Rheumatology Advance Access originally published online on July 28, 2006
Rheumatology 2007 46(2):232-237; doi:10.1093/rheumatology/kel226
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Does HLA-B27 influence the monocyte inflammatory response to lipopolysaccharide?
Department of Medicine, School of Clinical Medicine, University of Cambridge, Addenbrookes Hospital and 1Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK
Correspondence to: Dr Jane Goodall, University of Cambridge, School of Clinical Medicine, Box 157, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. E-mail: jcg23{at}medschl.cam.ac.uk
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Abstract |
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Objectives. How human leucocyte antigen B27 (HLA-B27) contributes towards arthritis susceptibility is still unclear, but effects on the response to bacteria unrelated to the classical antigen presenting role of B27 have been suggested. This study investigated whether HLA-B27 modulates the innate response to lipopolysaccharide (LPS), a component shared between all Gram negative bacteria that can trigger reactive arthritis.
Methods. Pools of U937 transfectants expressing either HLA-B27, HLA-A2 or the expression plasmid alone were differentiated with phorbol 12-myristate 13-acetate and stimulated with LPS. Supernatants were analysed for tumour necrosis factor-
(TNF-) secretion and the gene expression profiles of unstimulated and LPS-stimulated cells were determined by microarray analysis. Changes in gene expression that are indicative of an unfolded protein response (UPR) were also analysed by quantitative polymerase chain reaction (PCR).
Results. TNF- secretion, a biological marker of the inflammatory response to LPS, was not significantly different between U937-B27 and U937-control. No differences in gene expression between unstimulated U937-B27 and U937-control lines were detected. Both U937-control and U937-B27 exhibited a stereotypic response to LPS. Only one gene, OAS2, was differentially expressed by these cell lines, and this was confirmed by quantitative PCR. Analysis of XBP-1 splicing suggested that the UPR is induced following the LPS stimulation, but this increase was seen in all transfectants.
Conclusions. The expression of B27 does not profoundly alter the gene expression following LPS stimulation. Therefore, additional signals, such as those provided by cytokines or intracellular infection, may be required to reveal any influence of B27 expression on the inflammatory response.
KEY WORDS: HLA-B27, Ankylosing spondylitis, U937, LPS, Unfolded protein response, Inflammation, Microarray
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Introduction |
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HLA-B27 is strongly associated with the spondyloarthropathies (SpAs) and experimental evidence from transgenic rats and mice suggest that HLA-B27 plays a direct role in disease pathogenesis [1, 2]. Epidemiological studies suggest that expression of HLA-B27 increases the risk that an individual will have more severe and prolonged arthritis following an enteric or gastrointestinal infection (reviewed in [3]). These studies have also shown that the increased risk is not due to increased susceptibility to infection, suggesting that reactive arthritis (ReA) patients respond differently to these bacteria.
The direct association of a bacterial infection with ReA suggests an important role for microorganisms in its pathogenesis. Furthermore, evidence from HLA-B27 transgenic rats and mice suggest that the bacterial flora plays a role in the disease, since animals raised in germ-free conditions do not develop arthritis [4]. How bacteria and HLA-B27 contribute towards arthritis susceptibility is not known and recently, theories have been proposed that are not centred on the classical antigen presentation function of this HLA molecule [5]. Studies have suggested that HLA-B27 expression modulates the host cells’ susceptibility to infection although there are a number of conflicting reports on this effect [6–8]. More recent studies have suggested that bacterial persistence and intracellular replication may be modified when HLA-B27 is expressed [9, 10]. Furthermore, HLA-B27 transfectants have also shown altered signalling responses following infection by salmonella, resulting in increased c-fos expression in HeLa cell lines [11].
The pathogens that are associated with ReA are Yersinia, Shigella, Campylobacter, Salmonella and Chlamydia. These organisms are either obligate or facultatitively intracellular Gram-negative bacteria, with a lipopolysaccharide (LPS)-containing outer membrane. A stereotypic pattern of gene expression is observed following the stimulation of peripheral blood mononuclear cells (PBMCs) with LPS or bacteria [12]. Genes involved in the induction of this response are known to be regulated by NF-
B. Analysis of the response of monocytes to LPS using microarray and Serial Analysis of Gene Expression (SAGE) techniques has revealed that a diverse programme of gene expression occurs, which could orchestrate the inflammatory response [12–14]. These genes are involved in cell activation and migration, angiogenesis, tissue remodelling and metabolism. HLA-B27 stable U937 transfectants have been reported to show altered activation of NF-B following LPS stimulation [15], suggesting that B27 modifies the response to bacterial infection. If differences in NF-B activation are evident then this should differentially affect a diverse set of genes which are downstream of NF-B signalling.
In addition to possible effects on the response to intracellular infection, recent evidence suggests that HLA-B27 expression is associated with activation of the unfolded protein response (UPR) in HLA-B27 transgenic rats [16]. This in turn could be associated with pro-inflammatory mechanisms. Protein mis-folding in the endoplasmic reticulum (ER) activates a cascade of signalling events that can be detected by the increased expression of molecules such as BiP and an altered splice variant of XBP-1. These sensors of the UPR were shown to be increased in macrophages from HLA-B27 transgenic rats with inflammatory disease, but were seen only in bone marrow and colon, suggesting that additional factors in combination with HLA-B27 expression affect the UPR activation.
The aim of this study is to determine whether HLA-B27 modulates the innate response to LPS, a component shared between all Gram-negative bacteria that are associated with susceptibility to ReA and secondly, to assess whether any differences in the UPR can be detected following the LPS stimulation.
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Plasmids
Prc/RSV B27 expression construct was provided by Simon Powis. HLA-A2 was amplified by polymerase chain reaction (PCR) and inserted into the Prc/RSV vector (Invitrogen, Paisley, UK). The full-length coding sequences of HLA-A2 and HLA-B27 were verified by DNA sequencing. The expression vector utilizes the Rous sarcoma virus long-terminal repeat that enables continous expression in mammalian cells. Integration of this vector into the genome is required to achieve stable long-term expression.
Cell lines
U937 is a human monocytic cell line that expresses HLA class I alleles A3, A26, B18, B51, CW1 and CW3.
Transfectants and stimulation with LPS
U937 cells were transfected with expression vector alone or constructs containing either HLA-B27, HLA-A2, using nucleofector reagent V (Amaxa Biosystems, Koeln, Germany). Expression of the HLA molecules by the transfectants was detected using either the HLA-B27-specific antibody, B27-ABC-m3 (Serotec, Oxford, UK) or HLA-A2-specific antibody, MA2.1 [17]. Transfectants were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Sigma, Poole, UK), 2 mM glutamine and 20 mM HEPES in a humidified 5% CO2 atmosphere at 37°C, These lines were screened regularly to exclude mycoplasma infection. The lines were maintained in G418 at 500 µg/ml (Calbiochem, Nottingham, UK). Pools of transfectants from a minimum of nine clones were prepared prior to phorbol 12-myristate 13-acetate (PMA) differentiation and cultured at a cell concentration of 1 million cells/ml. U937 cells were differentiated with PMA (Sigma) (10 ng/ml) for 24 h followed by 4 h culture with or without Salmonella enteritidis LPS at 500 ng/ml (Sigma).
Differentiation of U937 and stimulation with LPS
Pools of transfectants were prepared (minimum nine per pool). U937 cells were differentiated for 24 h with PMA followed by a 4-h culture with or without 500 ng/ml LPS.
Quantification of TNF- secretion by U937 cells
Doubling dilutions of LPS were prepared and added to the pooled U937 cells in 96 U wells at 0.1 million cells per well. Cells were incubated for 5 h and cell culture supernatants were removed. Tumour necrosis factor- (TNF-) secretion was detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Biosource, Nivelles, Belgium).
Statistical analysis
Statistical analysis of TNF- secretion by transfectants from the multiple experiments was performed using a non-parametric Friedman test followed by a Dunn post test.
Statistical analysis of quantitative PCR and semiquantitative PCR data was performed using a one-way analysis of variance, followed by a Tukey post test.
Isolation of RNA, global amplification using template-switching PCR, and labelling of cDNA probes
Total RNA was extracted from U937 cell lines using RNA STAT60TM (AMS Biotechnology, Abingdon, UK) (Tel-Test ‘B’ Inc.) and further purified using RNeasyTM columns (Qiagen, Crawley, UK) as described by the manufacturers. cDNA was amplified from total RNA using template-switching PCR and labelled with Cy3 or Cy5 dyes as previously described [18]. The amplified cDNA was purified on an AutoSeq G-50 column (Amersham Biosciences, Little Chalfont, UK) according to the supplied protocol. The amplified cDNA of 1 µg was labelled using the BioPrimeTM DNA labelling system (Invitrogen) in a 100 µl reaction as described by the manufacturer, CyDyeTM5-dCTP or CyDyeTM3-dCTP (Amersham Biosciences). The labelled products were purified on an AutoSeq G-50 column; the Cy5 and Cy3 samples were then pooled and ethanol precipitated.
Hybridization of labelled probes to oligonucleotide arrays
The arrays utilized in this study are the Compugene human 19K oligonucleotide array that has been printed on two glass slides by the Microarray group at the Medical Research Council-Human Genome Mapping Project (MRC-HGMP), Hinxton. Each gene is represented in duplicate spots, genes 1–9500 printed on slide 1 and genes 9501–19 000 on slide 2. Microarray analysis of U937–B27 and U937–control was performed as follows; the different RNA samples were compared the transfectant control (B27 vs C and CLPS vs C) and also B27LPS vs CLPS. For each set of conditions tested, duplicate and dye-swap hybridizations were performed as described previously [19].
Scanning of arrays and data analysis
Arrays were scanned on an Agilent G2565 scanner (Warrington, UK) according to manufacturer's instructions. Raw image data were extracted using BlueFuse (BlueGnome Ltd, Cambridge, UK). Data were imported into GeneSpringTM 7.2 (Agilent) for analysis and normalization was performed using the Loess algorithm. Identification of genes with ratios statistically significantly different from 1 was performed using a t-test at the 95% confidence level. Data were deposited at the GEO repository http://www.ncbi.nlm.nih.gov/geo/info/linking.html, accession number GSE4579
[NCBI GEO]
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Semiquantitative and quantitative real-time PCR
XBP-1, BiP and OAS2 mRNAs were measured using the one-step real-time quantitative PCR and the ABI PRISM 7 (PE Applied Biosystems) according to the manufacturer's instructions.
Relative mRNA levels were determined by the comparative threshold cycle method (user bulletin 2; PE Applied Biosystems, Warrington, UK). The expression of each test gene was normalized to the expression of HPRT mRNA. XBP-1 amplification utilized the Applied Biosystems primer set, HS002931936 (Applied Biosystems, Warrington, UK). Oligonucleotides used for OAS2, BiP and HPRT quantitative PCR are avaliable upon request. Total XBP-1 cDNA was amplified by PCR using the following oligonucleotides spanning the differentially spliced region; 5'-GTTGCTGAAGAGGAGGCGGAAG-3' and 5'-CCAAGTTGTCCAGAATGCCCAAC-3'. U937 cells were incubated in the presence of 2 mM DTT for 4 h to stimulate XBP-1 splicing. Total RNA isolated from these cells was utilized as a positive control for XBP-1 splicing analysis. The relative expression of XBP-1 to the XBP-1 splice variant was determined by electrophoresis of XBP-1 PCR products on 3.5% MS agarose gels (Roche, Lewes, UK). The proportions of the unspliced (186 bp) to spliced (160 bp) products were determined using image analysis software, AlphaEase (Alpha Inotech, USA).
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U937-B27, U937-A2 and U937-control secrete identical quantities of TNF-
following stimulation with LPSTo investigate a possible modulatory effect of HLA-B27 on the monocyte response to LPS, we utilized the monocytic cell line U937 as the host cell line for the transfected HLA molecules. U937 cells were transfected with HLA-B27, HLA-A2 or expression vector alone. To reduce potential artifacts created by the analysis of single transfectants, multiple U937 transfectants from two independent transfections were isolated. The transfectants were pooled (minimum of nine transfectants per pool) immediately prior to differentiation with PMA to enable expression of the CD14 LPS co-receptor. The pooled cell lines expressed the appropriate HLA class I molecules and the control pool was negative for HLA-B27 and HLA-A2 (Fig. 1A). The staining levels of the transfectants are not indicative of the differences in levels of HLA class I between HLA-A2 and HLA-B27, since different antibodies were used with different staining protocols and staining efficiencies. Following PMA differentiation, the cells were responsive to LPS stimulation as detected by the production of TNF-
. The dose response relationship for TNF- secretion in response to LPS stimulation was determined for each pooled cell line (Fig. 2A–C). In addition, we analysed TNF- production by a second pool of independently derived U937 HLA class I transfectants (Fig. 2D). No significant differences were detected between the pooled cell lines with the exception of U937-A2 at the highest concentration of LPS 500 ng/ml, where lines secreted slightly less TNF- compared with the control U937 (U937-C) pooled line (P 
0.05).
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B27 does not modulate the gene expression of differentiated U937 cells
To investigate a direct modulatory effect of HLA-B27 on PMA-differentiated U937 cells, we performed microarray analysis of mRNA expression in the pooled lines. It is important to note that only genes with expression levels significantly different between B27 and control are represented in Fig. 3A (P
0.05). Only five genes from the 19 000 genes analysed exhibited >2-fold increase or decrease compared with control, suggesting that they were differentially regulated by the expression of HLA-B27. This finding was not supported by quantitative PCR analysis of each of the five genes (data not shown), suggesting that B27 does not modulate the gene expression profile of unstimulated U937 cells.
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Comparison of the LPS response signatures in U939 transfectants
Both U937-C and U937-B27 pooled cell lines responded in a stereotypical manner to the LPS stimulation previously described for freshly isolated peripheral blood mononuclear cells, monocytes and monocyte cell lines [12–14]. Only the genes significantly different from 1 are represented on Fig. 3 (i.e. differentially expressed between U937 control and U937-B27). One hundred and eighty-seven of the differentially expressed genes exhibited a 2-fold or greater increase in gene expression compared with the unstimulated cells. The LPS-induced transcripts consisted predominantly of immune activation genes involved in cell-to-cell signalling and proinflammatory mediators that induce systemic effects. These molecules included the cytokines, IL-6, IL-1
and TNF- and molecules involved in chemoattraction and differentiation such as CXCL3, GM-CSF, CCL3, CCL4 and CCL5 (Table 1). Thirty genes were found to be down-regulated more than 2-fold following LPS stimulation, including the previously reported genes such as TGF-ß SLAP and PCM1 [12]. Only two genes were found to be differentially regulated by U937-B27 compared with U937-C following LPS stimulation, Galanin receptor 1 (GalR1) and oligoadenylatesynthease 2 (OAS2). Quantitative PCR analysis corroborated the finding that OAS2 (Fig. 4) but not GalR1 (data not shown) gene expression was significantly different in U937-B27 compared with U937-C (P < 0.05). A second panel of transfectants was prepared and exhibited similar differences in OAS2 mRNA levels following LPS stimulation. However, this effect was not specific to the expression of HLA-B27 since U937-A2 transfectants exhibited similar differences to the U937-C, suggesting that the expression of an additional HLA molecule may modify OAS2 gene expression levels.
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B27 does not modify the unfolded protein response in transfected U937 cells
It has been shown that the UPR is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory diseases. Microarray analysis did not reveal any upregulation of UPR-associated genes such as BiP, CHOP, GADD34, GRP94, TRIB3 and XBP-1 in U937-B27. Quantitative PCR, which is a more sensitive and accurate measure of relative mRNA levels, was also used to analyse BiP expression and investigate more subtle differences between the transfectants (Fig. 4). U937 cells were treated with DTT to induce a UPR and were used as a positive control for PCR analysis. No differences in gene expression of BiP were detected between the transfectants. As an alternative sensor of the UPR, we analysed the expression of total XBP-1 (Fig. 5A) and an XBP-1 splice variant that has a 26 nucleotide region excised by IRE1 following the stimulation of UPR [20]. The amount of the splice variant can be expressed as a proportion of total XBP-1 as shown in Fig. 5B. Although total XBP-1 and the XBP-1 splice variant from all U937 transfectants showed a significant increase following LPS stimulation (P < 0.05), all U937 transfectants showed a similar response to LPS. In addition, no differences in XBP-1 splicing between the transfectants were observed at the later time point of 8 h (data not shown).
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Discussion |
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In this study, we investigated whether HLA-B27 modulates the innate immune response to LPS. Although the monocytic cell line, U937, exhibited a stereotypical response to LPS, we did not detect differences in gene expression when U937 was transfected with HLA-B27 as compared with the transfection with HLA-A2 or control vector. These results suggest that HLA-B27 does not modify the cellular environment to the extent that changes in gene transcription in U937 cells following LPS stimulation could be detected.
Penttinen et al. [15] suggested that HLA-B27 enhances NF-B activation following the stimulation of U937 with LPS. Genes known to be responsive to NF-B activation, such as pro-inflammatory cytokines, chemokines, toll-like receptors, signalling proteins and enzymes and molecules with microbicidal activity (reviewed in [21]) were well-represented in our analyses. Therefore, any regulatory effects of HLA-B27 on NF-B signalling should have been readily detectable, but in fact no differences were observed. Comparable quantities of TNF- with those reported by Penttinen et al. were secreted by the U937 cells in our study suggesting that a similar degree of stimulation by LPS was achieved. Methodological differences in expression constructs and the origin of the U937 cells used may account for these discrepancies.
HLA-B27 exhibits a number of unusual characteristics, including an increased propensity to misfold and form heavy chain homodimers [22]. It has been suggested that aberrant folding of HLA-B27 results in the stimulation of the UPR [5]. The potential involvement of the UPR in HLA-B27-associated disease has been highlighted recently in a study that demonstrated an increase in the UPR in bone marrow macrophages from B27 transgenic rats with inflammatory disease [16]. No differences in the UPR were detected between the long-term transfectants, which is in agreement with Penttinen et al. [9], although a UPR has been detected in cells transiently transfected with HLA-B27 [21]. This raises the question whether the U937 stable transfectants have adapted to HLA-B27 expression. Although the U937 cells exhibited a stereotypical macrophage response to LPS, it is important to consider that the U937 cells may not respond in the same manner as bone marrow-derived rat macrophages since they are transformed cells. Futhermore, the expression of B27 was not responsive to proinflammatory stimuli since the native HLA-B27 promoter was not present. Future studies investigating the effects of high level HLA-B27 expression in U937 cells could reveal different responses to other HLA transfectants. Although we identified a significant increase in the UPR following LPS stimulation of U937 cells, detected by an increase in both total XBP-1 expression and the XBP-1 splice variant, no HLA-B27-specific effect was observed. The lack of a detectable UPR in pre-morbid HLA-B27 rats suggests that the ability of B27 to exert this effect may be complex, and require additional factors such as those provided by intracellular bacteria infection or exogenous cytokines such as interferon-
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Whilst HLA-B27 expression may influence the nature of the response to intracellular infection, this study shows that the influence is likely to be rather subtle, since there was no modification of the response to LPS. A recent study has highlighted the synergy which can occur when different toll-like-receptors (TLRs) are stimulated on DC at the same time (e.g. LPS via TLR4 and CpG DNA via TLR9) [23]. Therefore, it remains possible that HLA-B27 might influence these interactions and synergistic responses.
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JCG is supported by a non-clinical fellowship from the Arthritis Research Campaign. This work was funded by the Arthritis and Research Campaign and the Medical Research Council. G.S.H.Y is supported by the Welcome Trust.
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