Fisher’s exact
test) (Table 1 and Figure 2). Lung disease was less
common, being present in only 2 (20%) of 10 recipients
of CD4⫹ cells from immunized mice. Transfer of 2
million CD4⫹ cells from naive mice to syngeneic donors
under the same protocol led to no lung or kidney disease
in any of the 5 recipients.
To ensure that the nephritis induction was mediated by anti-RNP–specific T cells, we isolated CD4⫹
splenocytes in the same manner as described above and
cultured the CD4⫹ cells with 70K antigen and irradiated
syngeneic APCs from naive donors for 2 weeks, followed
by separation of the T cells using Histopaque densitycentrifugation of the nonadherent cells. The T cells thus
isolated from the recipients remained homologous to
those isolated directly from immunized donors, with
regard to RNP epitope responses and TCR V␤-region
sequence (results not shown).
After adoptive transfer of 2 million of these T
cells from short-term culture into recipient mice, we
found that the same manifestations of nephritis without
Figure 3. Persistence of adoptively transferred myeloid dendritic cells (DCs) at 48 hours and 7 days after transfer, with variable tissue specificity
to the lungs. Myeloid DCs were purified from the spleens of immunized mice, labeled with 5,6-carboxyfluorescein succinimidyl ester (CFSE), and
transferred into 10-week-old naive syngeneic mice by tail-vein injection. At 48 hours and 1 week after cell transfer, single-cell suspensions of the
spleens and lungs were prepared, treated with OptiPrep centrifugation to enhance for identification of DCs, stained with fluorochrome-labeled
anti-CD11c and allophyocyanin, and assessed by flow cytometry. Histograms show CFSE fluorescence in relation to CD11c staining. Identical gating
conditions were used for all experiments. A, Transfer of myeloid DCs from immunized donors without proteinuria resulted in ⬎30% of the gated
cells from the lung (versus 1% from the spleen) showing CD11c⫹/CFSE⫹ staining consistent with that of donor-derived myeloid DCs, at both 48
hours and 1 week after transfer, with approximately equal intensity of CFSE staining at each time point, suggesting that there was minimal
proliferation or other dilution of these CD11c⫹ cells between 48 hours and 7 days. B, Transfer of myeloid DCs from immunized nephritic donors
resulted in fewer than 4% CD11c⫹/CFSE⫹ cells appearing in the lungs at 48 hours or at 1 week after transfer, while the migration of labeled myeloid
DCs to the spleen was similar to that shown in A.
lung disease had developed as was observed with direct
CD4⫹ cell transfers (Table 1 and Figure 2, showing
results representative of 1 of 2 separate experiments).
The results revealed that CD4⫹ cells from 70K⫹ donors
without renal disease expanded significantly in vitro,
after a total of 11 recipients (6 from one experiment and
5 from the other) had received 2 million T cells each.
Lung disease was observed in 3 (27%) of 11 recipients,
while renal disease was seen in 8 (73%) of 11 recipients
(P ⫽ 0.005 versus whole splenocyte transfer recipients
with renal disease, by Fisher’s exact test).
The renal lesions seen after CD4⫹ cell transfers
were typified by histologic findings of glomerular proliferation, mesangial hypertrophy, and hematoxylin body
formation, which are characteristic of lupus nephritis,
although immune complex deposition and complement
depletion were generally not observed (results not
shown). Among both the recipients of fresh CD4⫹ cells
and the recipients of short-term cultured cells, the small
group of mice with lung disease was equally divided
between those without renal disease (1 of 10 fresh
CD4⫹ cell recipients and 1 of 11 cultured CD4⫹ cell
recipients) and those that also had renal disease (1 of 10
fresh CD4⫹ cell recipients and 2 of 11 cultured CD4⫹
cell recipients). Recipients of CD4⫹ cells from immunized mice showed anti-RNP T cell reactivity, as previously described (8).
These results suggest that the effects of antiRNP–reactive T cells are sufficient to transfer anti-RNP
autoimmunity from immunized donors to otherwise
naive recipients. Moreover, the results demonstrate that
in the absence of other signals, RNP-reactive T cells can
direct the induction of glomerulonephritis.
Effects of myeloid DC adoptive transfers from
RNPⴙ donors. The divergence in tissue targeting between recipients of whole splenocyte transfers (frequent
lung disease, less renal disease) and CD4⫹ cell transfers
(frequent renal disease, less lung disease) from donor
mice without proteinuria suggests that non-CD4⫹
splenocytes participate in the development of the lungtargeting pattern observed with whole splenocyte transfers. Based on our previous observations that Toll-like
receptor 3 (TLR-3)–null mice were more susceptible to
renal disease and less susceptible to lung disease, as
compared with TLR-3–intact mice, after direct immunization with 70K plus U1 RNA (4,13), we considered
whether, in a similar manner, TLR-3–expressing splenocytes could protect against nephritis induction. We
therefore investigated myeloid DCs, a major known
TLR-3–expressing immune cell type in the spleen (15).
We separated myeloid DCs from RBC-depleted
spleens by a 3-step process of OptiPrep density-gradient
centrifugation, negative selection for plasmacytoid DCs
with anti-murine PDCA-1 magnetic beads, and then
positive selection for CD11c surface expression with
magnetic beads. We found that this yielded ⬃200,000
cells per spleen from the immunized mice, with a population of myeloid DCs that was ⬎95% pure, as determined
by flow cytometry using the CD11c and anti-murine
PDCA-1 selection markers (results not shown).
To establish that myeloid DC adoptive transfers
from immunized mice would lead to engraftment in
syngeneic naive recipients, we adoptively transferred
naive mice with 100,000 myeloid DCs from immunized
mice without proteinuria; the cells were labeled with
CFSE prior to infusion. Recipient mice were then killed
Figure 4. Effect of myeloid dendritic cell (DC) adoptive transfers.
Syngeneic naive recipient mice received 100,000 myeloid DCs from
RNP-immunized mice without renal disease (A and B) or the same
cells along with 2 million CD4⫹ cells from immunized mice with renal
disease (C and D). Representative hematoxylin and eosin–stained
sections of the lung (A and C) and kidney (B and D) of 2 different mice
are shown. Adoptive transfer of myeloid DCs alone was associated
with development of pulmonary infiltrates but not renal disease,
whereas adoptive transfer of myeloid DCs plus CD4⫹ cells typically
resulted in neither lung disease nor renal disease. (Original magnification ⫻ 10 in A and C; ⫻ 40 in B and D.)
either 48 hours or 7 days after cell transfer. In analyses
of cultures at 48 hours and 7 days after cell transfer,
substantial and stable numbers of CFSE-expressing
CD11c⫹ cells could be detected in the lungs (but not the
spleens) of recipient mice (Figure 3A). However, the
accumulation of lung myeloid DCs was insufficient to
manifest as histologically significant lung inflammation
at either the 48-hour or 7-day time points (results not
shown). Using CFSE-labeled myeloid DCs from identically immunized donors with proteinuria (and no lung
disease), we observed dramatically less trafficking of
donor myeloid DCs to the lungs of naive recipient mice
but comparable trafficking to the spleen (Figure 3B).
Among the mice receiving 100,000 myeloid DCs
from RNP⫹ mice with lung disease that were followed
up for 2 months, 3 (60%) of 5 developed lung disease,
whereas none of the 5 developed renal disease (Table 1
and Figure 4). These results were similar to the frequency of end-organ manifestations in recipients of
whole splenocytes.
To assess the ability of myeloid DCs to inhibit
CD4⫹ cell–induced nephritis, we performed cotransfers. When the same 100,000 myeloid DCs from RNP⫹
mice were transferred along with 2 million CD4⫹ cells
from RNP⫹ mice without renal disease, a dramatic drop
Figure 5. Induction of nephritis after cotransfer of splenocytes from nephritic and non-nephritic immunized mice. Syngeneic HLA–DR4–transgenic
B6 mice were immunized with U1–70-kd small nuclear RNP fusion protein plus U1 RNA adjuvant. CD4⫹ and CD4⫺ splenocytes from a typical
mouse with mixed connective tissue disease and lung disease but no renal lesions (A and B) were mixed with CD4⫹ and CD4⫺ splenocytes from
a mouse with active urinary sediment and histologic manifestations of nephritis but no lung disease (C and D). Representative sections from an
adoptive transfer recipient of mixed cells are shown, demonstrating nephritis in the absence of lung lesions (E and F), which was typical of the
findings in recipient mice in this experiment. (Original magnification ⫻ 10 in A, C, and E; ⫻ 40 in B, D, and F.)
in the incidence of nephritis (none of 5 mice) occurred,
as compared with that in mice receiving CD4⫹ cells
alone (7 [70%] of 10; P ⫽ 0.026, by Fisher’s exact test)
(Table 1 and Figure 4). In addition, among mice cotransferred with CD4⫹ cells and myeloid DCs, lung disease
was observed in only 1 (20%) of 5 recipients. These
results support the hypothesis that myeloid DCs from
immunized mice can mediate a nephritis-protective effect in anti-RNP autoimmunity.
In contrast, when we cotransferred CD4⫹ cells
from immunized mice without renal disease along with
myeloid DCs from nonimmunized syngeneic mice, we
observed that nephritis developed in all 5 (100%) of the
recipients, and lung disease developed in only 1 (20%) of
the 5 recipients. The protective effect of myeloid DCs on
CD4⫹ cell–mediated kidney disease was thus not
present with myeloid DCs from nonimmunized mice,
which were subjected to the same separation protocol.
Adoptive transfer from nephritic donors. Directly immunized RNP⫹ DR4 mice that had active
urinary sediment were also studied. The histologic lesions that were identified in these mice, similar to those
observed in the T cell recipient mice described above,
consisted of glomerular proliferation, mesangial hypertrophy, and hematoxylin body formation, without prominent immune complex deposition or complement depletion (results not shown).
For adoptive transfer studies, mice with renal
disease and no lung disease (determined on the basis of
active urinary sediment and subsequent confirmatory
histologic findings) were killed 2 months after immunization, and RBC-depleted splenocytes were purified as
described above. Mixing studies were then performed, in
which naive syngeneic DR4 recipient mice each received
5 million splenocytes from an RNP⫹ donor with lung
disease and no kidney disease, and each also received
5 million splenocytes from an RNP⫹ donor with renal
disease but no lung disease. When these recipients were
killed 2 months after adoptive transfer, renal disease was
found to be present in 4 (80%) of the 5 mice, and lung
disease was found in none of the 5 mice (Figure 5).
Using DC subsets derived from immunized donors with active urinary sediment, 4 (80%) of 5 recipients of plasmacytoid DCs developed renal disease, and
none of these 5 recipients developed lung disease. In
contrast, none of the 4 recipients of myeloid DCs from
nephritic donors developed renal disease. Notably, lung
disease also was not evident in any of the 4 recipients of
myeloid DCs from immunized donors with proteinuria
and no lung disease.
We were able to detect, by ELISA and/or immunoblotting, anti-RNP antibody responses in recipient mice
after transfer of immunized myeloid DCs alone from
donor mice without proteinuria or after cotransfer of
myeloid DCs along with CD4⫹ cells. Similarly, we
detected anti-RNP antibodies after transfer of plasma-
cytoid DCs alone from nephritic mice (results not
shown). Anti-RNP antibodies were not detected, however, in recipients of myeloid DCs from immunized
nephritic donors.
These studies demonstrate that whole splenocytes, CD4⫹ T cells, or purified DC subsets from
RNP-immunized mice can be sufficient to convey antiRNP autoimmunity to naive syngeneic mice. Furthermore, anti-RNP–reactive T cells can lead to either
lupus-like nephritis or MCTD-like lung disease, depending on the cell types that are transferred or cotransferred. Effects mediated by TLR-3–expressing myeloid
DCs on tissue targeting of anti-RNP responses appear
to potentially account for the previously reported divergence in pulmonary and renal manifestations in
directly immunized mice with anti-RNP immunity (4).
Consistent with the reported roles of plasmacytoid DCs
in lupus pathogenesis (16), adoptive transfer of a relatively small number of purified plasmacytoid DCs from
immunized mice was also able to induce the development
of glomerulonephritis in recipients.
The lung disease observed in our mice appears to
be indistinguishable from cases of lung disease in human
MCTD, as determined by conventional hematoxylin and
eosin staining. We did not observe substantial fibrosis
in the mouse lungs, in contrast to the findings in the
majority of cases of interstitial lung disease in patients
with MCTD (17). Further studies using immunohistochemistry to characterize the murine and human infiltrates are warranted.
The renal lesions in the mice in our study differed
from those typically found in lupus nephritis, in that
immunoglobulin or complement component deposits at
the glomerular basement membrane were rare. Nevertheless, diffuse or focal proliferative patterns, mesangial
involvement, and frequent development of hematoxylin
bodies were evident in our mice, as in lupus nephritis.
Reports have linked TLR-3 signals to exacerbated lung inflammation and, conversely, TLR-7 signals
to reduced lung inflammation, in models of asthma, viral
infection, and direct TLR agonist challenge (18–23). We
hypothesize that in our system, lung-predominant disease similarly develops when TLR-3–activated myeloid
DCs direct anti-RNP T cells to pulmonary, rather than
renal, targets. The mechanism of action of myeloid DCs
in mediating lung-specific tissue targeting has not yet
been defined, and further study will be needed to
establish the role, if any, of TLR-3 in this process.
Conversely, we hypothesize that renal disease
develops when anti-RNP autoimmunity is augmented at
an early stage by a TLR-7 signal, rather than being
modulated by a strong TLR-3 signal. The inability of
70K-immunized splenocytes from non-nephritic mice to
inhibit established nephritis in cotransfer experiments
may reflect the involvement of immune mediators downstream of the point where myeloid DCs are able to
inhibit nephritis induction, or may reflect the elaboration of factors that inhibit (putatively) TLR-3–induced
myeloid DC activity. The observation that anti-RNP
CD4⫹ T cell–induced nephritis was able to be inhibited
by myeloid DCs in our studies suggests that 1) development of anti-RNP immunity can occur prior to a commitment of an autoimmune process to renal injury, and
2) cells other than CD4⫹ T cells emerge after the
induction of anti-RNP immunity, which can drive the
renal targeting in a manner that is resistant to reversal by
activated myeloid DCs. Given the associations identified
between lupus nephritis and TLR-7 (24), TLR-7–
expressing cells such as plasmacytoid DCs or B cells
appear to be logical candidates for playing roles in the
development of renal disease downstream of anti-RNP
T cells, and myeloid DCs cannot reverse this process.
Since MCTD was first proposed clinically as a
diagnostic entity, debate has occurred regarding whether
MCTD is truly distinct from other established rheumatic
diseases, including lupus (25–27). Consistent with this
experience, the results of our study indicate that subtle
shadings of difference exist in a mouse model of antiRNP autoimmunity between MCTD-like (lung disease)
and lupus-like (renal disease) manifestations of tissue
injury. In mice, the same T cells appear to be relevant to
either lung disease or renal disease induction, with the
tissue targeting determined by accessory cell types,
including myeloid DCs and plasmacytoid DCs. Our
finding that in adoptive transfer mixing studies, cells
from mice with established renal disease are dominant
over cells from mice with established lung disease may
inform the clinical observation that MCTD can evolve
into systemic lupus erythematosus, but that systemic
lupus erythematosus seldom evolves into MCTD.
Although our adoptive transfer studies show that
either T cells or DCs can spark persistent autoimmunity in a naive host, there is no guarantee that either of
these cell types participate in the early stages of actual
spontaneously developing clinical disease. Studies in
humans are needed to support the relevance of the
observations made in mice.
This study demonstrates, for the first time, that
elements of the same immune responses to a rheumatic
disease autoantigen can lead to divergent tissue targeting based on differences in innate immune effectors.
Improved understanding of the cell and molecular biology underlying this process may allow for the development of new diagnostic and therapeutic approaches to
systemic autoimmune diseases in this spectrum.
Dr. Greidinger had full access to all of the data in the study
and takes responsibility for the integrity of the data and the accuracy
of the data analysis.
Study design. Greidinger, Hoffman.
Acquisition of data. Greidinger, Zang, Fernandez, Berho, Nassiri,
Analysis and interpretation of data. Greidinger, Zang, Fernandez,
Berho, Nassiri, Martinez, Hoffman.
Manuscript preparation. Greidinger, Zang, Fernandez, Martinez,
Statistical analysis. Greidinger.
1. Sharp GC, Irvin WS, May CM, Holman HR, McDuffie FC, Hess
EV, et al. Association of antibodies to ribonucleoprotein and Sm
antigens with mixed connective-tissue disease, systematic lupus
erythematosus and other rheumatic diseases. N Engl J Med
2. Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis
GJ, James JA, et al. Development of autoantibodies before the
clinical onset of systemic lupus erythematosus. N Engl J Med
3. Greidinger EL, Hoffman RW. The appearance of U1 RNP
antibody specificities in sequential autoimmune human antisera
follows a characteristic order that implicates the U1–70 kd and
B⬘/B proteins as predominant U1 RNP immunogens. Arthritis
Rheum 2001;44:368–75.
4. Greidinger EL, Zang YJ, Jaimes K, Hogenmiller S, Nassiri M,
Bejarano P, et al. A murine model of mixed connective tissue
disease induced with U1 small nuclear RNP autoantigen. Arthritis
Rheum 2006;54:661–9.
5. Keith MP, Moratz C, Egan R, Zacharia A, Greidinger EL,
Hoffman RW, et al. Anti-ribonucleoprotein antibodies mediate
enhanced lung injury following mesenteric ischemia/reperfusion in
Rag-1⫺/⫺ mice. Autoimmunity 2007;40:208–16.
6. Fatenejad S, Mamula MJ, Craft J. Role of intermolecular/intrastructural B- and T-cell determinants in the diversification of
autoantibodies to ribonucleoprotein particles. Proc Natl Acad Sci
U S A 1993;90:12010–4.
7. Wang D, Hill JA, Jevnikar AM, Cairns E, Bell DA. Induction of
transient arthritis by the adoptive transfer of a collagen II specific
Th1 clone to HLA-DR4 (B1*0401) transgenic mice. J Autoimmun
8. Greidinger EL, Zang YJ, Jaimes K, Martinez L, Nassiri M,
Hoffman RW. CD4⫹ T cells target epitopes residing within the
RNA binding domain of the U1-70kD small nuclear ribonucleoprotein autoantigen and have restricted TCR diversity in an
HLA-DR4 transgenic murine model of mixed connective tissue
disease. J Immunol 2008;180:8444–54.
9. Hoffman RW, Takeda Y, Sharp GC, Lee DR, Hill DL, Kaneoka
H, et al. Human T cell clones reactive against U-small nuclear
ribonucleoprotein autoantigens from connective tissue disease
patients and healthy individuals. J Immunol 1993;151:6460–9.
10. Holyst MM, Hill DL, Hoch SO, Hoffman RW. Analysis of human
T cell and B cell responses against U small nuclear ribonucleoprotein 70-kd, B and D polypeptides among patients with systemic
lupus erythematosus and mixed connective tissue disease. Arthritis
Rheum 1997;40:1493–503.
Greidinger EL, Gazitt T, Jaimes KF, Hoffman RW. Human T cell
clones specific for heterogeneous nuclear ribonucleoprotein A2
autoantigen from connective tissue disease patients assist in autoantibody production. Arthritis Rheum 2004:50:2216–22.
Greidinger EL, Foecking MF, Schafermeyer KR, Bailey CW,
Primm SL, Lee DR, et al. T cell immunity in connective tissue
disease patients targets the RNA binding domain of the U1-70kDa
small nuclear ribonucleoprotein. J Immunol 2002;169:3429–37.
Greidinger EL, Zang YJ, Martinez L, Jaimes K, Nassiri M,
Bejarano P, et al. Differential tissue targeting of autoimmunity
manifestations by autoantigen-associated Y RNAs. Arthritis Rheum
Greidinger EL, Foecking MF, Magee J, Wilson L, Ranatunga S,
Ortmann RA, et al. A major B cell epitope present on the
apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein autoantigen. J Immunol 2004;172:709–16.
Edwards AD, Diebold SS, Slack EM, Tomizawa H, Hemmi H,
Kaisho T, et al. Toll-like receptor expression in murine DC
subsets: lack of TLR7 expression by CD8 alpha⫹ DC correlates
with unresponsiveness to imidazoquinolines. Eur J Immunol 2003;
Banchereau J, Pascual V. Type I interferon in systemic lupus
erythematosus and other autoimmune diseases. Immunity 2006;
Bodolay E, Szekanecz Z, Devenyi K, Galuska L, Csipo I, Vegh J,
et al. Evaluation of interstitial lung disease in mixed connective
tissue disease (MCTD). Rheumatology (Oxford) 2005;44:656–61.
Le Goffic R, Pothlichet J, Vitour D, Fujita T, Meurs E, Chignard
M, et al. Cutting edge: influenza A virus activates TLR3-dependent inflammatory and RIG-I-dependent antiviral responses in
human lung epithelial cells. J Immunol 2007;178:3368–72.
Rudd BD, Smit JJ, Flavell RA, Alexopoulou L, Schaller MA,
Gruber A, et al. Deletion of TLR3 alters the pulmonary immune
environment and mucus production during respiratory syncytial
virus infection. J Immunol 2006;176:1937–42.
Camateros P, Tamaoka M, Hassan M, Marino R, Moisan J,
Marion D, et al. Chronic asthma-induced airway remodeling is
prevented by Toll-like receptor-7/8 ligand S28463. Am J Respir
Crit Care Med 2007;175:1241–9.
Demedts IK, Bracke KR, Maes T, Joos GF, Brusselle GG.
Different roles for human lung dendritic cell subsets in pulmonary
immune defense mechanisms. Am J Respir Cell Mol Biol 2006;
Kato A, Favoreto S Jr, Avila PC, Schleimer RP. TLR3- and Th2
cytokine-dependent production of thymic stromal lymphopoietin
in human airway epithelial cells. J Immunol 2007;179:1080–7.
Jeon SG, Oh SY, Park HK, Kim YS, Shim EJ, Lee HS, et al. TH2
and TH1 lung inflammation induced by airway allergen sensitization with low and high doses of double-stranded RNA. J Allergy
Clin Immunol 2007;120:803–12.
Christensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RA,
Shlomchik MJ. Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory
roles in a murine model of lupus. Immunity 2006;25:417–28.
Smolen JS, Steiner G. Mixed connective tissue disease: to be or not
to be? [review]. Arthritis Rheum 1998;41:768–77.
Sharp GC, Hoffman RW. Clinical, immunologic, and immunogenetic evidence that mixed connective tissue disease is a distinct
entity: comment on the article by Smolen and Steiner [letter].
Arthritis Rheum 1999;42:190–1.
Isenberg D, Black C. Naming names! Comment on the article by
Smolen and Steiner [letter]. Arthritis Rheum 1999;42:191–3.
Без категории
Размер файла
335 Кб
dendriticum, myeloid, mode, murine, rnp, anti, tissue, autoimmunityeffects, targeting, cells
Пожаловаться на содержимое документа