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* William S. Rowe Division of Rheumatology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, 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 Medical Center, Dallas, TX 75390
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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 pathogenesis of inflammatory diseases associated with this MHC class I allele. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The development of spondyloarthritis-like disease in HLA-B27/human 2-microglobulin (h2m)3 transgenic rats suggests a direct role for HLA-B27 in pathogenesis (3). Although the primary function 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 gastrointestinal inflammation in HLA-B27 transgenic rats (4). In addition, HLA-B27 transgenic, 2m-deficient mice develop arthritis (5), despite a dramatic reduction in class I-restricted peptide presentation and CD8+ T cell populations resulting from the absence of 2m (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 marrow (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) expressing HLA-B27 may be critical. Development of the inflammatory disease is not merely due to HLA class I overexpression because HLA-B7/h2m transgenic rats do not develop the spondyloarthritis-like phenotype (11).
A number of reports indicate that HLA-B27 H chains exhibit aberrant features, including a tendency to misfold (reviewed in Ref. 12). This occurs in the endoplasmic reticulum (ER), and involves inefficient folding of, and delayed 2m binding to, newly synthesized H chains (13, 14). Misfolding can result in the ER-associated degradation (ERAD) of HLA-B27 (13), and is associated with prolonged binding of H chains to the ER chaperone, BiP (Grp78) (14, 15, 16). The formation of high m.w. disulfide-linked complexes of HLA-B27 in the ER, including H chain homodimers and oligomers (14, 15, 16), appears to be a feature of misfolding. Most of these aberrant characteristics have been observed when HLA-B27 is expressed in rat as well as human cells (14, 15, 16).
Dimerization of HLA-B27 was first observed when H chains were refolded in vitro, particularly in the absence of 2m (17). Dimers have also been observed on the surface of cells (14, 15, 18, 19, 20). Cell surface dimers do not appear to originate in the ER, at least in cells with an intact class I assembly pathway (14). Instead, they appear to derive from cell surface H chain/2m/peptide complexes that have lost 2m (19). Consistent with this observation, relatively stable 2m-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), preventing their activation. Many proteins that misfold bind and sequester 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 misfolding 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Inbred HLA-B27/h2m 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/h2m (L.33-3 line), HLA-B7/h2m (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 contains 55 copies of the HLA-B27 gene and 66 copies of the h2m gene, while the 120-4 locus contains 26 copies of HLA-B7 and 5 copies of h2m. 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 h2m). The HLA-B (B27 and B7) and h2m 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/streptomycin, 50 µg/ml gentamicin sulfate (Sigma-Aldrich) (D-10), and L929 supernatant (30% day 0–5 and 2.5% day 5–6). L929 supernatant (containing 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 enteridis 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 molecules that is largely dependent on 2m and peptide binding (33), and HC10 recognizes free (unfolded or misfolded) HLA B and C allele H chains without 2m (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 µg/ml), washed, and incubated in fresh Met/Cys-deficient R-10 medium for 1 h at 37°C. Cells (2 x 107 per time point) were labeled for 1 h with [35S]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 rearrangement. Nuclei were removed, and then lysates were precleared with Formalin-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 µg per 2 x 107 cells in 500 µl), with immune complexes removed by incubation with protein A-Sepharose (100 µl of 50 mg/ml suspension per 500 µl of lysate) (Sigma-Aldrich) for 1 h at 4°C following each step. Protein A-Sepharose pellets were washed extensively and stored at –20°C until electrophoresis.
Electrophoresis, phosphorimaging, and immunoblotting
Immunoprecipitated proteins separated by SDS-PAGE were visualized either by phosphor imaging ([35S]Met/Cys labeled) of dried gels or immunoblotting after transfer to polyvinylidene difluoride membranes (Westran; Schleicher & Schuell Microscience), as described (14). For immunoblotting, anti-GRP78 (StressGen Biotechnologies) and HC10 (anti-2m-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 appropriate alkaline phosphatase-conjugated secondary Ab (Southern Biotechnology Associates). Proteins were visualized with 5-bromo-4-chloro-3-indolyl phosphate/NBT substrate (Sigma-Aldrich).
BMDM culture and RNA preparation
Rat BM cells were cultured in 30% L929-conditioned D-10 with gentamicin (50 µg/ml) in 75-cm2 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 nonadherent cells, TRIzol (Invitrogen Life Technologies) was added directly to the plate-bound adherent cells (BMDM), followed by RNA isolation, according to the manufacturer’s instructions. In some experiments, macrophages were cultured for additional lengths of time with IFN- (24 h) or TM (7 h) before RNA isolation. Culture medium contained <0.08 endotoxin U/ml based on the QCL-1000 chromogenic Limulus amebocyte lysate 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.
Quantitative real-time PCR and semiquantitative RT-PCR
Total RNA was reverse transcribed using oligo(dT) primers and the Superscript 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 (Amersham Biosciences) and ImageQuant software, and is expressed as a percentage 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 GeneChip 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) guidelines (www.mged.org/Workgroups/MIAME/miame.html). Differences between experimental conditions across all samples (e.g., WT vs HLA-B27 transgenic) were determined using GeneSpring 6.1 software (Silicon Genetics). 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). Supplemental information describing microarray sample preparation is provided in the online version of this article.
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Statistical analysis was performed using Student’s t test. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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 during a 19-h chase using HC10 and W6/32. HC10 recognizes H chains that are not completely folded and either have not yet acquired or have lost 2m (34), while W6/32 recognizes folded H chains typically associated with 2m and peptide (33). Immunoprecipitates 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 recognized by HC10, but not W6/32 (Fig. 1A). These complexes are qualitatively indistinguishable from what we have previously described in human cells and are referred to as ER dimers (14). ER dimers decay considerably by 6 h of chase. 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, indicating 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 immunoblotting 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).
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2m 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 coprecipitation 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.
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 comparison, the effect of TM, which inhibits N-linked glycosylation and causes global misfolding of newly synthesized glycoproteins, is shown (Fig. 2).
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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).
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To gain further insight into gene expression differences in BMDM from HLA-B27 transgenic rats, we used microarray analysis. Several genes known to be up-regulated during the UPR are overexpressed 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 differentially expressed in this experiment. Together with the evidence 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 evidence that these cells are exhibiting signs of ER stress. Microarray 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 observations). 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, compared with untreated or IFN--treated WT macrophages, respectively (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.
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treatment on BM macrophages by microarray analysis. UPR target gene expression is significantly 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 degradation-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), provides 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-expressing 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-expressing 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 experiments (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 were performed using rats with an F344 background, we also examined macrophages from HLA-B27 transgenic and WT rats on the Lewis background. As expected, IFN- induces expression of HLA-B and h2m mRNA in both HLA-B27 and HLA-B7 transgenic macrophages (Fig. 8A), which is accompanied by increased cell surface expression (our unpublished observations). Endogenous rat class I and rat 2m transcripts are also increased in WT and transgenic cells treated with IFN- (our unpublished observations). Although there are no differences in BiP expression between unstimulated WT, HLA-B27, and HLA-B7 transgenic cells, IFN- treatment leads to a several-fold up-regulation of BiP expression (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 differences. These data demonstrate that UPR activation in HLA-B27 transgenic rat macrophages in response to IFN- stimulation occurs 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.
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and UPR target gene expression in HLA-B27 transgenic rat colon
One of the earliest and most consistent manifestations of the inflammatory phenotype of HLA-B27 transgenic rats is colitis. Cytokine profiles from colon tissue suggest a prominent Th1 response, including increased expression of IFN- (11, 45), and intense staining for HLA-B27 has been demonstrated on cells infiltrating 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 inflammatory 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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HLA-B27 misfolding
Our studies demonstrate that HLA-B27, expressed in cells from F344 transgenic rats, exhibits biochemical characteristics of misfolding, 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 expression in human cells and subsequently in cells from HLA-B27 transgenic Lewis rats and rat cell lines (13, 14, 15, 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 protein 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 misfold can activate the UPR, although this is not a universal consequence, 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 inflammatory 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, thymus, and macrophages from premorbid rats, all of which express HLA-B27, suggests that ongoing UPR activation is not a widespread 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 h2m genomic DNAs used to make these transgenic rats contain 5' flanking sequence that enables tissue-specific and IFN--inducible expression (3, 31), as demonstrated by up-regulation in response to IFN- (Figs. 5 and 8). Therefore, these data are consistent with the idea that UPR activation in cells treated with IFN- is due to up-regulation of HLA-B27 expression. An alternative possibility, that h2m up-regulation contributes to UPR activation, seems unlikely because this is not observed in cells from HLA-B7/h2m transgenic rats. Although misfolding is an intrinsic property of the HLA-B27 H chain (13, 14), the ratio of H chain to h2m might influence folding efficiency. Although h2m is highly expressed and also up-regulated by IFN-, we cannot rule out the possibility that insufficient h2m expression enhances UPR activation by HLA-B27. In this regard, it should be noted that the ratio of HLA-B to h2m transcripts is higher for HLA-B27 than HLA-B7 transgenic macrophages. It seems unlikely that slightly greater h2m expression in HLA-B7 transgenic rat cells prevents it from misfolding, because human cells transfected with HLA-B7 and no additional h2m show no evidence that this occurs (14). Finally, it is possible that other gene products up-regulated by IFN- contribute to UPR activation, 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/2m/peptide complexes.
Although our studies establish a link between HLA-B27 misfolding 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 oligomers 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, ultrastructural analysis of these cells did not reveal abnormal cytosolic bodies, and thus, it seems unlikely that HLA-B27 forms aggresomes (49). The relationship between the expanded ER-Golgi compartment and Russell bodies is less clear, but based on ultrastructural descriptions, it appears distinct (50). We have also not observed differential cell death in HLA-B27 transgenic BM macrophages. 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-
B as a consequence of ER stress (51). It was initially unclear what signaling pathway was involved and whether it was distinct from the UPR. Although this has not been resolved, it is clear that during ER stress, PERK-mediated eukaryotic initiation factor 2- phosphorylation can lead to NF-B activation (52, 53) via down-regulation of IB synthesis (53). Our gene expression analyses of BM macrophages do not reveal a strong NF-B-dependent transcriptional 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 misfolding 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 prolonged 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 macrophages 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, increases 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 macrophages 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 minimal increases in BiP and CHOP in BM macrophages from premorbid 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. Although little is known about adaptation mechanisms, modest up-regulation of ER chaperones and ERAD components may be sufficient 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 relatively 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, 59, 60, 61, 62), suggesting that they are fundamentally different. It will be important to carefully examine cells from HLA-B27 transgenic rats for properties 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. Macrophages derived from the BM of transgenic rats with inflammatory disease exhibit UPR activation and display evidence of IFN exposure (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 macrophages 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 potentiate the IFN response. Lee et al. (37) have shown low-level induction of IFN- expression in mouse embryo fibroblasts undergoing 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 correlates 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 cytokines 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 expression in macrophages and other APC populations. We speculate that this process might in turn cause UPR activation in these cell populations and alter their function or survival, promoting the transformation 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 minimal (16), and in experiments with transfected cells, we find minimal BiP coprecipitating with HLA-B7, -B8, or -A2 (our unpublished observations). In contrast, BiP binding is prominent with HLA-B27 (14, 15, 16) (our unpublished observations). Structural features of HLA-B27 that contribute to misfolding include three B pocket residues (Glu45, Cys67, and Lys70) that together are virtually unique to HLA-B27 (14), although other conserved residues (e.g., Cys164) may also be involved (15). Taken together, these data support the idea that class I H chain misfolding may be specific 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 of subtype polymorphisms on HLA-B27 misfolding remains to be investigated.
Although CD8+ T cells are not required for arthritis or colitis, 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+ monocytes 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 reported 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 additional factors may be involved in up-regulating CD8 in macrophages derived from HLA-B27 transgenic rats with inflammatory 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 differentiating B cells (68). However, ER stress and UPR activation can have pathological consequences (69), and thus, have the potential to adversely affect immune function when they occur in APCs. Our studies have to date focused on macrophages. However, 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 findings do not rule out other possible mechanisms, including immunological 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.
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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 supported 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{at}cchmc.org
3 Abbreviations used in this paper: h2m, 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 degradation; 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.
Received for publication January 12, 2005. Accepted for publication May 30, 2005.
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2-m: an animal model of HLA-B27-associated human disorders. Cell 63: 1099-1112. [Medline]
T cells are not essential to the pathogenesis of arthritis or colitis in HLA-B27 transgenic rats. J. Immunol. 170: 1099-1105. 2-microglobulin: a model of human spondyloarthropathies. J. Exp. Med. 182: 1153-1158. 2-microglobulin-free heavy chain homodimer structure. J. Immunol. 162: 5045-5048. 2-microglobulin-free heavy chains of HLA-B2705 on the cell surface. J. Immunol. 169: 4379-4387. 
