Apolipoprotein E Deficient Mice Are Resistant to the Development of Collagen-Induced Arthritis

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1 ARTHRITIS & RHEUMATISM Vol. 62, No. 2, February 2010, pp DOI /art , American College of Rheumatology Apolipoprotein E Deficient Mice Are Resistant to the Development of Collagen-Induced Arthritis Darren L. Asquith, Ashley M. Miller, Axel J. Hueber, Foo Y. Liew, Naveed Sattar, and Iain B. McInnes Objective. To determine whether elevated serum lipid levels resulting from feeding animals a high-fat diet can affect the inflammatory process in C57BL/6 (B6) wild-type (WT) and B6 ApoE / mouse models of collagen-induced arthritis (CIA). Methods. Male B6 WT or ApoE / mice were fed either a normal chow diet or a high-fat diet. CIA was induced in mice at 12 weeks of age using type II chicken collagen, Freund s complete adjuvant, and, on occasion, a lipopolysaccharide boost. Expression levels of autoantibodies and cytokines were measured using enzymelinked immunosorbent assay and multiplex assay, respectively. Results. Whereas B6 WT mice developed severe articular inflammation after collagen immunization, ApoE / mice developed no clinical or histologic evidence of disease regardless of whether they had been fed a high-fat diet or a normal chow diet. The fact that arthritis was not present in ApoE / mice did not result from inadequate production of serum IgG2a collagen antibodies, since levels observed in ApoE / mice were similar to those observed in arthritic B6 WT control mice. Critically, development of atherosclerosis in ApoE / mice was not affected by the CIA protocol. Supported by grants from the Medical Research Council UK and the Wellcome Trust. Dr. Miller s work was supported by a British Heart Foundation fellowship (FS/08/035/25309). Dr. Hueber s work was supported by a German Research Foundation fellowship. Darren L. Asquith, MSc, Ashley M. Miller, PhD, Axel J. Hueber, MD, Foo Y. Liew, PhD, Naveed Sattar, MD, PhD, Iain B. McInnes, MD, PhD: University of Glasgow, Glasgow, UK. Mr. Asquith and Dr. Miller contributed equally to this work. Address correspondence and reprint requests to Iain B. McInnes, MD, PhD, Division of Immunology, Infection, and Inflammation, Glasgow Biomedical Research Centre, 120 University Place, University of Glasgow, Glasgow G12 8TA, UK. ibmi1w@ clinmed.gla.ac.uk. Submitted for publication June 25, 2009; accepted in revised form October 9, Conclusion. Our findings suggest that ApoE / mice are resistant to the development of CIA. Intriguingly, induction of host autoimmunity in the absence of articular inflammation had no effect on atherosclerosis progression, suggesting that articular inflammatory load may be a critical risk factor in vascular pathology. Rheumatoid arthritis (RA) is a chronic inflammatory disease, and patients with RA present with articular pain, stiffness, and synovitis, which leads to tissue destruction. RA is associated with significant morbidity and increased mortality primarily resulting from increased atherosclerosis and premature coronary heart disease, unexplained by traditional cardiovascular risk factors (1). Increased carotid intima-media thickness, aortic stiffness, calcification, and impaired endothelial function are observed in RA patients (2). Many immunologic and metabolic factors have been implicated in accelerated atherosclerosis, including proinflammatory cytokines, immune complexes, anti cyclic citrullinated peptide antibodies, rheumatoid factor, homocysteine, oxidative stress, and insulin resistance (3). The relative contribution of primary autoimmunity and innate responses to vascular lesions is unknown. Patients with RA also have complex lipid abnormalities that depend on disease activity and may contribute to increased risk of atherosclerosis. In patients with severe disease or with disease that has gone untreated, paradoxical findings of lower levels of cholesterol but also lower levels of high-density lipoprotein (HDL) cholesterol have been observed, which contrasts with the increases observed in both parameters once treatment using biologic agents to suppress inflammation is begun (4). Thus, there is an interaction of inflammation with lipids. 472

2 RESISTANCE TO CIA IN ApoE / MICE 473 To examine this issue further, we sought to investigate whether elevated lipid levels could accelerate the inflammatory processes in a mouse model of RA and whether articular inflammation could alter vascular lesion development. Apolipoprotein E (Apo E) is a glycoprotein synthesized predominantly in the liver and brain and is a constituent of all lipoproteins except low-density lipoproteins. It functions as a ligand for receptors that clear chylomicrons and very low-density lipoprotein remnants from blood in the liver. Using the ApoE / mouse model of elevated serum lipids and atherosclerosis (5), we demonstrate that, in contrast to our prediction of accelerated collagen-induced arthritis (CIA) and atherosclerosis, we were unable to induce CIA in ApoE / mice. Moreover, the development of collagen-specific immunity in the absence of articular inflammation in ApoE / mice mediated no effect on atherosclerosis development. MATERIALS AND METHODS Animals. Male ApoE / mice, backcrossed for 10 generations to a C57BL/6 (B6) H-2 b background, were bred in-house in a pathogen-free facility, and male B6 (H-2 b ) wild-type (WT) mice were obtained from Harlan (Indianapolis, IN). Experiments were carried out according to the guidelines of the UK Home Office. At 6 weeks of age, mice were randomly allocated to receive either a normal chow diet or an atherogenic diet (0.15% cholesterol and 21% lard; Special Diet Services, Edinburgh, UK) ad libitum for 12 weeks. Induction and scoring of arthritis. CIA was induced in B6 WT or ApoE / mice at 12 weeks of age (day 0), according to a previously described method (6). Briefly, on day 0, mice were injected intradermally with a type II chicken collagen/ Freund s complete adjuvant emulsification (200 g) (MD BioSciences, Zurich, Switzerland). On day 21, type II chicken collagen/phosphate buffered saline (200 g) was injected intraperitoneally. Then, mice were monitored for clinical signs of disease using microcaliper measurements and a clinical score based upon disease severity, where 1 swollen digit(s), 2 erythema, 3 swollen paw/ankle, and 4 loss of function. In some experiments, if no disease was evident in mice by day 26, a boost of lipopolysaccharide (LPS) (20 40 g) was administered intraperitoneally. For histologic assessment, rear paws were fixed for 3 weeks in 10% neutral buffered formalin, and for subsequent decalcification, paws were fixed for 3 weeks in 10% nitric acid. Paws were then cut into 7- m sections and stained with hematoxylin and eosin (H&E). Serum analysis. Levels of IgG2a anticollagen antibodies in sera were measured using enzyme-linked immunosorbent assay. Briefly, 96-well plates were coated with type II chicken collagen (0.4 g/well). Serially diluted serum samples were incubated for 1 hour, which was followed by the addition of biotin anti-mouse IgG2a (BD PharMingen, San Diego, CA) and then ExtrAvidin peroxidase (Sigma, St. Louis, MO). Mean optical density was read at 450 nm. Cytokine levels in serum samples were measured using a 20-plex mouse cytokine assay (Invitrogen, San Diego, CA). Total cholesterol, HDL cholesterol, and triglycerides (mmoles/liter) in serum samples were measured using enzymatic assay (Roche Diagnostics, Mannheim, Germany). Atherosclerotic lesion analysis. Atherosclerosis was quantified in the aortic sinus of 18-week-old ApoE / mice. Briefly, the heart was sectioned parallel to the atria and fixed, processed, and embedded in paraffin wax. Five 7- m sections starting from the 3 valve cusps of the aortic sinus at 35- m intervals were cut and stained with H&E for plaque measurement using Scion Image software (Scion, Frederick, MD). Statistical analysis. Data are expressed as the mean SEM, or as the median, where appropriate. Statistical analysis was performed by Student s unpaired t-test, log rank test (survival curves), or analysis of variance, using GraphPad Prism software (San Diego, CA). RESULTS Resistance to CIA in ApoE / mice. We compared the clinical progression of arthritis in B6 WT mice and ApoE / mice after collagen immunization. While B6 WT mice fed either a normal diet or a high-fat diet developed arthritis as expected, ApoE / mice were completely resistant to the development of arthritis (Figure 1A). The incidence of disease was 0 in ApoE / mice receiving both diets and at all time points, whereas 75% of B6 WT mice receiving a normal diet and 54% of B6 WT mice receiving a high-fat diet developed disease by day 42. B6 WT mice had mean arthritis severity scores of up to 3 (Figure 1B) and swollen paws peaking in size on day 30 (Figure 1C), whereas the paws in ApoE / mice exhibited no change in size throughout the experiment. Bacterial LPS, previously shown to boost the development of arthritis, had no effect on clinical score, incidence of arthritis, or paw size in ApoE / mice (data not shown). To check for evidence of subclinical disease, sections of hind paws were stained with H&E. A severe infiltration of inflammatory cells into the joint space and synovial tissue was observed in B6 WT mice that underwent the CIA protocol (Figure 1D). In contrast, ApoE / mice receiving either a normal diet or a high-fat diet exhibited no histologic evidence of inflammation after the CIA protocol. Induction of collagen antibodies and cytokines in sera of ApoE / mice. Titers of IgG2a anticollagen antibodies were measured to investigate antigen-specific

3 474 ASQUITH ET AL Figure 1. Resistance of ApoE / mice to collagen-induced arthritis (CIA). CIA was initiated in 12-week-old male C57BL/6 (B6) wild-type (WT) or ApoE / mice, animals were fed either a normal chow diet or a high-fat diet (HFD), and disease development was monitored for the indicated period. A C, The incidence of CIA (A), the severity of arthritis, as indicated by the mean clinical score (B), and paw swelling, as indicated by the percentage increase in paw size (C), are shown. D, On day 42 after induction of CIA, the mice were killed, and their paws were removed, decalcified, sectioned, and stained with hematoxylin and eosin. Images shown are representative of 2 sections per mouse (original magnification 10). Values in A C are the mean of mice per group from 2 3 independent experiments. P by log rank test in A or by two-way analysis of variance in B and C. adaptive immunity. As expected, B6 WT mice fed a normal diet developed high titers of collagen antibodies (Figure 2A). In contrast, B6 WT mice that were fed a high-fat diet had significantly reduced titers of collagen antibodies (P 0.05). Interestingly, despite complete resistance to the development of articular inflammation, ApoE / mice fed either a normal diet or a high-fat diet developed collagen antibody titers similar to those in B6 WT mice that were fed a high-fat diet and that developed clinical disease. As expected, control ApoE / mice that did not undergo the CIA protocol had no collagen antibodies (Figure 2). The immunologic profile of the mice was further investigated by assessing cytokine levels in serum samples ex vivo on day 42 in the groups receiving the high-fat diet. ApoE / mice in which CIA induction was attempted had serum levels of interleukin-1 (IL-1 ) and IL-12 that were similar to those observed in B6 WT mice that developed CIA (Figure 2B). However, IL-17 levels were significantly lower in ApoE / mice in which CIA induction was attempted versus B6 WT mice that developed CIA (P 0.05). Control ApoE / mice that did not undergo the CIA protocol had lower serum levels of all 3 cytokines. The levels of other cytokines measured either were below the limit of detection for the assay (tumor necrosis factor ) or were no different between the groups (IL-6) (data not shown). Lipid profiles in ApoE / mice with CIA. Serum lipid profiles were assessed in mice on day 42 after attempted CIA induction. Concentrations of total cho-

4 RESISTANCE TO CIA IN ApoE / MICE 475 Figure 2. Serum profiles in ApoE / mice with collagen-induced arthritis (CIA). Following CIA induction in ApoE / and C57BL6 (B6) wild-type (WT) mice, the animals were fed either a normal chow diet or a high-fat diet (HFD). A separate group of ApoE / mice were fed a high-fat diet but did not undergo CIA induction. Sera were collected on day 42 after CIA induction. A, Levels of IgG2a anticollagen antibodies in serially diluted serum samples, as determined by enzyme-linked immunosorbent assay. Values are the mean optical density (OD) of mice per group. P B, Levels of the cytokines interleukin-1 (IL-1 ), IL-12, and IL-17 in serum samples from age-matched control ApoE / mice without CIA that were fed a high-fat diet and from ApoE / mice and B6 WT mice with CIA, as determined by Luminex assay. Each data point represents a single mouse (n 8 per group); horizontal lines show the median. P C, Levels of total triglycerides, cholesterol, and high-density lipoprotein (HDL) cholesterol in serum samples. P 0.01 versus ApoE / mice with CIA fed a normal chow diet; P versus B6 WT mice with CIA fed a normal chow diet, by Student s unpaired t-test. Values are the mean and SEM of mice per group from 2 3 independent experiments. lesterol, HDL cholesterol, and triglycerides in serum samples are shown in Figure 2C. As expected, ApoE / mice receiving a high-fat diet had higher total cholesterol levels than mice fed a normal diet (P 0.01). However, we could detect no significant difference in the levels of either total cholesterol or HDL cholesterol in ApoE / mice that underwent the CIA procedure compared with controls. B6 WT mice fed a high-fat diet had significantly higher total cholesterol and HDL cholesterol levels than those fed a normal diet (P 0.001). Atherosclerosis assessment. Our data suggested that ApoE / mice were immunocompetent to respond to antigen/adjuvant, and therefore, we assessed atherosclerosis formation in each group. We quantified intimal atherosclerotic lesion size in the aortic sinus of 18-weekold ApoE / mice by cross-sectional computerized planimetry. There was no difference in atherosclerotic lesion size in the aortic sinus of ApoE / controls fed a high-fat diet (mean SEM mm 2 ) compared with ApoE / mice fed a high-fat diet that underwent the CIA protocol (mean SEM mm 2 ) (Figure 3A). In B6 WT mice fed either a normal diet or a high-fat diet, no detectable atherosclerotic lesions were found in the vasculature, regardless of whether the mice had undergone CIA induction (data not shown).

476 ASQUITH ET AL Figure 3. No change in the development of atherosclerotic plaques in the aortic sinus of ApoE / mice with collagen-induced arthritis (CIA).

5 476 ASQUITH ET AL Figure 3. No change in the development of atherosclerotic plaques in the aortic sinus of ApoE / mice with collagen-induced arthritis (CIA). On day 42 after induction of CIA, 18-week-old ApoE / mice that had been fed a high-fat diet were killed, and their hearts were removed, serially sectioned, and stained with hematoxylin and eosin (H&E) for assessment of atherosclerosis development. Hearts of mice that did not undergo the CIA protocol were removed at the same time point for comparison. A, Plaque size in the aortic sinus. Values are the mean and SEM of 5 mice per group. B, Representative photomicrographs of H&E-stained aortic sections (original magnification 10). M media; P plaque; L lumen; A adventitia. DISCUSSION In order to explore both the relationship between RA and accelerated atherosclerosis and the associated molecular mechanisms, we sought to induce CIA in ApoE / mice. However, our results indicate that ApoE / mice are resistant to the development of articular inflammation in CIA, despite exhibiting serum cytokine and anticollagen antibody responses that are similar to those observed in B6 WT mice that developed CIA. This serendipitous observation allows us to conclude that the development of joint-specific (auto)immunity per se is not sufficient to induce an increased atherosclerotic burden. By implication, the inflammatory load implicit in polyarticular disease may be a necessary prerequisite risk factor for vascular disease acceleration. Future studies using other arthritis induction protocols and a longer time course of atherosclerosis assessment will be necessary to formally test this intriguing possibility. Susceptibility to CIA in mice has been linked to strains that have class II major histocompatibility complex I-A q haplotypes; however, many mouse strains have variable degrees of susceptibility to CIA and to collagen derived from different species. Several other strains, such as B6 (H-2 b ), generate T cell and B cell immunity to type II collagen, yet do not develop CIA. However, the development of a refined protocol for the induction of CIA in B6 mice using chicken collagen has made arthritis studies possible in genetically modified mice (6). We therefore consider it unlikely that the genetic background of ApoE / (H-2 b ) mice is responsible for the resistance toward the development of arthritis. Furthermore, the Apo E protein is generally understood to exert antiinflammatory effects in inflammation and tissue repair (7). Several studies have already shown that chronic inflammatory disease in lupussusceptible mice can enhance atherosclerosis in ApoE / mouse models of atherosclerosis (8,9). In addition, because Apo E is a constituent of some plasma lipoproteins, it may enter inflammatory lesions due to increased vascular permeability. Indeed, the concentration of Apo E was elevated in synovial fluid relative to the concentration in plasma, in patients with inflammatory arthropathies (10). Hence, Apo E is found in inflamed joints, but its role in RA pathology is unclear. In humans, the gene for Apo E is polymorphic, with 3 common alleles (Apo E2, Apo E3, Apo E4). Although genetic risk factors for RA are wellestablished (most notably HLA DRB1 and PTPN22), the Apo E4 genotype may be a risk factor for amyloidosis and bone loss in RA (11,12). It is possible that changes to serum proteins or other unknown factors, as a consequence of the deletion

6 RESISTANCE TO CIA IN ApoE / MICE 477 of the Apo E gene, may have inhibited development of CIA. For example, we found significantly lower IL-17 levels in the sera of ApoE / mice than in the sera of B6 mice after CIA induction. Other studies have shown that IL-17 plays a crucial role in the development of CIA (13), and therefore reduced IL-17 production may explain our observations. In addition, ApoE / mice have severely dysregulated lipids, which may also help to explain our findings. Commensurate with this, we found significantly higher levels of IgG2a collagen antibodies in B6 WT mice fed a normal diet compared with both B6 WT mice fed a high-fat diet and ApoE / mice fed either a normal diet or a high-fat diet. Also, we did not observe a significant change in the clinical arthritis severity score or in the incidence of disease between B6 mice fed either diet, but the lipid levels in these mice were significantly lower than in ApoE / mice. It is possible that the high lipid levels seen in the ApoE / mice inhibited development of CIA. However, injected 3 H-cholesterol has been previously shown to accumulate on the synovial membrane at sites of damage in DBA/1J mice and has been shown to be associated with the most severe CIA (14). These data contrast with our observations of reduced collagen antibody titers and no disease in ApoE / mice with high cholesterol. Hence, it is unclear whether cholesterol, cholesterol fractions, or the various associated apolipoproteins influence RA pathology in a positive or negative way. Future experiments in an ApoE-independent mouse model of hypercholesterolemia (LDLR / mice) will be needed to make this assessment. Indeed, it is now clear that patients with RA also have complex lipid abnormalities that appear to depend on disease activity and may contribute to an increased risk of atherosclerosis (4). In summary, we have demonstrated that ApoE / mice are resistant to CIA, and the molecular pathways influenced by lipids and apolipoproteins in normal and inflamed joints merit further investigation. Moreover, we conclude that the development of immunity to articular-specific antigens per se is insufficient to modulate the atherogenic burden in this model. We thank Dr. L. Cherry for the serum lipid measurements. ACKNOWLEDGMENT AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. McInnes had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Asquith, Miller, Hueber, Liew, Sattar, McInnes. Acquisition of data. Asquith, Miller, Hueber. Analysis and interpretation of data. Asquith, Miller, Hueber, Liew, Sattar, McInnes. REFERENCES 1. Del Rincon I, Williams K, Stern MP, Freeman GL, Escalante A. High incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors. Arthritis Rheum 2001;44: Gonzalez-Juanatey C, Llorca J, Martin J, Gonzalez-Gay MA. Carotid intima-media thickness predicts the development of cardiovascular events in patients with rheumatoid arthritis. Semin Arthritis Rheum 2009;38: Sattar N, McCarey DW, Capell H, McInnes IB. Explaining how high-grade systemic inflammation accelerates vascular risk in rheumatoid arthritis. Circulation 2003;108: Choy E, Sattar N. Interpreting lipid levels in the context of high-grade inflammatory states with a focus on rheumatoid arthritis: a challenge to conventional cardiovascular risk actions. Ann Rheum Dis 2009;68: Daugherty A. Mouse models of atherosclerosis. Am J Med Sci 2002;323: Inglis JJ, Criado G, Medghalchi M, Andrews M, Sandison A, Feldmann M, et al. Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen. Arthritis Res Ther 2007;9:R Mahley RW, Rall SC Jr. Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet 2000;1: Feng X, Li H, Rumbin AA, Wang X, La Cava A, Brechtelsbauer K, et al. ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia. J Lipid Res 2007;48: Ma Z, Choudhury A, Kang SA, Monestier M, Cohen PL, Eisenberg RA. Accelerated atherosclerosis in ApoE deficient lupus mouse models. Clin Immunol 2008;127: Terkeltaub RA, Dyer CA, Martin J, Curtiss LK. Apolipoprotein (apo) E inhibits the capacity of monosodium urate crystals to stimulate neutrophils: characterization of intraarticular apo E and demonstration of apo E binding to urate crystals in vivo. J Clin Invest 1991;87: Hasegawa H, Nishi S, Ito S, Saeki T, Kuroda T, Kimura H, et al. High prevalence of serum apolipoprotein E4 isoprotein in rheumatoid arthritis patients with amyloidosis. Arthritis Rheum 1996; 39: Lee SI, Lee SY, Yoo WH. Association of apolipoprotein E polymorphism with bone mineral density in postmenopausal women with rheumatoid arthritis. Rheumatology (Oxford) 2005; 44: Nakae S, Nambu A, Sudo K, Iwakura Y. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J Immunol 2003;171: Hamer ER, Apfel MI, Carvalho JJ, Pereira MJ, Levy RA. Evaluation of the cholesterol influence in type II collagen-induced arthritis in DBA/1J mice: an autoradiographic study. J Cell Mol Med 2002;6:

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