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HLA-B27 Misfolding in Transgenic Rats Is Associated with
Activation of the Unfolded Protein Response
1
Matthew J. Turner,
†
Dawn P. Sowders,* Monica L. DeLay,* Rajashree Mohapatra,* Shuzhen Bai,*
Judith A. Smith,* Jaclyn R. Brandewie,* Joel D. Taurog,
‡
and Robert A. Colbert
2
*
The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast
to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the
endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant char-
acteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of
HLA-B27 were reported in cells from HLA-B27/human

2
-microglobulin transgenic (HLA-B27 transgenic) rats, an animal model
of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response
(UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease.
Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats
do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats
with IFN-
␥
increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone
HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27
misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with
biological effects that are independent of immunological recognition, but nevertheless may play an important role in the patho-
genesis of inflammatory diseases associated with this MHC class I allele. The Journal of Immunology, 2005, 175: 2438 –2448.
T
he MHC class I allele, HLA-B27, is strongly implicated
in susceptibility to the chronic inflammatory disease an-
kylosing spondylitis, and related spondyloarthritides (re-
viewed in Ref. 1). Although genetic susceptibility to ankylosing
spondylitis is complex (1, 2), close to 95% of Caucasians with this
disease carry HLA-B27 compared with 7– 8% of healthy controls,
making this one of the strongest HLA disease associations known.
Despite recognition of the relationship between HLA-B27 and
spondyloarthritis for over 30 years, the role of this MHC class I
molecule in pathogenesis remains undefined.
The development of spondyloarthritis-like disease in HLA-B27/
human

2
-microglobulin (h

2
m)
3
transgenic rats suggests a direct
role for HLA-B27 in pathogenesis (3). Although the primary func-
tion of MHC class I molecules is to present peptides to CD8
⫹
T
cells, classical T cell recognition of HLA-B27 does not appear to
be required for pathogenesis. For example, depletion of CD8
␣
⫹
T cells has no effect on the onset or severity of arthritis or gastro-
intestinal inflammation in HLA-B27 transgenic rats (4). In addi-
tion, HLA-B27 transgenic,

2
m-deficient mice develop arthritis
(5), despite a dramatic reduction in class I-restricted peptide pre-
sentation and CD8
⫹
T cell populations resulting from the absence
of

2
m (6). Although there is some evidence for autoreactive
CD8
⫹
T cells in humans with spondyloarthritis (7, 8), it remains
unclear whether autoreactivity is limited to HLA-B27, and whether
it is necessary for disease.
Although CD8
␣
⫹
T cells are not necessary for development of
the inflammatory phenotype in HLA-B27 transgenic rats, there is
a T cell requirement. For example, HLA-B27 transgenic athymic
(rnu/rnu) rats remain healthy unless reconstituted with T cells,
particularly CD4-enriched populations (9), although it is not
known whether this requires recognition of HLA-B27. Bone mar-
row (BM) transfer experiments with this model indicate that high-
level HLA-B27 expression in the hemopoietic compartment is
both necessary and sufficient for disease to develop (10). Because
high-level expression of HLA-B27 in T cells is not required (9),
these findings suggest that another leukocyte subpopulation(s) ex-
pressing HLA-B27 may be critical. Development of the inflam-
matory disease is not merely due to HLA class I overexpression
because HLA-B7/h

2
m transgenic rats do not develop the spon
-
dyloarthritis-like phenotype (11).
A number of reports indicate that HLA-B27 H chains exhibit ab-
errant features, including a tendency to misfold (reviewed in Ref. 12).
This occurs in the endoplasmic reticulum (ER), and involves ineffi-
cient folding of, and delayed

2
m binding to, newly synthesized H
chains (13, 14). Misfolding can result in the ER-associated degrada-
tion (ERAD) of HLA-B27 (13), and is associated with prolonged
*William S. Rowe Division of Rheumatology, Department of Pediatrics, Cincinnati
Children’s Hospital Medical Center and University of Cincinnati College of Medi-
cine, Cincinnati, OH 45229;
†
Physician Scientist Training Program, University of
Cincinnati College of Medicine, Cincinnati, OH 45267; and
‡
Rheumatic Diseases
Division, Department of Internal Medicine, University of Texas Southwestern Med-
ical Center, Dallas, TX 75390
Received for publication January 12, 2005. Accepted for publication May 30, 2005.
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 National Institutes of Health Grants R01 AR46177,
AR48372, and AR38319. M.J.T. was supported by a Functional Genomics Fellowship
granted through the University of Cincinnati College of Medicine. D.P.S. was sup-
ported by National Institutes of Health Training Grant T32 AR07594, and J.A.S. was
supported by an Arthritis Foundation Postdoctoral Fellowship.
2
Address correspondence and reprint requests to Dr. Robert A. Colbert, William S.
Rowe Division of Rheumatology, ML4010, Cincinnati Children’s Hospital Medical
Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address:
bob.colbert@cchmc.org
3
Abbreviations used in this paper: h

2
m, human

2
-microglobulin; ATF6, activating
transcription factor-6; BM, bone marrow; BMDM, BM-derived macrophage; CHOP,
C/EBP homologous protein; ER, endoplasmic reticulum; ERAD, ER-associated deg-
radation; IRE1, inositol-requiring 1 homologue; PERK, protein kinase, IFN-inducible
double-stranded RNA activated-like ER kinase; TM, tunicamycin; UPR, unfolded
protein response; WT, wild type; XBP-1, X box-binding protein-1; XBP-1s, spliced
XBP-1.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
binding of H chains to the ER chaperone, BiP (Grp78) (14 –16). The
formation of high m.w. disulfide-linked complexes of HLA-B27 in
the ER, including H chain homodimers and oligomers (14 –16), ap-
pears to be a feature of misfolding. Most of these aberrant character-
istics have been observed when HLA-B27 is expressed in rat as well
as human cells (14 –16).
Dimerization of HLA-B27 was first observed when H chains
were refolded in vitro, particularly in the absence of

2
m (17).
Dimers have also been observed on the surface of cells (14, 15,
18 –20). Cell surface dimers do not appear to originate in the ER,
at least in cells with an intact class I assembly pathway (14). In-
stead, they appear to derive from cell surface H chain/

2
m/peptide
complexes that have lost

2
m (19). Consistent with this observa
-
tion, relatively stable

2
m-free HLA-B27 H chains have been
found on the cell surface (21). Thus, cell surface dimer formation
appears to be mechanistically distinct from HLA-B27 misfolding.
Recognition of these aberrant characteristics of HLA-B27 has
led to new hypotheses about its role in disease pathogenesis. One
idea is that cell surface dimers may interact with killer Ig receptors
or leukocyte Ig-like receptors in humans (18, 22), paired Ig-like
receptors in rodents (20), or possibly CD4
⫹
T cells (23), resulting
in immunomodulation (24). Distinct from mechanisms invoking
immune recognition, HLA-B27 misfolding could lead to cellular
dysfunction via the activation of ER stress signaling pathways
(25). Protein misfolding in the ER can disrupt homeostasis (ER
stress) and activate signal transduction pathways that orchestrate
the unfolded protein response (UPR) (reviewed in Ref. 26). One
important sensor of ER stress is the chaperone BiP (27). In the
absence of protein misfolding, BiP binds to the proximal UPR
effector proteins inositol-requiring 1 homologue (IRE1), protein
kinase, IFN-inducible double-stranded RNA-activated-like ER
kinase (PERK), and activating transcription factor-6 (ATF6), pre-
venting their activation. Many proteins that misfold bind and se-
quester free BiP, titrating it away from the UPR effectors. As a
result, IRE1 and PERK autoactivate through homodimerization
and trans-autophosphorylation (reviewed in Ref. 28), and ATF6
translocates to the Golgi, where a transcriptionally active subunit
is released by proteolytic cleavage (29, 30). Activation of IRE1,
PERK, and ATF6 leads to translational and transcriptional changes
that initially decrease protein load on the ER, and then enhance
folding and secretory capacity, ERAD, and ultimately, resolution
of ER stress.
In this study, we provide the first evidence that HLA-B27 mis-
folding is associated with UPR activation in BM-derived cells
from the transgenic rat model of human disease. Furthermore, we
show that proinflammatory cytokines such as IFN-
␥
, which up-
regulate HLA-B27, may play a key role in this process. IFN-
␥
and
UPR target gene overexpression is found in the inflamed colon of
HLA-B27 transgenic rats, indicating potential involvement of the
UPR in the pathogenesis of HLA-B27-associated disease.
Materials and Methods
Rats
Inbred HLA-B27/h

2
m transgenic rats on the F344 background (F344.33-3
line) (11), and wild-type (WT) control F344 rats were purchased from
Taconic Farms and maintained in the conventional animal facility at the
Cincinnati Children’s Hospital Research Foundation. Inbred HLA-B27/
h

2
m (L.33-3 line), HLA-B7/h

2
m (120-4 line), and WT rats on the Lewis
background (11) were bred in the specific pathogen-free animal facility at
the University of Texas Southwestern Medical Center and then shipped to
Cincinnati Children’s Hospital Research Foundation. The 33-3 locus con-
tains 55 copies of the HLA-B27 gene and 66 copies of the h

2
m gene,
while the 120-4 locus contains 26 copies of HLA-B7 and 5 copies of h

2
m.
All HLA-B27 transgenic rats were hemizygous for the transgene locus,
while HLA-B7 transgenics were homozygous (i.e., 52 copies of H chain
and 12 copies of h

2
m). The HLA-B (B27 and B7) and h

2
m transgenes
consist of 6.5 and 13.6 kb of human genomic DNA sequence, respectively,
and all three contain promoter sequences that are responsive to induction
by IFNs (3, 31). The HLA-B27 transgene encodes the B*2705 subtype, and
the HLA-B7 transgene encodes the B*0702 subtype. Experiments were
performed based on protocols reviewed and approved by the Cincinnati
Children’s Hospital Research Foundation Institutional Animal Care and
Use Committee.
Chemical reagents and cell lines
BM-derived macrophage (BMDM) culture medium consisted of DMEM
supplemented with 10% FCS, 2 mM L-glutamine, 10 U/ml penicillin/strep-
tomycin, 50
g/ml gentamicin sulfate (Sigma-Aldrich) (D-10), and L929
supernatant (30% day 0–5 and 2.5% day 5– 6). L929 supernatant (contain-
ing M-CSF) was prepared as described (32). Splenocytes were cultured in
RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and 10 U/ml
penicillin/streptomycin (R-10). Tunicamycin (TM) and Salmonella enteri-
dis LPS were purchased from Sigma-Aldrich and diluted in DMSO and
RPMI 1640, respectively. Rat rIFN-
␥
was purchased from R&D Systems
and diluted in PBS. L929 cells (CCL-1) were purchased from American
Type Culture Collection.
Antibodies
W6/32 recognizes a conformation-dependent epitope on HLA class I mol-
ecules that is largely dependent on

2
m and peptide binding (33), and
HC10 recognizes free (unfolded or misfolded) HLA B and C allele H
chains without

2
m (34). Both W6/32 and HC10 are mouse mAbs. The
anti-GRP78 Ab used to detect BiP in immunoblots was purchased from
StressGen Biotechnologies.
Pulse-chase analysis and immunoprecipitation
Freshly isolated rat splenocytes were cultured for 24 h with LPS (20
g/
ml), washed, and incubated in fresh Met/Cys-deficient R-10 medium for
1 h at 37°C. Cells (2 ⫻ 10
7
per time point) were labeled for 1 h with
[
35
S]Met/Cys (Valeant Pharmaceuticals), and chased in 10-fold excess of
medium containing 2 mM nonradioactive Met/Cys for 0, 3, 6, and 19 h, as
described previously (14). At the end of each chase period, cells were
placed in ice-cold PBS containing 20 mM N-ethylmaleimide for 10 min
before lysis, to prevent postlysis disulfide bond formation and rearrange-
ment. Nuclei were removed, and then lysates were precleared with For-
malin-fixed Staphylococcus aureus (Sigma-Aldrich). Folded and unfolded
class I H chains were sequentially immunoprecipitated with W6/32 and
HC10 (1 h at 4°C), respectively (15
g per 2 ⫻ 10
7
cells in 500
l), with
immune complexes removed by incubation with protein A-Sepharose (100
l of 50 mg/ml suspension per 500
l of lysate) (Sigma-Aldrich) for 1 h
at 4°C following each step. Protein A-Sepharose pellets were washed ex-
tensively and stored at ⫺20°C until electrophoresis.
Electrophoresis, phosphorimaging, and immunoblotting
Immunoprecipitated proteins separated by SDS-PAGE were visualized ei-
ther by phosphor imaging ([
35
S]Met/Cys labeled) of dried gels or immu
-
noblotting after transfer to polyvinylidene difluoride membranes (Westran;
Schleicher & Schuell Microscience), as described (14). For immunoblot-
ting, anti-GRP78 (StressGen Biotechnologies) and HC10 (anti-

2
m-free
class I H chain) were used as primary Abs reacting with BiP and class I H
chain, respectively. After washing, blots were incubated with the appro-
priate alkaline phosphatase-conjugated secondary Ab (Southern Biotech-
nology Associates). Proteins were visualized with 5-bromo-4-chloro-3-in-
dolyl phosphate/NBT substrate (Sigma-Aldrich).
BMDM culture and RNA preparation
Rat BM cells were cultured in 30% L929-conditioned D-10 with gentami-
cin (50
g/ml) in 75-cm
2
flasks for 5 days. Nonadherent progenitor cells
were then collected by washing and pipetting and replated in 2.5% L929-
conditioned D-10 for 24 h. Following washing with PBS to remove non-
adherent cells, TRIzol (Invitrogen Life Technologies) was added directly to
the plate-bound adherent cells (BMDM), followed by RNA isolation, ac-
cording to the manufacturer’s instructions. In some experiments, macro-
phages were cultured for additional lengths of time with IFN-
␥
(24 h) or
TM (7 h) before RNA isolation. Culture medium contained ⬍0.08 endo-
toxin U/ml based on the QCL-1000 chromogenic Limulus amebocyte ly-
sate test kit (BioWhittaker).
Whole spleen and thymus tissues from rats were placed in RNAlater
(Ambion) at 4°C. Following homogenization, total RNA was isolated using
RNA-Stat 60 (Tel-Test), according to the manufacturer’s instructions.
2439The Journal of Immunology
Quantitative real-time PCR and semiquantitative RT-PCR
Total RNA was reverse transcribed using oligo(dT) primers and the Su-
perscript one-step RT-PCR system (Invitrogen Life Technologies). Real-
time PCR was performed using SYBR Green I and either a LightCycler
(Roche Diagnostics) or an iCycler (Bio-Rad). X box-binding protein-1
(XBP-1) splicing was determined by separating XBP-1 PCR products on
4% agarose gels (Cambrex) and measuring the relative amounts of the long
(unspliced) and short (spliced) products using a phosphor imager (Amer-
sham Biosciences) and ImageQuant software, and is expressed as a per-
centage of the total determined by RT-PCR. The relative expression of
spliced XBP-1 (XBP-1s) was determined using the following calculation:
(relative expression XBP-1s ⫽ (% XBP-1s)(relative expression XBP-1)),
where the relative expression of XBP-1 is determined by real-time PCR.
Primer sequences are available upon request.
Microarray hybridization and analysis
RNA quality was first verified using an Agilent Bioanalyzer 2100. Then
samples were reverse transcribed, converted to biotinylated cRNA, and
hybridized to Affymetrix microarrays, according to standard protocols used
in the Cincinnati Children’s Hospital Research Foundation Affymetrix Ge-
neChip Core (35). Initial experiments were performed using the RGU34A
GeneChip (Fig. 4), which was replaced by the new generation RAE230A
(Fig. 7). After hybridization, microarrays were washed and stained with
streptavidin-PE using an automated fluidics system, and scanned with a
Hewlett-Packard GeneArray Scanner (Hewlett-Packard). Following global
scaling of each microarray to allow interchip comparisons, gene transcript
levels were determined from data image files using Microarray Analysis
Suite (version 5.0) software (Affymetrix). Relative gene transcript levels
are measured by one or more probe sets (depending on the gene), and are
based on average difference values determined from perfect and single
mismatch oligonucleotide probes. The methodology is in accord with the
MIAME (Minimum Information About a Microarray Experiment) guide-
lines (www.mged.org/Workgroups/MIAME/miame.html). Differences be-
tween experimental conditions across all samples (e.g., WT vs HLA-B27
transgenic) were determined using GeneSpring 6.1 software (Silicon Ge-
netics). To produce Figs. 4 and 7, expression values for each gene were
normalized to the average of three controls (i.e., WT samples), and are
expressed as ratios representing fold increase (⬎1) or decrease (⬍1). Sup-
plemental information describing microarray sample preparation is pro-
vided in the online version of this article.
Statistical analysis
Statistical analysis was performed using Student’s t test. A value of p ⬍
0.05 was considered significant.
Results
HLA-B27 misfolding in rat splenocytes
To look for biochemical evidence of HLA-B27 misfolding in cells
from HLA-B27 transgenic rats, we pulse labeled splenocytes for
1 h, and then immunoprecipitated HLA-B27 at various times dur-
ing a 19-h chase using HC10 and W6/32. HC10 recognizes H
chains that are not completely folded and either have not yet ac-
quired or have lost

2
m (34), while W6/32 recognizes folded H
chains typically associated with

2
m and peptide (33). Immuno
-
precipitates were separated by SDS-PAGE under nonreducing and
reducing conditions, revealing newly synthesized HLA-B27 H
chains (0 h) in high m.w. disulfide-linked complexes that are rec-
ognized by HC10, but not W6/32 (Fig. 1A). These complexes are
qualitatively indistinguishable from what we have previously de-
scribed in human cells and are referred to as ER dimers (14). ER
dimers decay considerably by6hofchase. In contrast, W6/32-
reactive (folded) dimers are first detectable at 3–6 h of chase and
are still present at 19 h. Both HC10- and W6/32-reactive dimers
are eliminated by sample reduction before electrophoresis, indi-
cating their dependence on disulfide bonds. Previously, folded
dimers were not detected with the mAb B9.12.1 after a 4-h
metabolic labeling period, but they were detected by immuno-
blotting with this Ab (16). This is consistent with the slow
appearance of W6/32-reactive dimers seen in this study and in
our earlier study (14).
The HC10 Ab immunoprecipitates an additional 78-kDa band
that is not eliminated by sample reduction (Fig. 1A). This is likely
to be the ER chaperone BiP, which immunoprecipitates and co-
purifies with HLA-B27 H chains expressed in human and rat cells
(14 –16) (Fig. 1B). The detection of newly synthesized BiP bound
to HLA-B27 H chains after a brief pulse suggests that this inter-
action is occurring in the ER. The persistence of H chain-BiP
complexes after several hours of chase indicates that the interac-
tion is prolonged. This early BiP binding appears to be specific for
unfolded or misfolded H chains, as no radioactive BiP is detected
in W6/32 immunoprecipitates. The absence of radioactive or im-
munoreactive bands in immunoprecipitates from WT cells con-
firms that formation of disulfide-linked complexes and BiP binding
are the result of HLA-B27/h

2
m expression.
Pulse-chase experiments were performed using splenocytes, as
they are abundant and easily isolated. We have also performed 1-h
labeling and immunoblotting experiments with BM macrophages
that reveal high m.w. disulfide-linked complexes and BiP copre-
cipitation using the HC10 Ab, similar to what is shown in Fig. 1
(our unpublished observations). Thus, HLA-B27 expressed in
macrophages displays characteristics similar to those observed in
splenocytes.
FIGURE 1. HLA-B27 exhibits biochemical characteristics of misfold-
ing in rat cells. A, Freshly isolated splenocytes from WT, and HLA-B27
transgenic rats were stimulated with LPS (20
g/ml) for 24 h, pulse labeled
for 1 h with [
35
S]Met/Cys, and chased for the times indicated. HC10 and
W6/32 immunoprecipitates were separated by SDS-PAGE under nonre-
ducing (top, NR) and reducing (bottom, R) conditions, and radiolabeled
proteins were visualized by phosphor imaging. Material from HLA-B27
transgenic F344.33-3 (B27) rat splenocytes is shown on the right, with
immunoprecipitates from WT F344 rat cells on the left. Monomeric HLA-
B27 H chains (HC) are indicated by solid arrowheads. High m.w. disulfide-
linked H chain complexes are indicated by brackets (ⴱ), and BiP is desig-
nated by arrows. Only 0-h immunoprecipitates are shown for WT F344
cells because only background radioactivity was detected. B, HC10 immu-
noprecipitates from LPS-stimulated WT and HLA-B27 transgenic rat
splenocytes were blotted for BiP (top) and HLA class I H chain (HC)
(bottom).
2440 HLA-B27 MISFOLDING AND UPR
UPR activation in HLA-B27 transgenic rat macrophages
To determine whether the UPR is activated in cells from HLA-B27
transgenic rats, we quantified expression of the canonical UPR
target genes, BiP and C/EBP homologous protein (CHOP), in
whole spleen and thymus, and BMDM using real-time RT-PCR
(Fig. 2). Differential expression of BiP and CHOP is not seen in
whole spleen or thymus, nor is it observed in BM macrophages
from 4-wk-old premorbid animals. In contrast, transcripts for both
of these genes are elevated in BM macrophages from 10-wk-old
HLA-B27 transgenic rats with inflammatory disease. For compar-
ison, the effect of TM, which inhibits N-linked glycosylation and
causes global misfolding of newly synthesized glycoproteins, is
shown (Fig. 2).
As an independent assessment of UPR activation, we quantified
XBP-1 mRNA splicing. XBP-1 is a transcription factor that plays
an important role in the induction of certain UPR target genes (36,
37). The XBP-1 mRNA is constitutively produced in an unspliced
form. However, when IRE1 is activated, it functions as an endo-
nuclease that removes 26 bp to produce a spliced form of XBP-1
(XBP-1s). This results in a frameshift and the synthesis of the
active XBP-1 transcription factor (36, 38, 39). In addition to splic-
ing, XBP-1 mRNA levels (unspliced form) are induced during the
UPR by the active forms of ATF6 and XBP-1 proteins (36). In BM
macrophages derived from 10-wk-old HLA-B27 transgenic rats,
total XBP-1 mRNA transcripts are increased ⬃2.6-fold above WT
(Fig. 3A). There is also a small, but consistent, increase in XBP-1
splicing (Fig. 3B). Taking into account the increase in total XBP-1
transcripts, the relative expression of XBP-1s is ⬃5-fold higher in
HLA-B27-expressing BM macrophages (Fig. 3C). Although the
induction of XBP-1 by TM is similar to that associated with HLA-
B27 expression (Fig. 3A), TM has a much more dramatic effect on
XBP-1 splicing (Fig. 3, B and C).
UPR and IFN response signatures in HLA-B27 transgenic rat
macrophages
To gain further insight into gene expression differences in BMDM
from HLA-B27 transgenic rats, we used microarray analysis. Sev-
eral genes known to be up-regulated during the UPR are overex-
pressed in HLA-B27-expressing macrophages (Fig. 4). These are
designated as UPR target genes in Fig. 4 based on the literature
(37, 40, 41), and experiments in which WT BM macrophages were
treated with TM (our unpublished observations). It should be noted
that for purposes of completeness, the list in Fig. 4 includes genes
that are up-regulated in TM-treated macrophages, but are not dif-
ferentially expressed in this experiment. Together with the evi-
dence of BiP, CHOP, and XBP-1 overexpression, and XBP-1
splicing (Figs. 2 and 3), these data reveal the presence of a UPR
signature in BM macrophages expressing HLA-B27, and provide
FIGURE 2. BiP and CHOP are overexpressed in HLA-B27-expressing
transgenic rat macrophages. RNA was isolated from whole thymus, spleen,
and BMDM, prepared as described in Materials and Methods, from F344
WT and F344.33-3 HLA-B27 transgenic (B27) rats. Thymus and spleen
were from 30- and 24-wk-old rats, respectively, while BMDM were pre-
pared from 4- and 10-wk-old animals, as indicated. On the far right,
BMDM from a WT rat (58 wk old) were treated for 7 h with vehicle (⫺)
or 10
g/ml TM. Relative expression of BiP, CHOP, GAPDH, and/or

-actin mRNA was determined by real-time PCR. BiP and CHOP tran-
script levels were normalized to either GAPDH (spleen, thymus, 10-wk
BMDM) or

-actin (4-wk BMDM) expression. In each case, material from
three WT and three HLA-B27 transgenic rats was used, and thus the col-
umns represent the mean of triplicate biological replicates, except for the
TM experiment in which three measurements were made from a single
experiment. Error bars represent SEM, and p ⬍ 0.02 for B27 vs WT mac-
rophage samples derived from 10-wk-old rat BM.
FIGURE 3. XBP-1 expression and splicing in HLA-B27-expressing
transgenic rat macrophages. RNA from the experiment described in Fig. 2
was used to quantitate XBP-1 transcripts and splicing for BMDM from
10-wk-old rats. A, Total XBP-1 expression in WT and HLA-B27 trans-
genic (B27) BMDM (left) and in vehicle (⫺) and TM-treated (7 h, 10
g/ml) WT BMDM (right). The relative expression of XBP-1 and GAPDH
was determined by real-time PCR, and transcript levels for total XBP-1
were normalized to GAPDH expression. B, Percentage of XBP-1s deter-
mined by RT-PCR for WT and B27 BMDM (left) and in vehicle (⫺) and
TM-treated WT BMDM (right). Representative gel images (inset) show
PCR products for unspliced (U) and spliced (S) XBP-1. C, Relative ex-
pression of XBP-1s is determined by multiplying the relative expression of
XBP-1 as shown in (A), by the percentage of XBP-1s in B. WT and ve-
hicle-treated samples are then set to 1, with the appropriate scaling factor
then applied to the B27 and TM samples. Quantitation of XBP-1 tran-
scripts in A and XBP-1 splicing in B for WT and B27 samples repre-
sents the average with SE, for three biological replicates. Quantitation
of TM-induced XBP-1 expression represents five determinations for a
single experiment. No error bars are included for XBP-1 splicing in
TM-treated samples, as this represents a single determination. Value of
p ⬍ 0.02 for B27 vs WT macrophage samples in A and B and for WT
(TM) vs WT (⫺)inA.
2441The Journal of Immunology
evidence that these cells are exhibiting signs of ER stress. Mi-
croarray analysis also revealed increased expression of many
known IFN-responsive genes (Fig. 4), identified from the literature
(42), and confirmed in experiments that will be discussed below
(see Fig. 7).
IFN-
␥
augments HLA-B27 expression and UPR activation
The cooccurrence of UPR and IFN response signatures in HLA-
B27-expressing BM macrophages raised the question of whether
the UPR might directly induce IFN target gene expression, or that
IFN might activate the UPR, either directly, or more likely via
up-regulation of the IFN-responsive HLA-B27 transgene. The first
possibility is excluded, as expression of IFN-responsive genes is
not increased in TM-treated macrophages (our unpublished obser-
vations). To determine whether IFN contributes to UPR target
gene induction, we stimulated BM macrophages from 4-wk-old
premorbid rats with IFN-
␥
. This results in up-regulation of BiP and
CHOP in HLA-B27-expressing macrophages, but not in WT cells
(Fig. 5). IFN-
␥
also increases HLA-B27 transcript levels ⬃3-fold
(Fig. 5). In addition, total XBP-1 mRNA is increased by ⬃50–
70% in IFN-
␥
-treated HLA-B27-expressing macrophages, com-
pared with untreated or IFN-
␥
-treated WT macrophages, respec-
tively (Fig. 6A). XBP-1 splicing is increased more dramatically,
with ⬃10% of XBP-1 transcripts being spliced in IFN-
␥
-treated
HLA-B27-expressing macrophages, compared with 2% or less in
the other samples (Fig. 6B). This corresponds to an 8- to 16-fold
increase for XBP-1s (Fig. 6C). Importantly, IFN-
␥
treatment does
not alter BiP, CHOP, or XBP-1 mRNA expression, or XBP-1
splicing, in WT macrophages.
We next assessed the effect of IFN-
␥
treatment on BM macro-
phages by microarray analysis. UPR target gene expression is sig-
nificantly elevated in IFN-
␥
-treated macrophages expressing
HLA-B27, whereas this is not observed in WT cells (Fig. 7). This
analysis also reveals a significant increase in EDEM (ER degra-
dation-enhancing mannosidase-like protein) transcripts. Increased
expression of this gene, which encodes a key protein involved in
ERAD (43, 44) and is completely dependent on XBP-1s (37), pro-
vides evidence that the elevated XBP-1 splicing shown in Fig. 6
has functional significance. There is also a low-level increase in
expression of some UPR target genes in untreated HLA-B27-ex-
pressing macrophages (e.g., 50% increase in BiP). WT and HLA-
B27-expressing BM macrophages responded similarly to IFN-
␥
based on comparable induction of the IFN target genes (Fig. 7). In
addition to the weak UPR signature in untreated HLA-B27-ex-
pressing BM macrophages, there are also some IFN-responsive
genes that are overexpressed (e.g., CCXL10, IFN regulatory factor
7, Best5). It should be noted that the new, more comprehensive,
Affymetrix rat GeneChip (RAE230A) was used for these experi-
ments (Fig. 7), whereas RGU34A was used previously (Fig. 4).
Consequently, Fig. 7 contains all of the genes shown in Fig. 4
(except RT1.S3) as well as additional UPR and IFN response
genes present on the new chip.
IFN-
␥
-mediated UPR activation is specific for HLA-B27
To determine whether UPR activation is specific for HLA-B27 and
not a result of overexpressing a class I H chain, we examined UPR
target gene expression in HLA-B7 transgenic macrophages (Lewis
background) in response to IFN-
␥
. Because previous experiments
FIGURE 4. HLA-B27-expressing transgenic rat
macrophages exhibit UPR and IFN gene expression
signatures. RNA from BM macrophages derived
from 10-wk-old rats was subjected to microarray
analysis using the Affymetrix Rat U34A (RGU34A)
GeneChip. Following standard normalization of
each array, values for individual genes were normal-
ized to the average of the controls (WT), which is
given a value of 1. The figure shows a heat map of
relative expression values with each column repre-
senting a biological replicate (3-F344 WT and
3-F344.33-3 HLA-B27), and each row representing
a different probe set on the microarray. (Note that in
some cases there is more than one probe set mea-
suring the expression of a single gene.) Genes are
clustered in the heat map based on expression pat-
tern. Relative expression levels are colored such that
blue represents low expression, yellow is set to 1,
and red represents high expression, covering a 10-
fold range (0.3–3.0). The abbreviated name for each
gene is shown (Gene), as well as the GenBank num-
ber and a brief description. The FC column shows
the average fold change based on three comparisons
of HLA-B27 vs WT, with the latter set equal to 1.
The asterisk (ⴱ) indicates differences that are statis-
tically significant (p ⬍ 0.05). The genes are divided
into two groups, in which UPR designates those that
are known to be up-regulated by ER stress, and IFN
designates genes known to be responsive to this cy-
tokine (see Fig. 7).
2442 HLA-B27 MISFOLDING AND UPR
were performed using rats with an F344 background, we also ex-
amined macrophages from HLA-B27 transgenic and WT rats on
the Lewis background. As expected, IFN-
␥
induces expression of
HLA-B and h

2
m mRNA in both HLA-B27 and HLA-B7 trans
-
genic macrophages (Fig. 8A), which is accompanied by increased
cell surface expression (our unpublished observations). Endoge-
nous rat class I and rat

2
m transcripts are also increased in WT
and transgenic cells treated with IFN-
␥
(our unpublished observa-
tions). Although there are no differences in BiP expression be-
tween unstimulated WT, HLA-B27, and HLA-B7 transgenic cells,
IFN-
␥
treatment leads to a several-fold up-regulation of BiP ex-
pression (Fig. 8A) and XBP-1 mRNA splicing (Fig. 8, C and D)in
cells expressing HLA-B27. It is interesting that IFN-
␥
causes a
small increase in BiP and XBP-1 transcripts in WT cells from
Lewis rats (Fig. 8, A and B) that was not observed in cells from
F344 animals (Figs. 5 and 6), and thus may reflect strain differ-
ences. These data demonstrate that UPR activation in HLA-B27
transgenic rat macrophages in response to IFN-
␥
stimulation oc-
curs on two disease-prone genetic backgrounds, F344 and Lewis.
Furthermore, comparable expression and up-regulation of
HLA-B7 are not associated with UPR activation, suggesting that
an intrinsic property of the HLA-B27 H chain is responsible. The
most likely explanation for these results is that the tendency of the
HLA-B27 H chain to misfold (43, 44) causes an acute ER stress
when it is up-regulated, and that this is sufficient to activate
the UPR.
IFN-
␥
and UPR target gene expression in HLA-B27 transgenic
rat colon
One of the earliest and most consistent manifestations of the in-
flammatory phenotype of HLA-B27 transgenic rats is colitis. Cy-
tokine profiles from colon tissue suggest a prominent Th1 re-
sponse, including increased expression of IFN-
␥
(11, 45), and
intense staining for HLA-B27 has been demonstrated on cells in-
filtrating the colonic lamina propria in tissue sections from these
rats (31). In this study, we confirm increased expression of IFN-
␥
mRNA (Fig. 9A) and find increases in BiP and CHOP expression
in distal colon RNA from HLA-B27 transgenic rats with inflam-
matory disease (Fig. 9B). Differences in expression of BiP and
CHOP in HLA-B27 transgenic and WT tissue are not as large as
those observed in isolated macrophages. This is consistent with the
idea that a UPR is occurring, but may be limited to certain cell
types expressing high levels of HLA-B27.
Discussion
In this study, we report that HLA-B27 H chain misfolding is as-
sociated with activation of the UPR in an animal model of spon-
dyloarthritis. Importantly, UPR activation is not merely due to H
chain overexpression, as it is not induced by HLA-B7. These find-
ings also reveal that the UPR is not continuously active in cells
expressing HLA-B27, but can be induced by increasing HLA-B27
expression. To our knowledge, this is the first demonstration that
HLA-B27 can exert cell autonomous effects through the activation
of ER stress signaling pathways.
FIGURE 5. IFN-
␥
activation increases HLA-B27, BiP, and CHOP ex-
pression in HLA-B27-expressing rat macrophages. BM macrophages were
prepared from 4-wk-old WT F344 and HLA-B27 transgenic F344.33-3
(B27) rats and treated with IFN-
␥
(IFN; 100 U/ml for 24 h), or left un-
treated (⫺). Relative expression of HLA-B27 (B27), BiP, CHOP, and

-actin mRNA was determined by real-time PCR using total RNA. Ex-
pression of HLA-B27, BiP, and CHOP was normalized to

-actin, and is
expressed relative to untreated WT samples. Columns represent the mean
from triplicate biological replicates for each sample, with bars representing
SE. The abbreviation ND (not detected) indicates the absence of HLA-B27
expression in WT samples. The asterisk (ⴱ) indicates p ⬍ 0.02.
FIGURE 6. IFN-
␥
activation results in XBP-1 splicing in HLA-B27-
expressing rat macrophages. BM macrophages were prepared from 4-wk-
old WT F344 and HLA-B27 transgenic F344.33-3 (B27) rats and treated
with IFN-
␥
(IFN; 100 U/ml for 24 h), or left untreated (⫺). A, Relative
expression of XBP-1 mRNA was determined by real-time PCR. Data are
expressed relative to untreated WT macrophages (set to 1). B, Percentage
of total XBP-1 mRNA present in the spliced form (XBP-1s). Inset, Shows
a representative gel image with PCR products for unspliced (U) and spliced
(S) XBP-1 mRNA indicated. C, Relative expression of XBP-1s is the prod-
uct of the relative expression of XBP-1 as shown in A multiplied by the
percentage of XBP-1s shown in B. Untreated WT sample is set to 1. Col-
umns represent the mean of biological triplicates, with bars indicating the
SE. Asterisks (ⴱ) indicate statistically significant differences (p ⬍ 0.02) for
B27 vs WT.
2443The Journal of Immunology
HLA-B27 misfolding
Our studies demonstrate that HLA-B27, expressed in cells from
F344 transgenic rats, exhibits biochemical characteristics of mis-
folding, including inefficient folding, prolonged BiP binding, and
formation of disulfide-linked complexes in the ER. These results
confirm what we and others have reported for HLA-B27 expres-
sion in human cells and subsequently in cells from HLA-B27
transgenic Lewis rats and rat cell lines (13–16). In contrast, the
HLA-B7 allele shows no evidence of misfolding when expressed
either in human (14) or rat cells (16). Thus, our data indicate that
the biochemical characteristics of misfolding are similar on two
disease-permissive genetic backgrounds (11, 16), and confirm that
the propensity of HLA-B27 to misfold occurs when the molecule
is expressed either in a homologous system (human cells) or in
heterologous (rat) cells.
Consequences of protein misfolding and activation of the UPR
The consequences of misfolding depend on the nature of the pro-
tein and where it is produced. ER-synthesized proteins are subject
to stringent quality control that generally prevents the expression
of misfolded forms or unassembled intermediates (reviewed in
Ref. 46). These proteins are typically dislocated into the cytosol
and degraded by proteasomes in a process known as ER-associated
degradation (ERAD). When not adequately eliminated, misfolded
proteins can form aggregates in the cytosol (aggresomes) or ER
(Russell bodies), resulting in toxicity (47). Certain proteins that
FIGURE 7. IFN-
␥
activation results in UPR gene expression signature in HLA-B27-expressing rat macrophages. BM macrophages derived from
4-wk-old WT and HLA-B27 transgenic (B27) rats were left untreated (⫺IFN), or were treated with 100 U/ml IFN-
␥
(⫹IFN) for 24 h. RNA was isolated
and subjected to microarray analysis using the Affymetrix Rat 230A (RAE230A) GeneChip. Note that the gene lists in Fig. 7 were generated using a more
recent and comprehensive version of GeneChip (RAE230A) than that used for Fig. 4 (RGU34A). Thus, the list in Fig. 7 includes all UPR and IFN genes
listed in Fig. 4 (except RT1.S3) and several others not measured by the RGU34A GeneChip. As in Fig. 4, genes are clustered in the heat map based on
expression pattern, and thus, the order of genes listed in Fig. 7 differs from that in Fig. 4. Following standard normalization of each array, values for
individual genes were normalized to the average of the untreated controls (WT, ⫺IFN). The figure shows a heat map of relative expression values with
each column representing a biological replicate (3 per condition), and each row representing a different probe set. (Note that for some genes there is more
than one probe set measuring expression.) Relative expression levels are colored, as described in the legend to Fig. 4. The abbreviated name for each gene
is shown (Gene), as well as the GenBank number and a brief description. The FC columns show the average fold change based on three comparisons of
B27 vs WT in the absence (⫺) and presence (⫹) of IFN-
␥
. The genes are divided into UPR and IFN-responsive genes, as described in the legend to Fig.
4. The asterisks (ⴱ) indicate differences that are statistically significant (p ⬍ 0.05) for B27 vs WT.
2444 HLA-B27 MISFOLDING AND UPR
misfold can activate the UPR, although this is not a universal con-
sequence, and may depend on binding to BiP (26).
We hypothesized that the tendency of HLA-B27 to misfold
might generate ER stress and activate the UPR or the ER overload
response (13, 25). Recently, we established a correlation between
HLA class I H chain misfolding and the development of inflam-
matory disease in transgenic rats (16). In this study, we now link
HLA-B27 misfolding with UPR activation in cells and tissues
from these animals. The absence of this response in spleen, thy-
mus, and macrophages from premorbid rats, all of which express
HLA-B27, suggests that ongoing UPR activation is not a wide-
spread phenomenon in these animals, and may depend on cell type
and/or exogenous factors. For example, the UPR is activated when
macrophages from premorbid HLA-B27 transgenic rats are treated
with IFN-
␥
, while this is not seen in WT cells (Figs. 5– 8). The
HLA-B (B27 and B7) and h

2
m genomic DNAs used to make
these transgenic rats contain 5⬘ flanking sequence that enables tis-
sue-specific and IFN-
␥
-inducible expression (3, 31), as demon-
strated by up-regulation in response to IFN-
␥
(Figs. 5 and 8).
Therefore, these data are consistent with the idea that UPR acti-
vation in cells treated with IFN-
␥
is due to up-regulation of HLA-
B27 expression. An alternative possibility, that h

2
m up-regula
-
tion contributes to UPR activation, seems unlikely because this is
not observed in cells from HLA-B7/h

2
m transgenic rats. Al
-
though misfolding is an intrinsic property of the HLA-B27 H chain
(13, 14), the ratio of H chain to h

2
m might influence folding
efficiency. Although h

2
m is highly expressed and also up-regu
-
lated by IFN-
␥
, we cannot rule out the possibility that insufficient
h

2
m expression enhances UPR activation by HLA-B27. In this
regard, it should be noted that the ratio of HLA-B to h

2
m tran
-
scripts is higher for HLA-B27 than HLA-B7 transgenic macro-
phages. It seems unlikely that slightly greater h

2
m expression in
HLA-B7 transgenic rat cells prevents it from misfolding, because
human cells transfected with HLA-B7 and no additional h

2
m
show no evidence that this occurs (14). Finally, it is possible that
other gene products up-regulated by IFN-
␥
contribute to UPR ac-
tivation, but only when HLA-B27 is present. In this context, it is
interesting that the UPR occurs despite up-regulation of class I
assembly components such as the peptide transporter (TAP) and
tapasin, which are expected to promote the assembly of H chain/

2
m/peptide complexes.
Although our studies establish a link between HLA-B27 mis-
folding and UPR activation, there may be other consequences of
HLA-B27 misfolding in transgenic rat cells. Newly synthesized
HLA-B27 H chains form disulfide-linked oligomeric complexes as
well as smaller H chain dimers (16). It is conceivable that oli-
gomers might form persistent aggregates such as Russell bodies or
aggresomes. Interestingly, the majority of BiP bound to HLA-B27
appears to be associated with oligomeric H chains (16), suggesting
that they may be of particular significance for UPR activation. In
peptide transporter (TAP1)-deficient mice, HLA-B27 accumulates
in an expanded ER-Golgi compartment with components of the
ubiquitin system, suggesting ongoing ERAD (48). However, ul-
trastructural analysis of these cells did not reveal abnormal cyto-
solic bodies, and thus, it seems unlikely that HLA-B27 forms ag-
gresomes (49). The relationship between the expanded ER-Golgi
compartment and Russell bodies is less clear, but based on ultra-
structural descriptions, it appears distinct (50). We have also not
observed differential cell death in HLA-B27 transgenic BM mac-
rophages. However, we cannot rule out subtle differences or the
possibility that this might occur under different stimulation
conditions.
The ER overload response was proposed to activate NF-
Basa
consequence of ER stress (51). It was initially unclear what sig-
naling pathway was involved and whether it was distinct from the
FIGURE 8. IFN-
␥
activation increases BiP expression and XBP-1 splic-
ing in HLA-B27-, but not HLA-B7-expressing rat macrophages. BM mac-
rophages were prepared from WT Lewis, HLA-B27 transgenic (B27), and
HLA-B7 transgenic (B7) rats and treated with IFN-
␥
(IFN; 100 U/ml for
24 h), or left untreated (⫺). A, Relative expression of HLA-B (B27, B7),
h

2
m, BiP, and

-actin mRNA was determined by real-time PCR using
total RNA. B, Relative expression of XBP-1 and

-actin mRNA was de-
termined by real-time PCR using total RNA. Expression of HLA-B, h

2
m,
BiP, and XBP-1 was normalized to

-actin, and is expressed relative to
untreated WT samples (set to 1). C, Percentage of total XBP-1 mRNA
present in the spliced form (XBP-1s). Inset, Shows a representative gel
image with PCR products for unspliced (U) and spliced (S) XBP-1 mRNA
indicated. D, Relative expression of XBP-1s is the product of the relative
expression of XBP-1 as shown in B multiplied by the percentage of
XBP-1s shown in C. Data represent the mean of three replicates for each
sample, with bars representing SE. Asterisks indicate statistically signifi-
cant differences (p ⬍ 0.005) for B27 vs WT and B7 (ⴱ).
FIGURE 9. IFN-
␥
, BiP, and CHOP are overexpressed in HLA-B27
transgenic rat colon. A, RNA was isolated from distal colon tissue from
three 24-wk-old WT and three HLA-B27 transgenic (B27) rats. RT-PCR
results for IFN-
␥
and GAPDH are shown. B, BiP and CHOP expression
was determined by real-time PCR. Columns represent the mean of tripli-
cate biological replicates with SEs shown. Asterisks (ⴱ) indicate statisti-
cally significant differences (p ⬍ 0.02) for B27 vs WT.
2445The Journal of Immunology
UPR. Although this has not been resolved, it is clear that during
ER stress, PERK-mediated eukaryotic initiation factor 2-
␣
phos-
phorylation can lead to NF-
B activation (52, 53) via down-reg-
ulation of I
B
␣
synthesis (53). Our gene expression analyses of
BM macrophages do not reveal a strong NF-
B-dependent tran-
scriptional response, although some activation cannot be ruled out.
Indeed, the overall magnitude of the UPR associated with HLA-
B27 misfolding is less than what is observed with pharmacologic
agents such as TM (Figs. 2 and 3) that can also activate NF-
B.
This is not surprising and is consistent with the idea that misfold-
ing of a single protein species is a more physiologic stress than
pharmacologic agents such as TM that completely disrupt ER
function by causing virtually all newly synthesized glycoproteins
to misfold, and with prolonged exposure can cause cell death. It is
possible that a more robust UPR with NF-
B activation might be
observed in HLA-B27 transgenic macrophages with more pro-
longed IFN-
␥
stimulation, or in the presence of other factors such
as inflammatory mediators and/or microbes.
Our data reveal subtle differences in UPR activation in macro-
phages derived from 10-wk-old rats with inflammatory disease
compared with cells from premorbid 4 wk olds activated with
IFN-
␥
. Although the magnitude of BiP induction is similar, in-
creases in CHOP and XBP-1 transcripts are greater in cells from
10-wk-old rats compared with the IFN-
␥
-activated macrophages
(Figs. 2, 3, 5, and 6). In contrast, XBP-1 splicing is greatest in the
IFN-
␥
-activated cells (Figs. 3 and 6). (Note that this quantitative
comparison is based on real-time PCR and not microarray analysis.
The use of different microarrays for the two experiments precludes
quantitative comparisons, because in some cases the probe sets are
different.) One factor that may contribute to these differences is
that the UPR occurs in phases related to the degree and duration of
ER stress (54, 55). In this context, our results from 10-wk-old rats
with inflammatory disease may reflect a population of macro-
phages at different stages of the UPR, while a more synchronized
response may occur after IFN-
␥
treatment.
The absence of a UPR in spleen and thymus (Fig. 2), and min-
imal increases in BiP and CHOP in BM macrophages from pre-
morbid rats (Figs. 5 and 7), despite ongoing expression of HLA-
B27, may be related to several factors. The amount of H chain
expression in unstimulated cells may be insufficient, and/or the
proportion of H chains that misfold may depend on cell type and
relative expression of class I assembly pathway components. It is
also conceivable that certain cells are more capable of handling an
increased load on the ER. Another possibility is that cells from
HLA-B27 transgenic rats have adapted to chronic ER stress. Al-
though little is known about adaptation mechanisms, modest up-
regulation of ER chaperones and ERAD components may be suf-
ficient to raise the threshold for UPR activation. This concept is
supported by several observations. First, cells genetically deficient
in general ER chaperones (calreticulin or calnexin) exhibit rela-
tively small increases in BiP (1.5- to 3-fold) (56). Second, cells
from patients with type I congenital disorder of glycosylation are
somewhat resistant to chemical inducers of the UPR (57). Finally,
although UPR activation occurs in cells transiently transfected
with HLA-B27, we do not observe this in stable transfectants (58)
(our unpublished observations). Nevertheless, stable transfectants
display altered responsiveness to exogenous stimuli (58 – 62), sug-
gesting that they are fundamentally different. It will be important
to carefully examine cells from HLA-B27 transgenic rats for prop-
erties distinguishing them from WT cells, even in the absence of
UPR activation.
The role of IFN
These experiments reveal a correlation between the presence of
IFN and UPR activation in HLA-B27 transgenic rats. Macro-
phages derived from the BM of transgenic rats with inflammatory
disease exhibit UPR activation and display evidence of IFN expo-
sure (Fig. 4). However, UPR activation is minimal in macrophages
from premorbid rats (Figs. 5–7), and there is little evidence of IFN
exposure, with ⬍2-fold elevation of a subset of IFN-responsive
gene transcripts (Fig. 7; e.g., CXCL10, IFN regulatory factor 7,
Best5). Although IFN-
␥
is sufficient to generate an IFN response
signature and UPR activation (Fig. 7) like that observed in mac-
rophages derived from HLA-B27 transgenic rats with disease (Fig.
4), the large overlap in target genes up-regulated by this and type
I(
␣
) IFNs (42, 63) precludes any conclusion about which IFN is
responsible. It is perhaps surprising that there is a prominent IFN
signature in macrophages derived over several days of culture in
vitro. One possible explanation is that the UPR itself may poten-
tiate the IFN response. Lee et al. (37) have shown low-level in-
duction of IFN-

expression in mouse embryo fibroblasts under-
going a UPR. This could create a positive feedback loop through
up-regulation of HLA-B27 expression and further exacerbation of
the UPR. Interestingly, IFN-
␥
overexpression in colon tissue cor-
relates with colitis and disease susceptibility in various strains of
transgenic rats (11, 45). In this study, we demonstrate that the
presence of this cytokine is also associated with increased BiP and
CHOP expression (Fig. 8). It is worth noting that germfree HLA-
B27 transgenic rats do not develop colitis or arthritis (64), and
disease can be induced with normal gastrointestinal flora (45). This
suggests that an immune response is necessary to initiate the
pathogenic process and is consistent with the possibility that cy-
tokines such as IFNs might serve as potential triggers. Thus, the
inciting event in pathogenesis may be the inflammatory response
that occurs in the gut when it becomes colonized with normal
microbial flora, which could in turn stimulate HLA-B27 expres-
sion in macrophages and other APC populations. We speculate that
this process might in turn cause UPR activation in these cell pop-
ulations and alter their function or survival, promoting the trans-
formation from what is usually a controlled process, into chronic
inflammation.
Implications for HLA-B27-associated disease
Several pieces of evidence suggest that misfolding is unusual, if
not unique to HLA-B27 (12, 15, 16). First, HLA-B7, -B8, and
-B53 do not undergo ERAD (13). Second, newly synthesized
HLA-B7 H chains do not form disulfide-linked ER dimers, either
when expressed in human (14) or rat cells (16), and HLA-A2 does
not form stable H chain dimers (15). Third, while BiP binding was
initially described for HLA-B7, it was difficult to detect without
cross-linking (65), suggesting this interaction is transient. Indeed,
stable BiP binding to HLA-B7 in cells from transgenic rats is min-
imal (16), and in experiments with transfected cells, we find min-
imal BiP coprecipitating with HLA-B7, -B8, or -A2 (our unpub-
lished observations). In contrast, BiP binding is prominent with
HLA-B27 (14–16) (our unpublished observations). Structural fea-
tures of HLA-B27 that contribute to misfolding include three B
pocket residues (Glu
45
, Cys
67
, and Lys
70
) that together are virtu
-
ally unique to HLA-B27 (14), although other conserved residues
(e.g., Cys
164
) may also be involved (15). Taken together, these
data support the idea that class I H chain misfolding may be spe-
cific to the HLA-B27 allele. However, there are over 1000 HLA-A,
-B, and -C alleles reported to date (www.ebi.ac.uk/imgt/hla/docs/
release.html), and thus whether this aberrant characteristic is
unique to HLA-B27 remains to be determined. In addition, the role
2446 HLA-B27 MISFOLDING AND UPR
of subtype polymorphisms on HLA-B27 misfolding remains to be
investigated.
Although CD8
␣
⫹
T cells are not required for arthritis or co
-
litis, the high level of CD8
␣
transcripts in BM macrophages from
HLA-B27 transgenic rats with inflammatory disease (Fig. 4) is
interesting for several reasons. First, May et al. (4) reported a
correlation between disease and the expansion of CD8
␣
⫹
mono
-
cytes and macrophages in the peripheral blood and spleen of these
rats. Second, partial depletion of these populations using an anti-
CD8
␣
Ab correlated with a reduction in the severity of arthritis,
implicating these cells in pathogenesis. Third, CD8
␣
has been re-
ported to be a marker of activated macrophages in rats, and CD8-
expressing monocytes and macrophages have been described in
rats in which inflammation has been induced (66, 67). Although
CD8
␣
mRNA levels are ⬃1.4-fold higher in macrophages from
premorbid HLA-B27 transgenic vs WT rats, this does not appear
to be affected by IFN-
␥
activation (Fig. 7). This implies that ad-
ditional factors may be involved in up-regulating CD8
␣
in mac-
rophages derived from HLA-B27 transgenic rats with inflamma-
tory disease.
In summary, these results indicate that the balance between
HLA-B27 folding, misfolding, and degradation can be tipped to
activate ER stress signaling pathways that result in a UPR. The
UPR can serve to restore homeostasis and is part of a physiological
mechanism that expands the folding and secretory capacity of dif-
ferentiating B cells (68). However, ER stress and UPR activation
can have pathological consequences (69), and thus, have the po-
tential to adversely affect immune function when they occur in
APCs. Our studies have to date focused on macrophages. How-
ever, other populations such as dendritic cells that have been
shown to be dysfunctional in HLA-B27 transgenic rats (70, 71)
could also be affected. We do not know as yet the extent to which
HLA-B27 misfolding may activate the UPR in individuals with
disease, but it is worth noting that BiP overexpression has been
reported in adherent synovial fluid mononuclear cells from HLA-
B27-positive spondyloarthritis patients (72). Although these find-
ings do not rule out other possible mechanisms, including immu-
nological recognition of either classical or aberrant forms of HLA-
B27 on the cell surface, the role that the UPR may play in
pathogenesis of HLA-B27-associated disorders warrants further
investigation.
Acknowledgments
We thank Hidde Ploegh for the HC10-producing hybridoma;
Yasmine Belkaid for the BM macrophage derivation protocol; Tom Griffin,
Krupakar Jayarapu, Sherry Thornton, and David Adams for critical eval-
uation of the manuscript; and Martha Dorris and Nimman Satumtira for
breeding and maintenance of transgenic rats.
Disclosures
The authors have no financial conflict of interest.
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