Organ Messenger Ribonucleic Acid and Plasma Proteome Changes in the Adjuvant-Induced Arthritis Model: Responses to Disease Inducti
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《内分泌学杂志》
Biological Technologies (M.T.F., L.W., A.J.D.) and Cardiovascular and Metabolic Diseases (J.C.K.), Wyeth Research, Cambridge, Massachusetts 02140
Caprion Pharmaceuticals (M.P., D.C., C.H., P.K., P.T., E.P.), Montreal, Quebec, Canada H4S 2C8
Women’s Health Research Institute (H.A.H.), Wyeth Research, Collegeville, Pennsylvania 19426
Abstract
Two receptors [estrogen receptor (ER) and ER] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ER in the classical effects of estrogen activity, the physiological role of ER is less well understood. A small-molecule ER selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.
Introduction
ALTHOUGH WELL ACCEPTED as obligatory for sexual development and function, estrogen receptors (ERs) are now appreciated to function in many nonreproductive tissues. Two ERs have been characterized and are known to act as ligand-activated transcription factors. Other, more rapid actions of estrogens have also been described (1, 2), although their role in mediating estrogens’ functions is less well understood. Studies with knockout mice or receptor-selective ligands show a clear role for ER in mediating estrogens’ activity in classic models such as uterine stimulation, skeletal maintenance, vasomotor instability, and ovulation (3, 4, 5). The physiological role of ER is less well understood, but recent work suggests a role for ER in modulation of immune system function (6, 7, 8, 9).
Nonselective estrogens, such as 17-estradiol and 17-ethinyl-17-estradiol, have long been appreciated to affect the immune system (reviewed in Refs.10 and 11). In some situations these compounds are beneficial, such as in the mouse experimental autoimmune encephalitis model in which estradiol (acting via ER) delays the onset of disease and reduces symptoms (12, 13). In other models such as systemic lupus erythematosus, however, estrogens negatively impact disease (14, 15). In addition, there is a large body of work showing that estrogens affect several immune system cell types such as macrophages, natural killer (NK) cells, T cells, and B cells. Therefore, the effects of estrogen on the immune system in vivo are complex and likely mediated via several cell types.
Numerous suggestive, but not definitive, preclinical and clinical links can be made between estrogens and arthritis. For example, an ER antagonist worsened disease in a mouse collagen-induced arthritis model (16), and idoxifene (a selective ER modulator) improved disease symptoms in rat adjuvant-induced arthritis (AIA) (17). In humans, conditions of high estrogens, including current oral contraceptive use, may be protective from developing rheumatoid arthritis (RA) (18), and RA remits in the majority of women during pregnancy (19). In addition, the peak incidence of RA occurs after menopause and estrogen/progestin hormone therapy ameliorates disease (20). Finally, analysis of clinical trial adverse events shows that letrozole (an aromatase inhibitor designed to suppress estrogen production) caused a significant increase in arthritis (21). It should be noted that literature reports are not unanimous on the subject and that factors other than estrogens (e.g. progestins) might explain some the results cited above.
ERB-041 is a selective ER agonist that binds to ER with more than 200-fold higher affinity relative to ER. It is inactive in several classic models of estrogen activity but has potent antiinflammatory activity in the HLA-B27 transgenic rat and the Lewis rat model of AIA (9). Because the inflammatory process is a systemic response and the target cell(s) for ERB-041 action in this model are unknown, we chose to monitor global mRNA changes in three organs (liver, popliteal lymph node, and spleen) as well as changes in the plasma proteome. The analysis allows an unbiased survey of complete Freund’s adjuvant (CFA)-induced and compound-mediated changes in compartments likely to be affected by disease. We find that the expression of a large number of genes and proteins are altered in the disease model and that the majority of these are partially or fully reversed by ERB-041. In addition to demonstrating the profound antiinflammatory effect that ERB-041 has in this model, this analysis identified biomarkers previously associated with RA (thus validating the system) as well as novel biomarkers requiring further study.
Materials and Methods
Lewis rat AIA
This model was performed essentially as previously described (9) with six rats/group. Briefly, 12-wk-old, gonad-intact, male Lewis rats were injected intradermally on the ventral side of the base of the tail with CFA. The degree of arthritis severity was monitored daily and scored according to the following disease indices: hindpaw erythema, hindpaw swelling, tenderness of the joints, and movements, and posture. An integer scale of 0 to 3 is used to quantify the level of erythema (0, normal paw; 1, mild erythema; 2, moderate erythema; 3, severe erythema) and swelling (0, normal paw; 1, mild swelling; 2, moderate swelling; 3, severe swelling of the hindpaw). The maximal possible score per day is 12, and this score was seen in all rats 8 d after CFA injection.
A cohort of Lewis rats not injected with CFA but receiving daily oral doses of vehicle (2% Tween 80/0.5% methylcellulose) for 21 d were designated normal controls. CFA-treated rats were dosed with vehicle for 7 d until full joint inflammation developed and then were randomized into two groups and treated for another 14 d. One group continued to be dosed with vehicle, designated CFA hereafter. The other group was treated with daily oral doses of ERB-041 (5 mg/kg), designated CFA + ERB-041. The daily joint scores for each rat treated with ERB-041 are shown in supplemental Fig. 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). At necropsy on d 21, blood was collected via cardiac puncture for serum as well as plasma preparation. The liver, popliteal lymph node, and spleen were excised and frozen on dry ice for RNA preparation. All procedures involving animals were conducted under institution-approved protocols. ERB-041 was obtained from the Wyeth compound library (22).
Expression profiling of organ mRNA
RNA preparation and microarray analysis.
Rat tissue RNA was purified using a Total RNA kit (Promega, Madison, WI). Each RNA sample was prepared individually (no samples were pooled) and hybridized to a different microarray chip (total number of chips used was 54). Amplification of cRNA, RNeasy spin column purification (QIAGEN, Valencia, CA), and cRNA fragmentation were performed as described (10). Standard curve normalization and conversion to RNA frequencies was as described (23, 24). cRNA was hybridized to REA230A oligonucleotide arrays (Affymetrix, Santa Clara, CA), stained with Streptavidin R-phycoerythrin (Molecular Probes, Eugene, OR) using the GeneChip Fluidics Station 400, and scanned with a GeneArray scanner (Agilent, Palo Alto, CA) following the manufacturer’s instructions. The REA230A oligonucleotide array contains 15,878 tiled probe sets that interrogate known rat genes and expressed sequence tags in addition to 45 bacterial control sequences enabling measurement of a spiked-in standard curve (24). Data were collected using MicroArray Suite 4.0 software (Affymetrix, Santa Clara, CA). Individual frequencies measured for each of the experimental groups were averaged and analyzed by pairwise comparison. Genes were initially selected by both average fold change (>2-fold) and Student’s t test (P < 0.01) criteria. In addition to magnitude of gene expression as reflected by average perfect match to mismatch oligonucleotide fluorescence, an independent algorithm provides an absolute present/absent call for each probe set as described in Lockhart et al. (23). The genes selected by the fold-change and statistical criteria were subsequently filtered to remove genes with low expression (average < 5 ppm) or that were not consistently called present within the entire cohort in which the gene was induced.
Proteomic analysis.
Plasma samples from 13 rats (four normal, four CFA, and five CFA + ERB-041) were selected for proteomic analysis. These were chosen without bias except that samples with high hemoglobin content (from hemolysis during collection) were excluded. These were depleted of albumin and immunoglobulins using Montage albumin depletion kits (Millipore, Billerica, MA) and protein G resin (Amersham, Piscataway, NJ). Depleted plasma quantification was performed with a Micro BCA kit (Pierce Chemicals, Rockford, IL). Forty micrograms of each depleted plasma sample were fractionated by electrophoresis in duplicate on 12% Bis-Tris NuPAGE minigels (Invitrogen, Carlsbad, CA) using 3[N-morpholino]propanesulfonic acid running buffer (Invitrogen). Gels were cut into 24 equal bands using a fixed razor blade assembly. The bands were placed into 96-well plates for oxidation and trypsin digestion, peptides were extracted from the gel bands with 0.2 M urea/50% acetonitrile, and vacuum dried. Peptides derived from the 24 bands were resolubilized in 10% acetonitrile and 0.2% trifluoroacetic acid. Peptides from adjacent bands were combined to create 10 pooled fractions of similar peptide number.
Each of the 10 fractions was analyzed on a single LC-MS instrument (capillary reverse-phase liquid chromatography coupled by electrospray to a QTOF Ultima mass spectrometer; Waters, Milford, MA). Samples were randomized, and the first fraction of all samples was completed before moving on to the next fraction. Detected peptide ions in each fraction were matched across all 18 samples by mass, charge, and retention time. Maximum peak intensity for each matched peptide ion was measured and the mean peak intensity across each group (normal, CFA, and CFA + ERB-041) was determined. The ratio of intensity for a given peptide between groups, IB/IA, was used to derive the relative abundance of the parent protein, AB/AA, using the experimentally derived relationship: AB/AA = 1.6 (IB/IA). The pairwise matching rate for each peptide across serial injections of the same sample was 98%. Under the same conditions, the median coefficient of variation of peak intensity across all peptide ions was 12%. If a plasma sample was divided and the replicates processed in parallel, the median coefficient of variation of intensity for any given peak was 27%.
Peptides showing a consistent differential abundance between CFA and normal groups of 5-fold or more were selected for sequencing. Replicate peptide digests were analyzed by capillary reverse-phase liquid chromatography by electrospray to a QTOF Ultima mass spectrometer operating in tandem mass spectrometry mode to obtain a fragmentation pattern for each selected peptide. Mascot (MatrixScience, Boston, MA) was used to match fragmentation spectra to the National Center for Biotechnology Information nonredundant protein database to obtain peptide sequences. Peptides with Mascot scores of 25 or above were clustered by parent protein and reported. A manual audit of the data supporting 53 representative peptides was performed. This quality control step checked for correct matching of peptides across all samples and assignment of peak intensity as well as sequence interpretation and association with the differentially expressed peptide.
Analysis of serum haptoglobin and orosomucoid.
Serum haptoglobin levels were measured using a kit from TriDelta Development Ltd. (Dublin, Ireland) according to the manufacturer’s directions except that a kinetic rather than an end point measurement was used. This assay measures the peroxidase activity of hemoglobin that is preserved at low pH when haptoglobin is present. The standard curve was linear from 0 to 2 mg/ml and all samples were within this range. The measured value for CFA rats treated with vehicle was consistent with our historical unpublished values.
Orosomucoid was measured by a radial immunodiffusion assay (Life Diagnostics, West Chester, PA) using 10 μl of undiluted serum. Precipitin rings were developed by incubation of the plates at room temperature for 48 h, and a standard curve was constructed by plotting the square of the precipitin ring diameter vs. orosomucoid concentration. The standard curve was linear from 60 μg/ml to 1 mg/ml and all samples were within this range.
Results
ERB-041 reduces joint swelling
Consistent with previous findings (9), CFA-treated rats developed maximally inflamed paws within 8 d after intradermal adjuvant injection. These rats had joint scores of 12 throughout the study, compared with joint scores of 0 for normal rats. ERB-041 treatment was initiated on d 8, which reduced the joint scores to approximately 2 after 4 d and stabilized scores at approximately 1 after 10 d of treatment (Fig. 1).
Expression profiling of liver, lymph node, and spleen
CFA-induced gene expression and response to ERB-041 therapy was monitored in three tissues of immunological relevance, the spleen and lymph node as direct sites of immune cell activity, and the liver as indirect responder to inflammation. Tissue RNA from each rat (n = 6) per treatment group was independently hybridized to Affymetrix REA230A oligonucleotide arrays. Average fold change (AFC) values were computed from mean frequency values comparing baseline and CFA-treated animals. CFA-regulated genes were selected by combined magnitude (AFC > 2-fold) and statistical significance (P < 0.01) parameters. The overall transcriptional responses are summarized in Table 1. Selected genes regulated by CFA treatment and their responses to ERB-041 are highlighted in Table 2. A comprehensive list of known genes is shown in supplemental Tables 1–5 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Variability among genes detected in each tissue but not judged as regulated is shown in supplemental Table 6 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The coefficient of variation of majority of genes in each tissue is less than 30%.
In the spleen, expression of 31 genes was increased by CFA treatment with AFCs ranging from 2- to 5-fold with no decreased gene expression identified. Regulated genes include immune and inflammatory mediators, innate immune-associated proteins, transcription factors, and proteases (supplemental Table 1). Graphical representation of the spleen CFA induced gene expression (Fig. 2A) demonstrates nearly complete resolution after ERB-041 treatment. The overall mean induction of the 31 CFA-induced genes was decreased 72% by ERB-041 treatment. As summarized in Table 1, 18 of the gene transcripts demonstrated greater than 70% reversal of expression with an additional 12 genes moderately reduced (30–70%) after ERB-041 treatment. In all, 29 of 31 genes induced in the CFA-disease model demonstrated a statistically significant reversal (P < 0.05) in response to ERB-041.
Lymph node mRNA demonstrated the most dramatic CFA-induced transcriptional response with expression of 148 genes increased 2- to 15-fold, and 155 genes decreased 2- to 12-fold. Transcripts up-regulated in the lymph node with CFA treatment included genes associated with an immune response in addition to genes representing a wide range of biological processes such as proliferation, protein secretion, cell adhesion, and metabolism (supplemental Table 2). The CFA-repressed genes demonstrated a striking decrease in metabolism-associated genes, 16 of which are directly related to lipid homeostasis (supplemental Table 3). The CFA-modulated gene expression in lymph node was ameliorated by ERB-041 treatment but, in general, did not completely return to normal levels (Fig. 2A). Of the CFA-induced lymph node gene expression, there was an overall 51% mean reduction after ERB-041 treatment with 36 genes reduced by greater than 70%, whereas the majority (84 genes) showed moderate reduction (30–70%). Similarly, ERB-041 treatment restored expression of CFA-repressed genes on average by 42%, with the majority of genes (n = 116) showing moderate level of recovery with only seven genes returned to near basal levels.
Liver gene expression responded to CFA treatment with 48 genes induced (2- to 49-fold), and 22 genes decreased with a range of 2- to 6-fold. As with the other tissues profiled, a majority of the CFA-induced genes have established roles in immune and acute phase responses (supplemental Table 4). Similar to spleen, the liver CFA-induced genes demonstrated nearly complete reversal after ERB-041 treatment (Fig. 2A), with a mean reduction of 82% of CFA-induced expression and statistically significant reduction (P < 0.05) in 42 of the 48 genes. In contrast, ERB-041 response for the CFA-repressed genes demonstrated a bimodal response with the expression of nine genes being nearly completely restored, whereas eight genes showed little or no response to drug and only five genes demonstrating an intermediate response. For the CFA-repressed gene set, there was a tendency for complete ERB-041-mediated recovery within genes associated with metabolism and ion transport, whereas genes associated with signal transduction and proliferation were generally less responsive (supplemental Table 5).
Plasma proteome analysis
Plasma peptides were matched across all samples according to their mass to charge ratio, chromatography retention time, charge, and peak intensity. A total of 18,567 peptides were matched and reproducibly observed. Pairwise comparison of peak intensities between groups for each of these study peptides identified 1632 with differential expression between at least two of the groups (P < 0.05). Thus, the robust joint swelling observed in the animals (Fig. 1) resulted in a change to only a small portion (9%) of the detectable plasma proteome. Graphical representation of the plasma concentrations of the disease-associated peptides (Fig. 2B) demonstrates a general reversal to normal levels after ERB-041 therapy coordinate with the tissue gene expression.
Of 61 disease-associated proteins (170 peptides) identified with high confidence by targeted tandem mass spectrometry, 42 (138 peptides) were selected as candidate biomarkers (Table 2) with specific peptides and concentrations detailed in supplemental Table 7 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Of these, 19 proteins increased in plasma abundance after CFA treatment, and 24 decreased. Nearly all (38 of 42) reverted by more than 50% toward control levels after ERB-041 treatment, with 26 proteins reverting by more than 85%. Only two were apparently unaffected by drug.
The identified plasma disease-related protein increases included positive acute phase proteins, complement proteins C3/C9, and both fibrinogen- and - chains. In addition to negative acute phase proteins, decreased hemoglobin- and - chains and four components of high-density lipoprotein (HDL) complexes [apolipoprotein (Apo)A1, ApoAIV, ApoM, and glycosyl-phosphatidylinositol-linked phospholipase D] were identified. The expression patterns of two proteins, orosomucoid and haptoglobin, were independently confirmed by standard biochemical assays (Fig. 3). The values for the change in abundance in response to CFA and the subsequent reversion after treatment with ERB-041 matched very closely between the biochemical assays and the mass spectrometer for both proteins.
Coordinate changes at the mRNA level in tissue were observed for four of the 42 differentially abundant plasma proteins. Statistically significant increases in haptoglobin, complement C3, -2-macroglobulin (A2M), and kininogen tissue mRNA expression are reported in Table 2. In addition, transcripts for several of the serum proteins reported in Table 2 demonstrated similar changes but were not included in the CFA-modulated gene set by virtue of the dual fold-change and statistical criteria chosen for inclusion. For example, liver mRNAs encoding ceruloplasmin increased 2-fold (P = 0.06), whereas orosomucoid increased 1.5-fold (P < 0.01). Similarly, liver expression of ApoAI decreased 1.4-fold (P < 0.01). Although consistent with the direction of change in the plasma proteins, the mRNA transcripts were measured only in a subset of the potentially responsive tissues and thus may not always fully reflect the overall magnitude serum protein changes. Kininogen and A2M tissue mRNA and serum peptides are illustrated in Fig. 4 as an example of where direct concordance between the transcriptional and proteomic profiling was observed.
Discussion
The discovery of a second form of the ER led to much optimism about its therapeutic utility (25), but translating its attractive tissue distribution into a clinical indication has proved a difficult task. The design of highly ER subtype selective ligands has allowed the dissection of ER- and ER-mediated activities (4, 9, 26, 27) and led to the discovery that selective ER activation reduces inflammation in two preclinical models. In the Lewis rat model of arthritis, for example, clinical signs are rapidly normalized over the course of 6–10 d of treatment, and histological scores (synovitis and Mankin) are significantly improved (9). Nonselective estrogens, such as 17-estradiol, have long been appreciated to affect the immune system (10, 11), yet the effects of estrogens are complex and likely mediated via several mechanisms involving multiple cell types.
RA is a systemic autoimmune disease of unknown etiology characterized by inflammation of the joint synovium leading to pain and gradual erosion of joint tissue. Several adjuvant- or antigen-induced disease models have been developed in rodents and share phenotypic responses common to the human disease. Increasingly in animal models, global proteomic or transcriptional profiling is being used to identify possible biomarkers of disease and drug response for clinical application. In the present study, the tissue transcriptional profiles of CFA-induced gene expression in the Lewis rat AIA model identify multiple genes and biological processes that would be anticipated in the course of an inflammatory response. In the tissues profiled, genes associated with both innate and adaptive immune function as well as direct immune mediators are induced. In addition, multiple genes associated with biological processes including proliferation, oxidative stress, protein trafficking and turnover, metabolism, adhesion and migration, and proteolysis show increased gene expression in the CFA-induced disease model. All of the tissues profiled demonstrated a clear therapeutic response to ERB-041 treatment with reversal of the majority of disease-associated transcription, consistent with amelioration of disease manifestations. Spleen and liver disease-associated transcription was substantially resolved at a time point at which the animals demonstrate complete phenotypic resolution of disease symptoms, whereas lymph node transcription was typically 50% reversed.
Included in the tissue expression profiles are several genes associated with increased eicosanoid synthesis, proinflammatory agents long implicated in RA pathophysiology. CFA stimulation increased secreted phospholipase A2 (sPLA2-IIA) mRNA 12-fold in lymph node and to a lesser degree in spleen. sPLA2 hydrolysis of phospholipids releases arachidonic acid, the precursor for prostaglandin and leukotriene synthesis. Increased sPLA2 has been identified in RA synovial fluid, and serum levels correlate with disease severity (28). Administration of a sPLA2-inhibitory peptide reduced bone erosion and cartilage destruction in a TNF transgenic model of disease (29). Increased 5-lipoxygenase-activating protein in the spleen mRNA profile further suggests activation of leukotriene arm of arachidonate metabolism, of which leukotriene B4 is most closely associated with RA. Mice deficient in 5-lipoxygenase-activating protein, required for presentation of arachidonic acid to 5-leukotriene oxidase, exhibit decreased disease activity in collagen-induced arthritis models (30). Additionally, increased expression of leukotriene C4 synthase is observed in the CFA-induced liver profile. In human RA patients, methotrexate therapy decreases neutrophil production of products downstream of 5-lipoxygenase, suggesting that inhibition of this pathway correlates to therapeutic benefit (31).
Anemia of chronic disease (ACD) is a frequent complication resulting from the chronic cycles of inflammation in RA. Many studies have focused on the role of TNF and its effect on erythropoiesis (32), yet a subgroup of ACD patients (46%) present with a coexistent iron deficiency (33). The proteomic and transcriptional profiles identify changes in both globin expression and genes central to iron homeostasis in the course of inflammatory responses. The plasma proteomic analysis identified a decrease in both globin- and globin- in response to CFA. LCN2/NGAL, a neutrophil-expressed lipocalin, has recently elucidated roles in both iron transport (34) and binding of iron siderophores as part of innate immune antimicrobial defense mechanism (35). LCN2 gene expression is dramatically increased in all three tissues profiled after CFA disease induction and demonstrates complete attenuation with ERB-041 treatment. The induction of iron-sequestering proteins as a protective response to infection may have pathologic consequences in chronic inflammatory diseases such as RA resulting from long-term alteration of iron homeostasis. At present there is no association of LCN2 in RA, but its role in iron homeostasis, expression by activated neutrophils, and induction in the Lewis rat AIA model suggest LCN2 as a candidate gene for further investigation in the etiology of ACD.
CFA-induced expression of both S100A8 and S100A9 was observed in all three of the tissue profiles. S100A8/A9 are members of the EF-hand homology family of calcium-binding proteins predominately expressed in neutrophils and monocytes. S100A8/A9 form both homodimers and heterodimers and have been implicated in a wide range of granulocyte activities including cytoskeletal rearrangements; arachidonate metabolism; and regulation of neutrophilic nicotinamide adenine dinucleotide phosphate reduced oxidase, adhesion, and transendothelial migration (36). Concentrations of S100A8/A9 reach 50% of soluble cytosolic protein in granulocytes and are released by a protein kinase C-dependent, nonclassical secretory process after contact with activated endothelium (37). Increased concentrations of S100A8/A9 complexes in both synovial fluid and blood have been identified as a biomarker of RA by a number of independent studies (38) and is correlated both to disease activity (39) and drug response (37, 40). The increase in both S100A8 and S100A9 mRNA in all three tissues profiled, ranging 2.4- to 13-fold, was consistently decreased 51–93% after ERB-041 treatment. The strong disease association and drug response in the animal model identify these S100 proteins as candidate biomarkers in clinical evaluation of ERB-041 in the treatment of RA.
The plasma proteomic profile of the Lewis rat AIA model identified a number of proteins altered in the course of CFA disease induction and amelioration in response to the ER agonist. As has been shown in a variety of global proteomic disease studies, the predominant response in blood plasma was an acute-phase response. Two of the proteins identified, A2M and T-kininogen, may serve as clinical biomarkers due to their direct correlation with RNA changes in liver gene expression, strong disease association, and full normalization by ERB-041. A2M functions both as a broad-spectrum protease inhibitor and oxidation-dependent regulator of cytokine activity (41). Carriers of a 5-bp deletion allele in the A2M gene have an increased risk of developing an early active severe form of RA (42). A2M mRNA is dramatically induced in the rat liver by CFA. Protein is detected in plasma by 34 independent peptides, 15 of which were validated by manual inspection of the data. Induction of liver mRNA and plasma protein is completely reversed after ERB-041 treatment. T-kininogen, measured in both liver mRNA profiles and plasma proteomics, follows a similar pattern as A2M and also identifies a protein of pathophysiological importance to RA that may be a potential drug-responsive clinical biomarker. Kininogens serve as substrate for kallikrein in the biosynthesis of vasoactive and proinflammatory kinins that have been associated with edema, pain, and proinflammatory responses (43). Direct inhibition of kallikrein activity or decreasing kininogen by monoclonal antibody titration provides therapeutic benefit in a Lewis rat models of reactive arthritis (44).
Several of the plasma proteomic changes identified in this animal model can be directly related to RA pathophysiology. We observed peptides representing both complement C3 and C9 increase, which is consistent with increases in C3- and C9-containing circulating immune complexes in serum and synovial fluid of RA patients (45). Both the - and -chains of fibrinogen increase in the rat model plasma. Elevated fibrinogen can enhance intercellular adhesion molecule and cytokine expression in synovial fibroblasts (46) and increase red blood cell adhesiveness/aggregation (47). Four proteins associated with HDL complexes decrease in rat plasma after CFA-induced disease. ApoA1 is a principal protein of HDL, and glycosylphosphatidylinositol-phospholipase D associates with ApoAI/ApoAIV-containing complexes (48). ApoAIV itself displays antiinflammatory activity in experimental colitis models (49). Lipid profiles of RA blood demonstrate decreased ApoA1-containing HDL complexes that are increased with successful treatment (50). The coordinate decrease in these proteins and 54–87% reversal by ERB-041 highlight an additional set of set of disease-related biomarkers to evaluate clinical activity.
Expression of a number of transcription factors was induced in the CFA disease model and was attenuated by ERB-041 treatment. CCAAT/enhancer-binding protein (C/EBP)-, C/EBP, or both were induced in all three tissues surveyed. Signal transducer and activator of transcription (STAT)-1, STAT-2, and STAT-3 were also induced in the lymph node. Janus kinase-3 and STAP-2, modulators of STAT activation, (51), were induced in the liver. C/EBP family members play a pivotal role in transcription of several of the inflammatory mediators and acute-phase proteins (52), many of which were identified in the CFA-induced tissue expression profiles. STAT1/STAT2 and STAT3, critical to interferon- and IL-6 signaling, respectively, are key to inflammation and inflammatory diseases (53) and responsible for IL-6-mediated up-regulation of C/EBP (54). Evidence of STAT1 activation in a subset of RA patients has been reported and may reflect an important parameter for stratification of clinical disease (55). ERB-041 treatment reduced each of these CFA-induced transcription factors and modulators by 51–100%. Whereas global gene or protein expression studies cannot in themselves reveal mechanism, the notable drug response in several transcription factors, in themselves fundamentally associated with immune and acute phase responses, indicates that the ERB-041 therapeutic activity in the Lewis rat AIA model lies at the core of the inflammatory response as opposed to modulation of symptomatic responses alone.
As can be seen from both the gene expression and plasma proteome data presented in this study, ERB-041 appears to act in an immunomodulatory fashion, dampening the inflammatory response evoked by the CFA stimulus without exerting a generalized immunosuppressive action. It is interesting to note that some immune-associated gene expression and plasma proteins are completely unaffected and in some cases are augmented over the response evoked by CFA treatment/vehicle treatment. For example, granzyme and NK cell group 7 sequence, two NK T cell transcripts, increase 3- and 4-fold, respectively, with CFA. The latter is unchanged by ERB-041 treatment, whereas granzyme B is increased an additional 75% over CFA by the administration of ERB-041 (Table 2). Although granzyme B contributes to the cytotoxic effects of NK T cells, the increased expression may represent the expansion of the NK T cell population. NK cells can also be immunoregulatory in nature. The NK cell group 7 sequence found on both NK cells and macrophages acts as an inhibitory membrane receptor (56). Recently NK cell dysfunction, i.e. deficiency, has been reported in patients with systemic onset juvenile RA (57). These patients exhibited a marked decrease in NK cell function and an absence of the CD56bright NK cell population, the immunoregulatory NK cell. It is interesting to note that this CD56bright NK cell population is expanded in both the decidua and peripheral blood of pregnant women and is thought to actively modulate the maternal immune response to the fetal allograft (58). As mentioned in the introductory text, RA often remits during pregnancy (19).
Fetuin, -fetoprotein, another pregnancy-associated negative acute-phase protein, is elevated 3-fold by CFA in the plasma and is unchanged by ERB-041. Fetuin has several functions in plasma and the body tissues. It can act as an inhibitor of dystrophic calcification (59), important in progression of the arthropathy of RA and perhaps contributing to the increased incidence of cardiovascular mortality in RA. In addition, fetuin may contribute to the immunomodulatory effects of ERB-041 by changing the expression of Fas/Fas ligand and TNF-related apoptosis-inducing ligand and its receptor, the net effect being a down regulation of the immune response and increased tolerance to the presence of neoantigens (60).
In summary, we show that Lewis rat AIA model bears similarity to human RA in tissue gene expression and plasma proteins that change during disease. Many of the dysregulated genes and proteins have well-established pathophysiological roles. The two data sets were highly concordant in the general response to disease and drug as well as complementary in that most changes were uniquely identified by a single technology. These observations highlight the importance of an integrated transcriptional and proteomic approach. ERB-041 significantly attenuates the majority of disease-induced changes, and thus, these data identify biomarkers that can be developed to monitor disease severity and drug response in clinical studies. Most importantly, these data support the possibility that ER agonists represent a new class of clinically useful antiinflammatory agents.
Acknowledgments
The authors thank Leo Albert and Yelena Leathurby for assistance with the Lewis rat adjuvant-induced model, Sean McLarney for the serum haptoglobin analysis, and Chris Chadwick for the orosomucoid analysis. Orest Hurko, Al Kotake, Don Raible, Bill Jacobson, and Darryl Webster provided valuable project support. Andrew Hill provided advice for the statistical analyses. We also thank Rick Winneker for helpful discussions about the project and manuscript.
Footnotes
The authors are full-time employees of the institutions listed on the title page.
First Published Online November 3, 2005
Abbreviations: ACD, Anemia of chronic disease; AFC, average fold change; AIA, adjuvant-induced arthritis; A2M, -2-macroglobulin; Apo, apolipoprotein; C/EBP, CCAAT/enhancer-binding protein; CFA, complete Freund’s adjuvant; ER, estrogen receptor; HDL, high-density lipoprotein; NK, natural killer; RA, rheumatoid arthritis; sPLA2, secreted phospholipase A2; STAT, signal transducer and activator of transcription.
Accepted for publication October 26, 2005.
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Caprion Pharmaceuticals (M.P., D.C., C.H., P.K., P.T., E.P.), Montreal, Quebec, Canada H4S 2C8
Women’s Health Research Institute (H.A.H.), Wyeth Research, Collegeville, Pennsylvania 19426
Abstract
Two receptors [estrogen receptor (ER) and ER] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ER in the classical effects of estrogen activity, the physiological role of ER is less well understood. A small-molecule ER selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.
Introduction
ALTHOUGH WELL ACCEPTED as obligatory for sexual development and function, estrogen receptors (ERs) are now appreciated to function in many nonreproductive tissues. Two ERs have been characterized and are known to act as ligand-activated transcription factors. Other, more rapid actions of estrogens have also been described (1, 2), although their role in mediating estrogens’ functions is less well understood. Studies with knockout mice or receptor-selective ligands show a clear role for ER in mediating estrogens’ activity in classic models such as uterine stimulation, skeletal maintenance, vasomotor instability, and ovulation (3, 4, 5). The physiological role of ER is less well understood, but recent work suggests a role for ER in modulation of immune system function (6, 7, 8, 9).
Nonselective estrogens, such as 17-estradiol and 17-ethinyl-17-estradiol, have long been appreciated to affect the immune system (reviewed in Refs.10 and 11). In some situations these compounds are beneficial, such as in the mouse experimental autoimmune encephalitis model in which estradiol (acting via ER) delays the onset of disease and reduces symptoms (12, 13). In other models such as systemic lupus erythematosus, however, estrogens negatively impact disease (14, 15). In addition, there is a large body of work showing that estrogens affect several immune system cell types such as macrophages, natural killer (NK) cells, T cells, and B cells. Therefore, the effects of estrogen on the immune system in vivo are complex and likely mediated via several cell types.
Numerous suggestive, but not definitive, preclinical and clinical links can be made between estrogens and arthritis. For example, an ER antagonist worsened disease in a mouse collagen-induced arthritis model (16), and idoxifene (a selective ER modulator) improved disease symptoms in rat adjuvant-induced arthritis (AIA) (17). In humans, conditions of high estrogens, including current oral contraceptive use, may be protective from developing rheumatoid arthritis (RA) (18), and RA remits in the majority of women during pregnancy (19). In addition, the peak incidence of RA occurs after menopause and estrogen/progestin hormone therapy ameliorates disease (20). Finally, analysis of clinical trial adverse events shows that letrozole (an aromatase inhibitor designed to suppress estrogen production) caused a significant increase in arthritis (21). It should be noted that literature reports are not unanimous on the subject and that factors other than estrogens (e.g. progestins) might explain some the results cited above.
ERB-041 is a selective ER agonist that binds to ER with more than 200-fold higher affinity relative to ER. It is inactive in several classic models of estrogen activity but has potent antiinflammatory activity in the HLA-B27 transgenic rat and the Lewis rat model of AIA (9). Because the inflammatory process is a systemic response and the target cell(s) for ERB-041 action in this model are unknown, we chose to monitor global mRNA changes in three organs (liver, popliteal lymph node, and spleen) as well as changes in the plasma proteome. The analysis allows an unbiased survey of complete Freund’s adjuvant (CFA)-induced and compound-mediated changes in compartments likely to be affected by disease. We find that the expression of a large number of genes and proteins are altered in the disease model and that the majority of these are partially or fully reversed by ERB-041. In addition to demonstrating the profound antiinflammatory effect that ERB-041 has in this model, this analysis identified biomarkers previously associated with RA (thus validating the system) as well as novel biomarkers requiring further study.
Materials and Methods
Lewis rat AIA
This model was performed essentially as previously described (9) with six rats/group. Briefly, 12-wk-old, gonad-intact, male Lewis rats were injected intradermally on the ventral side of the base of the tail with CFA. The degree of arthritis severity was monitored daily and scored according to the following disease indices: hindpaw erythema, hindpaw swelling, tenderness of the joints, and movements, and posture. An integer scale of 0 to 3 is used to quantify the level of erythema (0, normal paw; 1, mild erythema; 2, moderate erythema; 3, severe erythema) and swelling (0, normal paw; 1, mild swelling; 2, moderate swelling; 3, severe swelling of the hindpaw). The maximal possible score per day is 12, and this score was seen in all rats 8 d after CFA injection.
A cohort of Lewis rats not injected with CFA but receiving daily oral doses of vehicle (2% Tween 80/0.5% methylcellulose) for 21 d were designated normal controls. CFA-treated rats were dosed with vehicle for 7 d until full joint inflammation developed and then were randomized into two groups and treated for another 14 d. One group continued to be dosed with vehicle, designated CFA hereafter. The other group was treated with daily oral doses of ERB-041 (5 mg/kg), designated CFA + ERB-041. The daily joint scores for each rat treated with ERB-041 are shown in supplemental Fig. 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). At necropsy on d 21, blood was collected via cardiac puncture for serum as well as plasma preparation. The liver, popliteal lymph node, and spleen were excised and frozen on dry ice for RNA preparation. All procedures involving animals were conducted under institution-approved protocols. ERB-041 was obtained from the Wyeth compound library (22).
Expression profiling of organ mRNA
RNA preparation and microarray analysis.
Rat tissue RNA was purified using a Total RNA kit (Promega, Madison, WI). Each RNA sample was prepared individually (no samples were pooled) and hybridized to a different microarray chip (total number of chips used was 54). Amplification of cRNA, RNeasy spin column purification (QIAGEN, Valencia, CA), and cRNA fragmentation were performed as described (10). Standard curve normalization and conversion to RNA frequencies was as described (23, 24). cRNA was hybridized to REA230A oligonucleotide arrays (Affymetrix, Santa Clara, CA), stained with Streptavidin R-phycoerythrin (Molecular Probes, Eugene, OR) using the GeneChip Fluidics Station 400, and scanned with a GeneArray scanner (Agilent, Palo Alto, CA) following the manufacturer’s instructions. The REA230A oligonucleotide array contains 15,878 tiled probe sets that interrogate known rat genes and expressed sequence tags in addition to 45 bacterial control sequences enabling measurement of a spiked-in standard curve (24). Data were collected using MicroArray Suite 4.0 software (Affymetrix, Santa Clara, CA). Individual frequencies measured for each of the experimental groups were averaged and analyzed by pairwise comparison. Genes were initially selected by both average fold change (>2-fold) and Student’s t test (P < 0.01) criteria. In addition to magnitude of gene expression as reflected by average perfect match to mismatch oligonucleotide fluorescence, an independent algorithm provides an absolute present/absent call for each probe set as described in Lockhart et al. (23). The genes selected by the fold-change and statistical criteria were subsequently filtered to remove genes with low expression (average < 5 ppm) or that were not consistently called present within the entire cohort in which the gene was induced.
Proteomic analysis.
Plasma samples from 13 rats (four normal, four CFA, and five CFA + ERB-041) were selected for proteomic analysis. These were chosen without bias except that samples with high hemoglobin content (from hemolysis during collection) were excluded. These were depleted of albumin and immunoglobulins using Montage albumin depletion kits (Millipore, Billerica, MA) and protein G resin (Amersham, Piscataway, NJ). Depleted plasma quantification was performed with a Micro BCA kit (Pierce Chemicals, Rockford, IL). Forty micrograms of each depleted plasma sample were fractionated by electrophoresis in duplicate on 12% Bis-Tris NuPAGE minigels (Invitrogen, Carlsbad, CA) using 3[N-morpholino]propanesulfonic acid running buffer (Invitrogen). Gels were cut into 24 equal bands using a fixed razor blade assembly. The bands were placed into 96-well plates for oxidation and trypsin digestion, peptides were extracted from the gel bands with 0.2 M urea/50% acetonitrile, and vacuum dried. Peptides derived from the 24 bands were resolubilized in 10% acetonitrile and 0.2% trifluoroacetic acid. Peptides from adjacent bands were combined to create 10 pooled fractions of similar peptide number.
Each of the 10 fractions was analyzed on a single LC-MS instrument (capillary reverse-phase liquid chromatography coupled by electrospray to a QTOF Ultima mass spectrometer; Waters, Milford, MA). Samples were randomized, and the first fraction of all samples was completed before moving on to the next fraction. Detected peptide ions in each fraction were matched across all 18 samples by mass, charge, and retention time. Maximum peak intensity for each matched peptide ion was measured and the mean peak intensity across each group (normal, CFA, and CFA + ERB-041) was determined. The ratio of intensity for a given peptide between groups, IB/IA, was used to derive the relative abundance of the parent protein, AB/AA, using the experimentally derived relationship: AB/AA = 1.6 (IB/IA). The pairwise matching rate for each peptide across serial injections of the same sample was 98%. Under the same conditions, the median coefficient of variation of peak intensity across all peptide ions was 12%. If a plasma sample was divided and the replicates processed in parallel, the median coefficient of variation of intensity for any given peak was 27%.
Peptides showing a consistent differential abundance between CFA and normal groups of 5-fold or more were selected for sequencing. Replicate peptide digests were analyzed by capillary reverse-phase liquid chromatography by electrospray to a QTOF Ultima mass spectrometer operating in tandem mass spectrometry mode to obtain a fragmentation pattern for each selected peptide. Mascot (MatrixScience, Boston, MA) was used to match fragmentation spectra to the National Center for Biotechnology Information nonredundant protein database to obtain peptide sequences. Peptides with Mascot scores of 25 or above were clustered by parent protein and reported. A manual audit of the data supporting 53 representative peptides was performed. This quality control step checked for correct matching of peptides across all samples and assignment of peak intensity as well as sequence interpretation and association with the differentially expressed peptide.
Analysis of serum haptoglobin and orosomucoid.
Serum haptoglobin levels were measured using a kit from TriDelta Development Ltd. (Dublin, Ireland) according to the manufacturer’s directions except that a kinetic rather than an end point measurement was used. This assay measures the peroxidase activity of hemoglobin that is preserved at low pH when haptoglobin is present. The standard curve was linear from 0 to 2 mg/ml and all samples were within this range. The measured value for CFA rats treated with vehicle was consistent with our historical unpublished values.
Orosomucoid was measured by a radial immunodiffusion assay (Life Diagnostics, West Chester, PA) using 10 μl of undiluted serum. Precipitin rings were developed by incubation of the plates at room temperature for 48 h, and a standard curve was constructed by plotting the square of the precipitin ring diameter vs. orosomucoid concentration. The standard curve was linear from 60 μg/ml to 1 mg/ml and all samples were within this range.
Results
ERB-041 reduces joint swelling
Consistent with previous findings (9), CFA-treated rats developed maximally inflamed paws within 8 d after intradermal adjuvant injection. These rats had joint scores of 12 throughout the study, compared with joint scores of 0 for normal rats. ERB-041 treatment was initiated on d 8, which reduced the joint scores to approximately 2 after 4 d and stabilized scores at approximately 1 after 10 d of treatment (Fig. 1).
Expression profiling of liver, lymph node, and spleen
CFA-induced gene expression and response to ERB-041 therapy was monitored in three tissues of immunological relevance, the spleen and lymph node as direct sites of immune cell activity, and the liver as indirect responder to inflammation. Tissue RNA from each rat (n = 6) per treatment group was independently hybridized to Affymetrix REA230A oligonucleotide arrays. Average fold change (AFC) values were computed from mean frequency values comparing baseline and CFA-treated animals. CFA-regulated genes were selected by combined magnitude (AFC > 2-fold) and statistical significance (P < 0.01) parameters. The overall transcriptional responses are summarized in Table 1. Selected genes regulated by CFA treatment and their responses to ERB-041 are highlighted in Table 2. A comprehensive list of known genes is shown in supplemental Tables 1–5 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Variability among genes detected in each tissue but not judged as regulated is shown in supplemental Table 6 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The coefficient of variation of majority of genes in each tissue is less than 30%.
In the spleen, expression of 31 genes was increased by CFA treatment with AFCs ranging from 2- to 5-fold with no decreased gene expression identified. Regulated genes include immune and inflammatory mediators, innate immune-associated proteins, transcription factors, and proteases (supplemental Table 1). Graphical representation of the spleen CFA induced gene expression (Fig. 2A) demonstrates nearly complete resolution after ERB-041 treatment. The overall mean induction of the 31 CFA-induced genes was decreased 72% by ERB-041 treatment. As summarized in Table 1, 18 of the gene transcripts demonstrated greater than 70% reversal of expression with an additional 12 genes moderately reduced (30–70%) after ERB-041 treatment. In all, 29 of 31 genes induced in the CFA-disease model demonstrated a statistically significant reversal (P < 0.05) in response to ERB-041.
Lymph node mRNA demonstrated the most dramatic CFA-induced transcriptional response with expression of 148 genes increased 2- to 15-fold, and 155 genes decreased 2- to 12-fold. Transcripts up-regulated in the lymph node with CFA treatment included genes associated with an immune response in addition to genes representing a wide range of biological processes such as proliferation, protein secretion, cell adhesion, and metabolism (supplemental Table 2). The CFA-repressed genes demonstrated a striking decrease in metabolism-associated genes, 16 of which are directly related to lipid homeostasis (supplemental Table 3). The CFA-modulated gene expression in lymph node was ameliorated by ERB-041 treatment but, in general, did not completely return to normal levels (Fig. 2A). Of the CFA-induced lymph node gene expression, there was an overall 51% mean reduction after ERB-041 treatment with 36 genes reduced by greater than 70%, whereas the majority (84 genes) showed moderate reduction (30–70%). Similarly, ERB-041 treatment restored expression of CFA-repressed genes on average by 42%, with the majority of genes (n = 116) showing moderate level of recovery with only seven genes returned to near basal levels.
Liver gene expression responded to CFA treatment with 48 genes induced (2- to 49-fold), and 22 genes decreased with a range of 2- to 6-fold. As with the other tissues profiled, a majority of the CFA-induced genes have established roles in immune and acute phase responses (supplemental Table 4). Similar to spleen, the liver CFA-induced genes demonstrated nearly complete reversal after ERB-041 treatment (Fig. 2A), with a mean reduction of 82% of CFA-induced expression and statistically significant reduction (P < 0.05) in 42 of the 48 genes. In contrast, ERB-041 response for the CFA-repressed genes demonstrated a bimodal response with the expression of nine genes being nearly completely restored, whereas eight genes showed little or no response to drug and only five genes demonstrating an intermediate response. For the CFA-repressed gene set, there was a tendency for complete ERB-041-mediated recovery within genes associated with metabolism and ion transport, whereas genes associated with signal transduction and proliferation were generally less responsive (supplemental Table 5).
Plasma proteome analysis
Plasma peptides were matched across all samples according to their mass to charge ratio, chromatography retention time, charge, and peak intensity. A total of 18,567 peptides were matched and reproducibly observed. Pairwise comparison of peak intensities between groups for each of these study peptides identified 1632 with differential expression between at least two of the groups (P < 0.05). Thus, the robust joint swelling observed in the animals (Fig. 1) resulted in a change to only a small portion (9%) of the detectable plasma proteome. Graphical representation of the plasma concentrations of the disease-associated peptides (Fig. 2B) demonstrates a general reversal to normal levels after ERB-041 therapy coordinate with the tissue gene expression.
Of 61 disease-associated proteins (170 peptides) identified with high confidence by targeted tandem mass spectrometry, 42 (138 peptides) were selected as candidate biomarkers (Table 2) with specific peptides and concentrations detailed in supplemental Table 7 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Of these, 19 proteins increased in plasma abundance after CFA treatment, and 24 decreased. Nearly all (38 of 42) reverted by more than 50% toward control levels after ERB-041 treatment, with 26 proteins reverting by more than 85%. Only two were apparently unaffected by drug.
The identified plasma disease-related protein increases included positive acute phase proteins, complement proteins C3/C9, and both fibrinogen- and - chains. In addition to negative acute phase proteins, decreased hemoglobin- and - chains and four components of high-density lipoprotein (HDL) complexes [apolipoprotein (Apo)A1, ApoAIV, ApoM, and glycosyl-phosphatidylinositol-linked phospholipase D] were identified. The expression patterns of two proteins, orosomucoid and haptoglobin, were independently confirmed by standard biochemical assays (Fig. 3). The values for the change in abundance in response to CFA and the subsequent reversion after treatment with ERB-041 matched very closely between the biochemical assays and the mass spectrometer for both proteins.
Coordinate changes at the mRNA level in tissue were observed for four of the 42 differentially abundant plasma proteins. Statistically significant increases in haptoglobin, complement C3, -2-macroglobulin (A2M), and kininogen tissue mRNA expression are reported in Table 2. In addition, transcripts for several of the serum proteins reported in Table 2 demonstrated similar changes but were not included in the CFA-modulated gene set by virtue of the dual fold-change and statistical criteria chosen for inclusion. For example, liver mRNAs encoding ceruloplasmin increased 2-fold (P = 0.06), whereas orosomucoid increased 1.5-fold (P < 0.01). Similarly, liver expression of ApoAI decreased 1.4-fold (P < 0.01). Although consistent with the direction of change in the plasma proteins, the mRNA transcripts were measured only in a subset of the potentially responsive tissues and thus may not always fully reflect the overall magnitude serum protein changes. Kininogen and A2M tissue mRNA and serum peptides are illustrated in Fig. 4 as an example of where direct concordance between the transcriptional and proteomic profiling was observed.
Discussion
The discovery of a second form of the ER led to much optimism about its therapeutic utility (25), but translating its attractive tissue distribution into a clinical indication has proved a difficult task. The design of highly ER subtype selective ligands has allowed the dissection of ER- and ER-mediated activities (4, 9, 26, 27) and led to the discovery that selective ER activation reduces inflammation in two preclinical models. In the Lewis rat model of arthritis, for example, clinical signs are rapidly normalized over the course of 6–10 d of treatment, and histological scores (synovitis and Mankin) are significantly improved (9). Nonselective estrogens, such as 17-estradiol, have long been appreciated to affect the immune system (10, 11), yet the effects of estrogens are complex and likely mediated via several mechanisms involving multiple cell types.
RA is a systemic autoimmune disease of unknown etiology characterized by inflammation of the joint synovium leading to pain and gradual erosion of joint tissue. Several adjuvant- or antigen-induced disease models have been developed in rodents and share phenotypic responses common to the human disease. Increasingly in animal models, global proteomic or transcriptional profiling is being used to identify possible biomarkers of disease and drug response for clinical application. In the present study, the tissue transcriptional profiles of CFA-induced gene expression in the Lewis rat AIA model identify multiple genes and biological processes that would be anticipated in the course of an inflammatory response. In the tissues profiled, genes associated with both innate and adaptive immune function as well as direct immune mediators are induced. In addition, multiple genes associated with biological processes including proliferation, oxidative stress, protein trafficking and turnover, metabolism, adhesion and migration, and proteolysis show increased gene expression in the CFA-induced disease model. All of the tissues profiled demonstrated a clear therapeutic response to ERB-041 treatment with reversal of the majority of disease-associated transcription, consistent with amelioration of disease manifestations. Spleen and liver disease-associated transcription was substantially resolved at a time point at which the animals demonstrate complete phenotypic resolution of disease symptoms, whereas lymph node transcription was typically 50% reversed.
Included in the tissue expression profiles are several genes associated with increased eicosanoid synthesis, proinflammatory agents long implicated in RA pathophysiology. CFA stimulation increased secreted phospholipase A2 (sPLA2-IIA) mRNA 12-fold in lymph node and to a lesser degree in spleen. sPLA2 hydrolysis of phospholipids releases arachidonic acid, the precursor for prostaglandin and leukotriene synthesis. Increased sPLA2 has been identified in RA synovial fluid, and serum levels correlate with disease severity (28). Administration of a sPLA2-inhibitory peptide reduced bone erosion and cartilage destruction in a TNF transgenic model of disease (29). Increased 5-lipoxygenase-activating protein in the spleen mRNA profile further suggests activation of leukotriene arm of arachidonate metabolism, of which leukotriene B4 is most closely associated with RA. Mice deficient in 5-lipoxygenase-activating protein, required for presentation of arachidonic acid to 5-leukotriene oxidase, exhibit decreased disease activity in collagen-induced arthritis models (30). Additionally, increased expression of leukotriene C4 synthase is observed in the CFA-induced liver profile. In human RA patients, methotrexate therapy decreases neutrophil production of products downstream of 5-lipoxygenase, suggesting that inhibition of this pathway correlates to therapeutic benefit (31).
Anemia of chronic disease (ACD) is a frequent complication resulting from the chronic cycles of inflammation in RA. Many studies have focused on the role of TNF and its effect on erythropoiesis (32), yet a subgroup of ACD patients (46%) present with a coexistent iron deficiency (33). The proteomic and transcriptional profiles identify changes in both globin expression and genes central to iron homeostasis in the course of inflammatory responses. The plasma proteomic analysis identified a decrease in both globin- and globin- in response to CFA. LCN2/NGAL, a neutrophil-expressed lipocalin, has recently elucidated roles in both iron transport (34) and binding of iron siderophores as part of innate immune antimicrobial defense mechanism (35). LCN2 gene expression is dramatically increased in all three tissues profiled after CFA disease induction and demonstrates complete attenuation with ERB-041 treatment. The induction of iron-sequestering proteins as a protective response to infection may have pathologic consequences in chronic inflammatory diseases such as RA resulting from long-term alteration of iron homeostasis. At present there is no association of LCN2 in RA, but its role in iron homeostasis, expression by activated neutrophils, and induction in the Lewis rat AIA model suggest LCN2 as a candidate gene for further investigation in the etiology of ACD.
CFA-induced expression of both S100A8 and S100A9 was observed in all three of the tissue profiles. S100A8/A9 are members of the EF-hand homology family of calcium-binding proteins predominately expressed in neutrophils and monocytes. S100A8/A9 form both homodimers and heterodimers and have been implicated in a wide range of granulocyte activities including cytoskeletal rearrangements; arachidonate metabolism; and regulation of neutrophilic nicotinamide adenine dinucleotide phosphate reduced oxidase, adhesion, and transendothelial migration (36). Concentrations of S100A8/A9 reach 50% of soluble cytosolic protein in granulocytes and are released by a protein kinase C-dependent, nonclassical secretory process after contact with activated endothelium (37). Increased concentrations of S100A8/A9 complexes in both synovial fluid and blood have been identified as a biomarker of RA by a number of independent studies (38) and is correlated both to disease activity (39) and drug response (37, 40). The increase in both S100A8 and S100A9 mRNA in all three tissues profiled, ranging 2.4- to 13-fold, was consistently decreased 51–93% after ERB-041 treatment. The strong disease association and drug response in the animal model identify these S100 proteins as candidate biomarkers in clinical evaluation of ERB-041 in the treatment of RA.
The plasma proteomic profile of the Lewis rat AIA model identified a number of proteins altered in the course of CFA disease induction and amelioration in response to the ER agonist. As has been shown in a variety of global proteomic disease studies, the predominant response in blood plasma was an acute-phase response. Two of the proteins identified, A2M and T-kininogen, may serve as clinical biomarkers due to their direct correlation with RNA changes in liver gene expression, strong disease association, and full normalization by ERB-041. A2M functions both as a broad-spectrum protease inhibitor and oxidation-dependent regulator of cytokine activity (41). Carriers of a 5-bp deletion allele in the A2M gene have an increased risk of developing an early active severe form of RA (42). A2M mRNA is dramatically induced in the rat liver by CFA. Protein is detected in plasma by 34 independent peptides, 15 of which were validated by manual inspection of the data. Induction of liver mRNA and plasma protein is completely reversed after ERB-041 treatment. T-kininogen, measured in both liver mRNA profiles and plasma proteomics, follows a similar pattern as A2M and also identifies a protein of pathophysiological importance to RA that may be a potential drug-responsive clinical biomarker. Kininogens serve as substrate for kallikrein in the biosynthesis of vasoactive and proinflammatory kinins that have been associated with edema, pain, and proinflammatory responses (43). Direct inhibition of kallikrein activity or decreasing kininogen by monoclonal antibody titration provides therapeutic benefit in a Lewis rat models of reactive arthritis (44).
Several of the plasma proteomic changes identified in this animal model can be directly related to RA pathophysiology. We observed peptides representing both complement C3 and C9 increase, which is consistent with increases in C3- and C9-containing circulating immune complexes in serum and synovial fluid of RA patients (45). Both the - and -chains of fibrinogen increase in the rat model plasma. Elevated fibrinogen can enhance intercellular adhesion molecule and cytokine expression in synovial fibroblasts (46) and increase red blood cell adhesiveness/aggregation (47). Four proteins associated with HDL complexes decrease in rat plasma after CFA-induced disease. ApoA1 is a principal protein of HDL, and glycosylphosphatidylinositol-phospholipase D associates with ApoAI/ApoAIV-containing complexes (48). ApoAIV itself displays antiinflammatory activity in experimental colitis models (49). Lipid profiles of RA blood demonstrate decreased ApoA1-containing HDL complexes that are increased with successful treatment (50). The coordinate decrease in these proteins and 54–87% reversal by ERB-041 highlight an additional set of set of disease-related biomarkers to evaluate clinical activity.
Expression of a number of transcription factors was induced in the CFA disease model and was attenuated by ERB-041 treatment. CCAAT/enhancer-binding protein (C/EBP)-, C/EBP, or both were induced in all three tissues surveyed. Signal transducer and activator of transcription (STAT)-1, STAT-2, and STAT-3 were also induced in the lymph node. Janus kinase-3 and STAP-2, modulators of STAT activation, (51), were induced in the liver. C/EBP family members play a pivotal role in transcription of several of the inflammatory mediators and acute-phase proteins (52), many of which were identified in the CFA-induced tissue expression profiles. STAT1/STAT2 and STAT3, critical to interferon- and IL-6 signaling, respectively, are key to inflammation and inflammatory diseases (53) and responsible for IL-6-mediated up-regulation of C/EBP (54). Evidence of STAT1 activation in a subset of RA patients has been reported and may reflect an important parameter for stratification of clinical disease (55). ERB-041 treatment reduced each of these CFA-induced transcription factors and modulators by 51–100%. Whereas global gene or protein expression studies cannot in themselves reveal mechanism, the notable drug response in several transcription factors, in themselves fundamentally associated with immune and acute phase responses, indicates that the ERB-041 therapeutic activity in the Lewis rat AIA model lies at the core of the inflammatory response as opposed to modulation of symptomatic responses alone.
As can be seen from both the gene expression and plasma proteome data presented in this study, ERB-041 appears to act in an immunomodulatory fashion, dampening the inflammatory response evoked by the CFA stimulus without exerting a generalized immunosuppressive action. It is interesting to note that some immune-associated gene expression and plasma proteins are completely unaffected and in some cases are augmented over the response evoked by CFA treatment/vehicle treatment. For example, granzyme and NK cell group 7 sequence, two NK T cell transcripts, increase 3- and 4-fold, respectively, with CFA. The latter is unchanged by ERB-041 treatment, whereas granzyme B is increased an additional 75% over CFA by the administration of ERB-041 (Table 2). Although granzyme B contributes to the cytotoxic effects of NK T cells, the increased expression may represent the expansion of the NK T cell population. NK cells can also be immunoregulatory in nature. The NK cell group 7 sequence found on both NK cells and macrophages acts as an inhibitory membrane receptor (56). Recently NK cell dysfunction, i.e. deficiency, has been reported in patients with systemic onset juvenile RA (57). These patients exhibited a marked decrease in NK cell function and an absence of the CD56bright NK cell population, the immunoregulatory NK cell. It is interesting to note that this CD56bright NK cell population is expanded in both the decidua and peripheral blood of pregnant women and is thought to actively modulate the maternal immune response to the fetal allograft (58). As mentioned in the introductory text, RA often remits during pregnancy (19).
Fetuin, -fetoprotein, another pregnancy-associated negative acute-phase protein, is elevated 3-fold by CFA in the plasma and is unchanged by ERB-041. Fetuin has several functions in plasma and the body tissues. It can act as an inhibitor of dystrophic calcification (59), important in progression of the arthropathy of RA and perhaps contributing to the increased incidence of cardiovascular mortality in RA. In addition, fetuin may contribute to the immunomodulatory effects of ERB-041 by changing the expression of Fas/Fas ligand and TNF-related apoptosis-inducing ligand and its receptor, the net effect being a down regulation of the immune response and increased tolerance to the presence of neoantigens (60).
In summary, we show that Lewis rat AIA model bears similarity to human RA in tissue gene expression and plasma proteins that change during disease. Many of the dysregulated genes and proteins have well-established pathophysiological roles. The two data sets were highly concordant in the general response to disease and drug as well as complementary in that most changes were uniquely identified by a single technology. These observations highlight the importance of an integrated transcriptional and proteomic approach. ERB-041 significantly attenuates the majority of disease-induced changes, and thus, these data identify biomarkers that can be developed to monitor disease severity and drug response in clinical studies. Most importantly, these data support the possibility that ER agonists represent a new class of clinically useful antiinflammatory agents.
Acknowledgments
The authors thank Leo Albert and Yelena Leathurby for assistance with the Lewis rat adjuvant-induced model, Sean McLarney for the serum haptoglobin analysis, and Chris Chadwick for the orosomucoid analysis. Orest Hurko, Al Kotake, Don Raible, Bill Jacobson, and Darryl Webster provided valuable project support. Andrew Hill provided advice for the statistical analyses. We also thank Rick Winneker for helpful discussions about the project and manuscript.
Footnotes
The authors are full-time employees of the institutions listed on the title page.
First Published Online November 3, 2005
Abbreviations: ACD, Anemia of chronic disease; AFC, average fold change; AIA, adjuvant-induced arthritis; A2M, -2-macroglobulin; Apo, apolipoprotein; C/EBP, CCAAT/enhancer-binding protein; CFA, complete Freund’s adjuvant; ER, estrogen receptor; HDL, high-density lipoprotein; NK, natural killer; RA, rheumatoid arthritis; sPLA2, secreted phospholipase A2; STAT, signal transducer and activator of transcription.
Accepted for publication October 26, 2005.
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