Involvement of Tumor Necrosis Factor-α in the Development of T Cell–Dependent Aortitis in Interleukin-1 Receptor Antagonist–Deficient Mice
Background— Interleukin-1 receptor antagonist–deficient (IL-1Ra−/−) mice on the BALB/c background spontaneously develop inflammatory arthropathy that resembles rheumatoid arthritis in humans. These mice also frequently develop aortitis at the root of the aorta, but the mechanism underlying the development of this disease has not been completely elucidated.
Methods and Results— Using IL-1Ra−/− mice (backcrossed 8 generations to the BALB/c background) and wild-type mice, we studied the histopathology and examined the immunologic mechanisms involved in the development of aortic inflammation by cell transplantation experiments. Half of the IL-1Ra−/− mice developed aortitis at the root of the aorta, with massive infiltration of macrophages and monocytes and loss of elastic lamellae in the aortic media. Left ventricular hypertrophy and mild aortic stenosis were also shown by transthoracic echocardiography. Transplantation of T cells from IL-1Ra−/− mice induced aortitis in recipient nu/nu mice. Bone marrow cell transplants from IL-1Ra−/− mice also induced aortitis in irradiated wild-type recipient mice. Furthermore, tumor necrosis factor (TNF)-α deficiency completely suppressed the development of aortitis in IL-1Ra−/− mice, whereas IL-6 deficiency did not affect pathology.
Conclusions— These observations suggest that IL-1Ra deficiency in T cells activates them excessively, resulting in the development of aortitis in IL-1Ra−/− mice in a TNF-α–dependent manner.
Received November 15, 2004; de novo received March 16, 2005; revision received May 25, 2005; accepted June 1, 2005;
Interleukin (IL)-1 is a major mediator of inflammation and plays important roles in host defense mechanisms through regulation of not only the immune system but also the neuronal and endocrine systems, which interface with the immune system.1,2 IL-1 consists of 2 molecular species, IL-1α and IL-1β, both of which exert similar but not completely overlapping biological functions through the IL-1 type I receptor (IL-1RI). Another IL-1R, the type II receptor (IL-1RII), has also been identified, but it is not involved in signal transduction; rather, it plays a regulatory role as a decoy. The IL-1R antagonist (IL-1Ra), another member of the IL-1 gene family, binds to IL-1Rs without exerting agonistic activity. IL-1Ra, IL-1RII, and the secreted forms of IL-1RI and IL-1RII are thought to be negative regulators of IL-1 signaling, participating in the complex regulation of IL-1 activity. Production of both IL-1 and IL-1Ra is induced by a number of other cytokines, bacterial and viral components, and mechanical stresses in a wide variety of cell types, including monocytes/macrophages, epithelial and endothelial cells, and glial cells.3
We previously reported that IL-1Ra gene–deficient (IL-1Ra−/−) mice on the BALB/c background spontaneously developed chronic inflammatory arthropathy.4 Histopathological analysis showed marked synovial and periarticular inflammation, with articular erosion caused by invasion of granulation tissues closely resembling rheumatoid arthritis in humans. Moreover, elevated levels of antibodies against IgG, type II collagen, and double-stranded DNA (dsDNA) were detected in the sera of these mice, suggesting the development of autoimmunity. Proinflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor (TNF)-α were overexpressed in the joints of these animals, indicating a regulatory role for IL-1Ra in the cytokine network. Therefore, it was suggested that IL-1Ra is crucial for homeostasis of the immune system.
Classic primary vasculitis syndromes such as Takayasu arteritis and giant-cell (temporal) arteritis involve massive recruitment of lymphocytes and macrophages into the vascular wall, destruction of the medial layer with concurrent fibrosis, and proliferation of smooth muscle cells in the intima, leading to neointima formation.5 Although a number of potential mechanisms, including microbial infection and autoimmune reactions, have been implicated in the development of inflammatory reactions in the vascular system, the precise mechanism underlying the development of vasculitis remains to be elucidated.
Nicklin et al6 reported that IL-1Ra−/− mice developed aortic inflammation on the 129/O1a×MF1 background. Arterial inflammation with massive transmural infiltration of neutrophils, macrophages, and CD4+ T cells was found at branch points and flexures of the aorta. IL-1β expression was observed mainly in macrophages that were associated with CD4+ cells deep within the vessel wall, suggesting the involvement of CD4+ cells in enhancing IL-1β production. Although the histological changes in the affected IL-1Ra−/− arteries were described in detail, the mechanism underlying the development of arteritis caused by IL-1Ra deficiency was not completely elucidated.
In this investigation, we examined the possibility that autoimmunity is involved in the development of spontaneous arterial inflammation in our IL-1Ra−/− mice on the BALB/c background by cell transplantation experiments. Furthermore, we investigated the role of the proinflammatory cytokines TNF-α and IL-6 in chronic arterial inflammation by generating cytokine-deficient IL-1Ra−/− mice.
IL-1Ra−/− mice were produced as described previously.7 TNF-α−/− and IL-6−/− mice were kindly provided by Dr K. Sekikawa (National Institute of Agrobiological Sciences, Tsukuba, Japan) and Dr M. Kopf (Swiss Federal Institute of Technology, Zurich, Switzerland), respectively. These mice were backcrossed to BALB/c or C57BL/6 mice for 8 generations and then intercrossed with IL-1Ra−/− mice to generate doubly deficient mice (IL-1Ra−/−×TNF-α−/− or IL-1Ra−/−×IL-6−/− mice)8. BALB/c, C57BL/6, and BALB/c-nu/nu mice were purchased from Japan Clea (Tokyo, Japan). A group of wild-type mice of the same age and sex as the test mice was used as a control in each experiment. Mice were housed under specific pathogen-free conditions in an environmentally controlled clean room at the Center for Experimental Medicine, Institute of Medical Science, University of Tokyo. Mice were housed at an ambient temperature of 24°C and a daily light/dark cycle of 12 hours each (light from 8 am to 8 pm). All experiments were carried out according to institutional ethics guidelines for animal experiments and safety guidelines for gene manipulation experiments.
Histological and Clinical Evaluation for Aortitis and Arthritis
For histological examination of aortitis, mice were anesthetized with pentobarbital and perfused with phosphate-buffered saline (PBS) followed by 10% formalin from an angiocatheter placed in the left ventricule (LV) of the heart. The aorta was fixed in 10% formalin for 48 hours and embedded in paraffin. Serial 10-μm sections of aorta were stained with hematoxylin/eosin for examination of cell infiltration. Masson’s trichrome stain was used to evaluate connective tissue damage.9,10 To detect calcification of the vessel, von Kossa staining, in which sections were treated with 3% AgNO3 and exposed to bright light for 30 minutes, was used. Sections were counterstained with hematoxylin/eosin. Lesion sizes were measured with NIH Image 1.55 software (public domain software). The severity of aortitis was graded on a scale of 0 to 3 by the degree of inflammation near the aortic valve, as follows: grade 0=normal and no infiltration; grade 1=infiltration and loss of elastic lamellae over less than one third of the media of the aortic sinus; grade 2=loss in one third to two thirds of the aortic sinus; and grade 3=loss over more than two thirds of the aortic sinus (see Figure 1).
The incidence and severity of arthritis were judged macroscopically and histologically, as previously described.4 In brief, each joint was examined weekly for swelling and redness, and severity was graded from 0 to 3 for each paw: grade 0=no special changes; grade 1-light swelling of the joint and/or redness of the foot pad; grade 2=obvious swelling of the joint; and grade 3=fixation of the joint. Severity score was calculated for the 4 legs for a total of 12 points for each mouse. For histological examination, joints were fixed with 10% phosphate-buffered formalin, decalcified in 10% EDTA-4Na, and embedded in paraffin. Sections (4 μm) were stained with hematoxylin/eosin.
To examine valve function, transthoracic echocardiography was performed with a Sonos 5500 unit (Phillips Co) equipped with 12-MHz and 15-MHz imaging transducers. Mice (female, 40 weeks old) were anesthetized with 2,2,2-tribromoethanol (250 mg/g IP), the chest was shaved, and ECG leads were attached to each limb with needle electrodes. Mice were imaged in a shallow left lateral decubitus position; short- and long-axis views of the LV were obtained by slight angulation and rotation of the transducer. Two-dimensional, targeted M-mode studies were generally taken from the short axis (at the level of the largest LV diameter).
Intraventricular septum thickness, end-diastolic LV internal diameter, end-systolic LV internal diameter, and LV posterior wall thickness were measured. Percent fractional shortening was calculated as [(end-diastolic LV internal diameter)−(end-systolic LV internal diameter)/(end-diastolic LV internal diameter×100)].11
Color flow Doppler measurements were used to identify areas of increased (aliased) velocities in the outflow tract from angulated parasternal long-axis views, and these were quantified by pulsed- and/or continuous-wave Doppler. Attempts were made to align the ultrasound beam as parallel as possible with the direction of flow and to record the highest velocities.12 Then the peak pressure gradient through the LV outflow tract was estimated according to the simplified Bernoulli equation.13
Blood Pressure and Heart Rate Measurements
To evaluate hemodynamics, blood pressure and heart rate were measured in nonanesthetized mice (female, 12 weeks old) by the tail-cuff method with a Softron BP-98A device (Softron Co) in the morning. Body and heart weights of these mice were also measured. Values were measured at least 3 times per mouse and were averaged for each individual.
Plasma Cytokine Levels
Proinflammatory cytokine levels in the plasma from 8-week-old male IL-1Ra−/− and wild-type mice were measured by ELISA.14 Hamster anti-mouse IL-1α monoclonal antibody, hamster anti-mouse IL-1β monoclonal antibody, and polyclonal goat anti-mouse TNF-α antibody (all from Genzyme) were used as capture antibodies. Polyclonal rabbit anti-mouse IL-1α, polyclonal rabbit anti-mouse IL-1β, and polyclonal biotinylated goat anti-mouse TNF-α antibodies (all from Genzyme) were used as secondary antibodies. Detection was performed with horseradish peroxidase–conjugated goat anti-rabbit IgG and horseradish peroxidase–streptavidin (Zymed). TMB substrate was purchased from Dako. IL-6 levels were measured with the OptEIASet mouse IL-6 kit (BD Pharmingen). All assays were performed in duplicate.
T Cell and Bone Marrow (BM) Cell Transplantation
To elucidate the role of T cells in the development of aortitis and arthritis, T-cell transplantation was performed.12 In brief, cells were prepared from the spleen and lymph nodes of IL-1Ra−/− (n=10, female, 6 to 8 weeks old) and wild-type (n=10, female, 6 to 8 weeks old) mice, and then the cells were treated with hemolysis buffer (17 mmol/L Tris-HCl and 140 mmol/L NH4Cl, pH 7.2) to remove red blood cells, washed, and passed through a nylon wool column. Then anti-mouse B220 and anti–Mac-1 magnetic bead (Miltenyi Biotec) –treated cells were passed through a MACS column (Miltenyi Biotec) to obtain T cells. The resulting purified T cells were resuspended in 0.2 mL PBS (2×107 cells/mouse) and transplanted intravenously into BALB/c-nu/nu mice (n=20, female, 6 weeks old). The development of aortitis in recipient mice was analyzed 10 weeks later.
For BM cell transplantation, BM cells were taken from femurs, tibias, and pelves of IL-1Ra−/− (n=17, female, 5 to 6 weeks old) and wild-type (n=14, female, 5 to 6 weeks old) mice and were treated with hemolysis buffer. T cells were removed by treating the BM cells with anti-mouse Thy1.2 magnetic beads and passing the cells through an MACS column. Purified BM cells (107 cells/mouse) in 0.2 mL PBS were transplanted intravenously into lethally irradiated (750 rad) recipient mice at 4 weeks of age (IL-1Ra−/−, n=12, female; wild-type mice, n=17, female). The recipient mice were histologically examined 12 and 24 weeks later.
All values were calculated as the mean±SD except where indicated. Fisher’s exact test was used for evaluation of the incidence of aortitis between unpaired groups. To compare the values between 2 independent groups, we used the Student t test for echocardiographic and hemodynamic values, tissue weights, and cytokine levels. To compare discontinuous values between 2 independent groups, such as aortitis severity score, we used the Mann-Whitney U test. A value of P<0.05 was considered significant.
Development of Aortitis in IL-1Ra−/− Mice
IL-1Ra−/− mice on the BALB/c background spontaneously developed arterial inflammation beginning at the age of 4 weeks, and ≈50% of them were affected by the age of 12 weeks (Table 1). Interestingly, on the C57BL/6J background, there were no signs of arterial inflammation (data not shown), suggesting the involvement of background genes in the development of aortitis; a similar observation has been made in the case of arthritis.4 Inflammation developed at several sites in the artery, including the region of the coronary artery ostium near the aorta (Figure 1). Arterial inflammation in IL-1Ra−/− mice was not influenced by sex (incidence of 58% [7/12] in male mice and of 45% [5/11] in female mice at 10 to 14 weeks old; P=0.42 by Fisher’s exact test). IL-1Ra−/− mice also developed mild myocarditis in the subepidermal pericardium at low incidence (data not shown).
Infiltration of monocytes and occasionally neutrophils was observed in the aorta and valve, and a loss of elastic lamellae in the aortic media was observed on histological examination. Monocytes/macrophages and some neutrophils infiltrated the inflammatory sites in the aortic sinus (Figure 2A). Thus, aortic inflammation may have characteristics of both the acute and chronic phases. We found numerous examples of neovascularization at sites of severe lesions (score 3; Figure 2B). Chondrocyte-like cells were observed in most of the aortas of IL-1Ra−/− mice, although no such cells were observed in the aortas of wild-type mice (Figure 2C). The chondrocyte-like cells were detected at sites of severe inflammation that also exhibited loss of elastic lamellae in the media. To determine whether calcification existed within arterial walls, the sections were stained with hematoxylin/eosin and von Kossa‘s technique after eosin staining. Calcification of the media of the aorta was observed in ≈30% of affected IL-1Ra−/− mice (Figure 2D and data not shown).
Correlation Between Aortitis and Arthritis
As shown in Table 2, 53% of IL-1Ra−/− mice developed aortitis by 14 weeks of age, whereas 95% of these mice developed arthritis at 14 weeks of age. However, the mutant mice developed aortitis as early as 4 weeks of age, a time when they had not yet developed arthritis (Table 2). Although most of the mice that developed aortitis also developed arthritis, a few of them developed aortitis only without any sign of arthritis at 14 weeks of age (Table 2). This observation was confirmed by histological examination of the joints of IL-1Ra−/− mice that had developed aortitis (data not shown). Thus, the development of aortitis is not necessarily correlated with the development of arthritis.
Development of Cardiac Hypertrophy in IL-1Ra−/− Mice
Because the aortic valve plays a crucial role in heart function and arterial inflammation in IL-1Ra−/− mice occurs specifically in the aortic sinus, we took echocardiograms of IL-1Ra−/− and wild-type mice to examine valve function under conditions of Avertin anesthesia (Table 3). The thickness of both the interventricular septum wall and the LV posterior wall was notably increased. In contrast, LV end-diastolic and end-systolic dimensions and fractional shortening, which are reported to be influenced by Avertin anesthesia,15 were unchanged, suggesting that the effect of anesthesia was low, if at all. Pressure gradient and flow velocity were significantly increased in IL-1Ra−/− mice. These results suggest that LV function is normal, that the pressure gradient is affected by mild aortic stenosis, and that LV hypertrophy may be induced by pressure overload.
Furthermore, we measured blood pressure and heart rate in 4 IL-1Ra−/− mice and compared these values with these of 4 wild-type mice (Table 4). IL-1Ra−/− mice showed normal blood pressure, but they also showed a small but significant decrease in heart rate under nonanesthetized conditions. The heart weight of IL-1Ra−/− mice was similar to that of wild-type mice, as was their body weight (Table 4).
Development of Aortitis in Mice That Received Transplants of IL-1Ra−/− T Cells or BM Cells
We have previously reported that IL-1Ra−/− mice showed increased levels of total IgG, IgG1, or IgE and autoantibodies against Igs, type II collagen, and dsDNA, suggesting involvement of an autoimmune mechanism in the development of disease in this mouse strain.4 The observation of abundant CD4+ T-cell infiltration at sites of arterial inflammation in IL-1Ra−/− mice also supports this notion.6 Thus, we examined the role of T cells in the development of aortitis by peripheral T-cell transplantation. Transplantation of T cells from wild-type mice induced mild aortitis at a low incidence in nu/nu mice. In contrast, T cells from IL-1Ra−/− mice induced aortitis at a much higher incidence. The severity score was also significantly increased in this experimental group, indicating that T cells are involved in the development of aortitis in IL-1Ra−/− mice (Figure 3A and 3B and Table 5). To determine whether IL-1Ra deficiency in T cells itself or T-cell sensitization in IL-1Ra−/− mice was important for the development of aortitis, we performed IL-1Ra−/− BM cell transplantation into wild-type recipients. Irradiated control mice without BM cell transplantation died within 2 weeks. Wild-type mice that received wild-type BM cells did not develop any arterial inflammation. A high incidence (100% and 71% at 12 and 24 weeks after transplantation, respectively) of aortitis was observed in wild-type mice that received BM cells from IL-1Ra−/− mice (Figure 3C and 3D and Table 5). When wild-type BM cells were transplanted into IL-1Ra−/− mice, no protective effect on the development of aortitis was observed (incidence of 100% and 33% at 12 and 24 weeks after transplantation, respectively). These results demonstrate that IL-1Ra deficiency in T cells is responsible for the development of aortitis.
Suppression of Aortitis in TNF-α–Deficient but Not IL-6–Deficient, IL-1Ra−/− Mice
It has been suggested that TNF-α and IL-6 are involved in the development of cardiovascular diseases.16 Therefore, we studied the roles of TNF-α and IL-6 in the development of aortitis in IL-1Ra−/− mice by generating doubly gene-deficient mice. The aortic valves of TNF-α−/−–IL-1Ra−/− or IL-6−/−–IL-1Ra−/− mice were histologically analyzed at 14 or 8 weeks of age, respectively. Interestingly, TNF-α−/−–IL-1Ra−/− mice showed no signs of arterial inflammation, whereas ≈50% of the IL-1Ra−/− mice developed aortitis (Figure 4 and Table 6). On the other hand, the incidence of aortitis was increased in IL-6−/−–IL-1Ra−/− mice, although the difference was not statistically significant (by Fisher’s exact test, P=0.09). The severity score was comparable to that in IL-1Ra−/− mice. These observations indicate that TNF-α is crucial for the development of aortitis in IL-1Ra−/− mice.
In IL-1Ra−/− mice, TNF-α protein levels in the blood were slightly higher than in wild-type mice, whereas the levels of IL-1α, IL-1β, and IL-6 were normal compared with wild-type mice (Table 7).
In this report, we have demonstrated that T cells play a crucial role in the pathogenesis of aortitis in IL-1Ra−/− mice on the BALB/c background and that TNF-α is essential for development of the disease. Inflammation of the cardiovascular system was preferentially observed at the aortic root of IL-1Ra−/− mice. As a result, these mice developed mild aortic stenosis and hyperplasia of both the interventricular septum wall and the LV posterior wall. However, the severity of these phenotypes seemed to be much milder on the BALB/c background than on the 129/O1a×MF1 background,6 in which not only the aortic root but also the main arteries were affected at a high incidence, especially at branch points. It is possible, however, that this apparent difference may reflect not that due to genetic backgrounds but to the ages of the mice, because exact ages of the mice were not known in the preceding report.6 Shepherd et al17 also recently reported that IL-1Ra−/− mice on the BALB/c background spontaneously develop aortitis. These authors reported that these mice also spontaneously develop cutaneous inflammation, and we also observed similar signs in our IL-1Ra−/− mice (authors’ unpublished observations). Shepherd et al reported that aortic inflammation was normally observed in IL-1Ra−/− (BALB/c×C57BL/6) F2 hybrid mice as in IL-1Ra−/−–BALB/c mice, whereas arthritis was rarely seen in the hybrid mice, suggesting that different background genes are involved in the development of aortitis and arthritis.
At the aortic root of IL-1Ra−/− mice, infiltration of monocytes and macrophages was observed frequently, but accumulation of foam cells, which are derived from macrophages and cause atherosclerosis, was not observed. Occasional infiltration of neutrophils was observed. Loss of elastic lamellae in the aortic media and occasional calcification of the media, signs of degenerative processes that mainly reflect degradation of smooth muscle cells,18,19 were observed in these mice. Neovascularization was also frequently observed, reflecting inflammation. These pathological findings resemble some aspects of Takayasu arteritis or polyarteritis nodosa in humans, in agreement with a previous report.6
We have demonstrated that peripheral T cells from IL-1Ra−/− mice can cause aortitis in nu/nu mice, suggesting that activated and/or memory T cells are generated and involved in the development of aortitis. Because IL-1Ra deficiency in BM cells could induce aortitis in wild-type recipient mice, it was suggested that T-cell intrinsic disjunction rather than abnormality of positive-negative selection of T cells in the thymus was responsible for the development of aortitis. With regard to this concept, we have shown that the development of arthritis in IL-1Ra−/− mice was also dependent on T cells.8 We showed that IL-1 signaling activates T cells by enhancing CD40L and OX40 expression on T cells and causes the development of autoimmunity.4,20,21 Furthermore, we showed that IL-1Ra is produced by CD4+ T cells and regulates the action of IL-1 in an autocrine manner.8 Thus, we suggest that IL-1Ra–deficient T cells are excessively activated even by physiological levels of IL-1 and may lose tolerance for aortic endothelial cell components, resulting in the development of autoimmunity and inflammation.
It is known that a small portion of mainstream aortic flow is intercepted during systole by the sinus ridge, or the downstream corner of the sinus of Valsalva; this fluid curls back toward the ventricle to form a large eddy, or vortex, that spins within the sinus cavity and generates turbulence.22 Hemodynamic force may affect structural and metabolic aspects of vascular endothelial cells,23 and high shear forces on the leaflet may lead to increased cell damage or turnover,24 resulting in production of IL-1 from these cells. Indeed, it is known that IL-1 release is increased at the aortic root or at the branch point of the aorta where cells are exposed to mechanical stress caused by blood flow.25 Therefore, in the absence of IL-1Ra, T cells near the areas where cells are exposed to mechanical stress may be excessively activated.
In IL-1Ra−/− mice, serum levels of myeloperoxidase anti-neutrophil cytoplasmic antibodies, which increase in some types of systemic vasculitis in humans, were not increased (data not shown), although the levels of other autoantibodies such as anti-IgG and anti–type II collagen were increased.4 These pathologies closely resemble human systemic vasculitis that is typically not associated with anti-neutrophil cytoplasmic antibodies (polyarteritis nodosa, Takayasu arteritis, and giant-cell arteritis). The pathogenic antigens in the aorta in this model remain to be elucidated.
We have shown that most of the mice that developed aortitis also developed arthritis, suggesting that these 2 diseases have a similar pathogenesis (or mechanism). Indeed, we have shown that both diseases are caused by a T cell–dependent mechanism. However, considering the facts that aortitis begins to develop earlier than arthritis and that a large proportion of mice develop only 1 of the diseases, either aortitis (5%) or arthritis (47%), at 14 weeks of age, the pathogenic processes underlying these diseases may be different in part.
Interestingly, we found that TNF-α deficiency suppressed the development of aortitis in IL-1Ra−/− mice. In contrast, IL-6 deficiency in IL-1Ra−/− mice showed pathological findings of aortitis. These results indicate that TNF-α plays an important role in the development of aortitis. TNF-α deficiency but not IL-6 deficiency also suppressed the development of arthritis in IL-1Ra−/− mice.8 Consistent with these observations, circulating levels of TNF-α but not of IL-6 were increased in IL-1Ra−/− mice. In this context, it is known that activation of antigen-presenting cells by activated T cells through interaction with CD40/CD40L induces TNF-α.26 Thus, TNF-α production may be enhanced in antigen-presenting cells through interaction with activated T cells in IL-1Ra−/− mice. Furthermore, we previously reported that TNF-α production was induced in T cells by IL-1 stimulation27 and that T cell–derived TNF-α played an important role in the pathogenesis of contact hypersensitivity27 and arthritis.8 TNF-α production by CD4+ T cells is also induced on stimulation with anti-CD3 monoclonal antibody, and IL-1Ra−/− T cells produce significantly higher levels of TNF-α together with IL-4 and interferon-γ than do wild-type T cells in culture supernatants.8 Other investigators have also reported the production of TNF-α in T cells28,29 and the presence of TNF receptors in aortic smooth muscle and endothelial cells.30 Thus, excess TNF-α produced by IL-1Ra−/− T cells and antigen-presenting cells may activate endothelial cells to produce excessive amounts of various inflammatory cytokines and chemokines, resulting in the development of inflammation.31 It is also known that TNF-α induces the expression of vascular cell adhesion molecule-1 in endothelial cells, which promotes early adhesion of mononuclear leukocytes to the arterial endothelium at sites of inflammation.32 Although transfer of TNF-α−/−–IL-1Ra−/− T cells into nu/nu mice will help evaluate the contribution of T cell–derived TNF-α to the development of aortitis separately from that of antigen-presenting cells, we were unable to address this question because of the difference in the major histocompatibility locus between TNF-α−/− mice (H-2 locus b/b) and BALB/c-nu/nu mice (H-2 locus d/d), even after 8 generations of backcrossing to BALB/c strain.
Taken together, our observations suggest that excessively activated T cells are responsible for the development of aortitis and that TNF-α mediates the inflammatory process. Autoimmune responses against specific antigens on vessel walls may thus be induced, as in the case of arthritis in these mice. However, further analysis is necessary to confirm this finding, because it is also possible that excessively activated T cells directly induce inflammation by producing inflammatory cytokines without the involvement of autoimmunity. Nonetheless, it is possible that both aortitis in IL-1Ra−/− mice and anti-neutrophil cytoplasmic antibody–associated systemic vasculitis in humans share a similar pathogenic process involving TNF-α. Consistent with this notion, it was recently reported that infliximab, an anti–TNF-α antibody, improved endothelial dysfunction in anti-neutrophil cytoplasmic antibody–associated systemic vasculitis in humans.33 These observations provide new insights into the pathogenesis of vasculitis, and the IL-1Ra−/− mouse should be a useful model to study the pathogenic mechanisms of vasculitis.
This work was supported by grants from the Ministry of Education, Science, Sport and Culture of Japan; the Ministry of Health and Welfare of Japan; and the Japan Society for the Promotion of Science. We thank Drs Kenji Sekikawa (National Institute of Animal Health, Japan) and Manfred Kopf (Swiss Federal Institute of Technology, Zurich) for kindly providing TNF-α−/− mice and IL-6−/− mice, respectively. We also thank Drs Katsuko Sudo and Aya Nambu for technical support and critical comments. We thank all members of our laboratory for their discussions and help in animal care.
Turnbull AV, Rivier CL. Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action. Physiol Rev. 1999; 79: 1–71.
Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, Ikuse T, Asano M, Iwakura Y. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med. 2000; 191: 313–320.
Nicklin MJ, Hughes DE, Barton JL, Ure JM, Duff GW. Arterial inflammation in mice lacking the interleukin 1 receptor antagonist gene. J Exp Med. 2000; 191: 303–312.
Horai R, Asano M, Sudo K, Kanuka H, Suzuki M, Nishihara M, Takahashi M, Iwakura Y. Production of mice deficient in genes for interleukin (IL)-1α, IL-1β, IL-1α/β, and IL-1 receptor antagonist shows that IL-1β is crucial in turpentine-induced fever development and glucocorticoid secretion. J Exp Med. 1998; 187: 1463–1475.
Horai R, Nakajima A, Habiro K, Kotani M, Nakae S, Matsuki T, Nambu A, Saijo S, Kotaki H, Sudo K, Okahara A, Tanioka H, Ikuse T, Ishii N, Schwartzberg PL, Abe R, Iwakura Y. TNF-α is crucial for the development of autoimmune arthritis in IL-1 receptor antagonist-deficient mice. J Clin Invest. 2004; 114: 1603–1611.
Isoda K, Nishikawa K, Kamezawa Y, Yoshida M, Kusuhara M, Moroi M, Tada N, Ohsuzu F. Osteopontin plays an important role in the development of medial thickening and neointimal formation. Circ Res. 2002; 91: 77–82.
Isoda K, Kamezawa Y, Ayaori M, Kusuhara M, Tada N, Ohsuzu F. Osteopontin transgenic mice fed a high-cholesterol diet develop early fatty-streak lesions. Circulation. 2003; 107: 679–681.
Hoit BD, Khoury SF, Kranias EG, Ball N, Walsh RA. In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency. Circ Res. 1995; 77: 632–637.
Hart CY, Burnett JC Jr, Redfield MM. Effects of avertin versus xylazine-ketamine anesthesia on cardiac function in normal mice. Am J Physiol Heart Circ Physiol. 2001; 281: H1938–H1945.
Nakae S, Asano M, Horai R, Sakaguchi N, Iwakura Y. IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells. J Immunol. 2001; 167: 90–97.
Peacock JA. An in vitro study of the onset of turbulence in the sinus of Valsalva. Circ Res. 1990; 67: 448–460.
Davies PF, Remuzzi A, Gordon EJ, Dewey CF Jr, Gimbrone MA Jr. Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro. Proc Natl Acad Sci U S A. 1986; 83: 2114–2117.
van Kooten C, Banchereau J. CD40-CD40 ligand. J Leukoc Biol. 2000; 67: 2–17.
Nakae S, Komiyama Y, Narumi S, Sudo K, Horai R, Tagawa Y, Sekikawa K, Matsushima K, Asano M, Iwakura Y. IL-1-induced tumor necrosis factor-α elicits inflammatory cell infiltration in the skin by inducing IFN-γ-inducible protein 10 in the elicitation phase of the contact hypersensitivity response. Int Immunol. 2003; 15: 251–260.
Ramshaw AL, Roskell DE, Parums DV. Cytokine gene expression in aortic adventitial inflammation associated with advanced atherosclerosis (chronic periaortitis). J Clin Pathol. 1994; 47: 721–727.
Booth AD, Jayne DR, Kharbanda RK, McEniery CM, Mackenzie IS, Brown J, Wilkinson IB. Infliximab improves endothelial dysfunction in systemic vasculitis: a model of vascular inflammation. Circulation. 2004; 109: 1718–1723.
Vasculitis syndromes such as Takayasu arteritis and giant-cell arteritis involve massive recruitment of lymphocytes and macrophages into the vascular wall, destruction of the medial layer with concurrent fibrosis, and proliferation of smooth muscle cells in the intima, leading to neointima formation. Although a number of potential mechanisms, including microbial infection and autoimmune reactions, have been implicated in the development of inflammatory reactions in the vasculature, the precise mechanism underlying the development of vasculitis remains to be elucidated. In this issue, we showed that IL-1Ra−/− mice, in which excess IL-1 signaling is induced under physiological conditions owing to deficiency of the antagonist, spontaneously develop aortitis at the root of the aorta, with massive infiltration of macrophages and monocytes and loss of elastic lamellae in the aortic media. LV hypertrophy and mild aortic stenosis were also shown by transthoracic echocardiography. These pathological findings resemble some aspects of Takayasu arteritis or polyarteritis nodosa in humans, indicating that IL-1Ra−/− mice are a good model for these vascular diseases. Interestingly, transplantation of T cells from IL-1Ra−/− mice induced aortitis in recipient nu/nu mice, suggesting involvement of T cells in pathogenesis. Furthermore, TNF-α deficiency completely suppressed the development of aortitis in IL-1Ra−/− mice, whereas IL-6 deficiency did not. These observations indicate that both IL-1 and TNF-α play crucial roles in the development of aortitis in IL-1Ra−/− mice. Therefore, control of either IL-1 or TNF-α activity may be beneficial for the treatment of vasculitis in humans.
T.M. is currently at the ERATO Yanagisawa Orphan Receptor Project, Japan Science and Technology Agency, Tokyo, Japan, and R.H. is currently at the National Human Genome Research Institute, National Institutes of Health, Bethesda, Md.