Elastic-Vessel Arteritis in Interleukin-1 Receptor Antagonist–Deficient Mice Involves Effector Th1 Cells and Requires Interleukin-1 Receptor
http://www.100md.com
《循环学杂志》
the Division of Genomic Medicine, University of Sheffield, Sheffield, United Kingdom.
Abstract
Background— In mice that lack interleukin-1 receptor antagonist (IL-1ra), transmural inflammation of the elastic arteries develops at sites of turbulent flow. We described late histopathology previously. Here, we investigate the cellular events in nonlethal arteritis at the aortic root and compare them with Takayasu’s arteritis and giant cell arteritis.
Methods and Results— IL-1ra–deficient mice were inbred from the original stocks and from BALB/c backcrosses. Disease was ascertained histologically and immunohistologically postmortem at the aortic root. Onset appeared to be stochastic and was not detectably age dependent; in our local Sf3 strain, the half-time of onset was 52 days. Loss of the type I IL-1 receptor suppressed the arteritis. Microvascular activation, as determined by absence of strong E-selectin expression, was absent from preaffected vessels. In mildly affected cases, infiltration was adventitial. In severely affected animals, infiltrates appeared to be active in destroying elastin, but resynthesis of disorganized elastin occurred at closely adjacent sites. Infiltrates consisted predominantly of macrophages but were rich in CD4+–interferon-+ cells, which are likely to represent Th1 cells. Dendritic cells accumulated in lesional areas.
Conclusions— The arteritic phenotype of IL-1ra deficiency is mediated by the interleukin-1 receptor and involves effector Th1 cells. The destructive pattern and many of the cellular features of arteritis in IL-1ra–deficient mice resemble the human elastic-vessel arteritides, for which these mice may be a useful animal model.
Key Words: immunohistochemistry ; immunology ; inflammation ; interleukins ; receptors
Introduction
The interleukin-1 (IL-1) receptor antagonist protein (IL-1ra; gene symbol Il1rn) is a homolog and an endogenous competitive antagonist of the proinflammatory cytokines IL-1 and IL-1; (collectively called IL-1) for binding to their single known functional (type I) IL-1 receptor (IL-1R1; see review in Dinarello1), which is widely distributed. IL-1ra is produced at a low basal level by many tissues and has been reported to be inducibly expressed by vascular smooth muscle and endothelial cells.2,3 It is most actively expressed, along with IL-1, by monocytes and macrophages that are subjected to a proinflammatory stimulus in vitro or in vivo.4 Cells respond strongly to partial occupancy of receptors by IL-1, despite an often large local excess of competing IL-1ra.5
IL-1ra deficiency in laboratory mice creates 3 apparently spontaneous inflammatory diseases, with strain selectivity. Humoral autoimmunity appears limited or absent. We have demonstrated transmural inflammation at sites of turbulence in the elastic arteries from mice derived from outbred 129xMF1 Il1rn–/– mice6 and BALB/c mice (Harlan)7 and localized psoriasiform cutaneous inflammation in Il1rn–/– BALB/c,7 whereas Horai and colleagues have demonstrated arthritis in the same strain, which we have confirmed.7,8
Chronic inflammation of the elastic arteries is seen in 2 rare but medically serious human disorders: giant cell or temporal arteritis (GCA)9 and Takayasu’s arteritis (TA).10 GCA is a disease of age, whereas TA affects young adults. GCA tends to focus on the upper extracranial branches of the aorta, whereas TA affects the thoracic and brachial branches. Both affect the aorta in a significant minority of cases. Both GCA and TA are regarded as medical emergencies because they can result in ischemia to vital organs, particularly the retina, brain, and myocardium. TA and GCA are treated with steroids but can be refractory and usually relapse when the drug is withdrawn. To avoid the side effects of chronic systemic use of steroids, steroid-sparing immunosuppression is often indicated, which carries other obvious risks. Consequently, new therapeutic strategies should be sought; however, the rarity of the diseases is an impediment to clinical trials, and animal models are likely to be valuable. In one currently available model, arteries from patients affected with GCA can be grafted into severe combined immunodeficient (SCID) mice, which allows manipulation of the transferred leukocyte populations.11 The other type of model can be induced by persistent infection, particularly by Herpesviridae in mice, but remains dependent on viral replication.12 We suggest that apparently spontaneous arteritis in Il1rn–/– mice might also be considered. We have already reported that there are pathological features of the mouse disease6 that are shared with TA13 and GCA9: lesions are sited in the high-pressure arterial system; inflammation extends throughout the vessel wall but is longitudinally delimited; and there is dense macrophage accumulation, elastin destruction (which sometimes leads to aneurysms), and smooth muscle proliferation into the vessel intima that causes vessel obstruction. Acute illness in Il1rn–/– mice also tends to be caused by vessel rupture or by ischemia resulting from vessel stenosis. At the cellular level, we also showed that the destruction appears to be the result of activated macrophages associated with T cells, although giant multinuclear cells are absent.
To identify some of the processes in the etiology of the arteritis in Il1rn–/–-deficient mice, we now report an immunohistological analysis of disease lesions in apparently healthy mice. We selected the aortic root as an easily identifiable and frequently affected site. We show genetically that arteritis is mediated by IL-1R1 and hence by IL-1.
Methods
Mice
BALB/c and C57BL/6 stocks were from Harlan (United Kingdom). The Sf3 line was originally derived from a cell line from a 129P2/Hsd mouse and the outbred line MF1 (Harlan)6 and has been sib-sib inbred. The line was rederived into specific pathogen-free conditions at generation 5. All Sf3 mice described here are descended from 1 inbred pair from the tenth generation (expected residual heterozygosity of 12%). Both alleles of Il1rn are retained in the stock. Wild-type and Il1rn–/– animals are from the same or closely related litters. The null allele of Il1rn was backcrossed onto BALB/c to N5 before inbreeding to generate Il1rn–/– and Il1rn+/+ littermates. All mice were housed behind a positive-pressure barrier; technical staff showered before entering the facility and wore full-body overalls. All material that came into the facility was sterilized. Mice were sampled periodically and found to be seronegative for specific pathogens, described previously.6 Mice were reared under UK Home Office Project and Personal Licenses and were usually killed by cervical dislocation. The work received approval from the appropriate ethics review panel of the University of Sheffield.
Sections and Inflammation Scoring
Frozen sections (6 μm) were prepared by standard methods and stored at –70°C. Alternatively, tissue was fixed for >24 hours in 3.7% neutral phosphate-buffered formaldehyde or zinc-Tris fixative or dissected from formaldehyde-fixed cadavers and processed to paraffin. At least 1 section per 120 μm was stained with hematoxylin and eosin to reveal sites of inflammation in the arterial wall. In time-course samples, in which frozen sections were examined, the degree of inflammation was graded by estimating the number of interferon (IFN)-+ cells present in the lesion on the most densely inflamed single 6-μm section. Grades were assigned thus: 0 (<10 cells), 1 (10 to 100 cells), 2 (100 to 1000 cells), and 3 (>1000 cells).
With the exception of some major histocompatibility complex II (MHC II) and F4/80 staining, immunohistological visualization of antigens was done, by necessity, on frozen sections. Primary monoclonal antibodies were detected with biotinylated secondary antibodies and then revealed with peroxidase or phosphatase-coupled avidin-biotin complexes (ABC reagent kits, Vector Laboratories). The peroxidase substrate was diaminobenzidine, and the phosphatase substrate was Vector Red (Vector Laboratories). All washes and incubations were performed with PBS/0.1% saponin; otherwise, standard methods were used in all cases. A list of the antibodies that we used in the study is available from the authors on request. Connective tissue remodeling and degradation was detected by elastin and van Gieson staining by standard procedures.
Results
Epistasis Between Il1rn and Il1r1
We bred together receptor-deficient (Il1rn–/– C57BL/6) and Il1rn–/– Sf3 mice. Progeny were crossed for 2 further generations against the Sf3 line. Double heterozygotes (Il1r1+/–-Il1rn+/–) were inbred, and Il1r1+/–- Il1rn–/– progeny were inbred again. Twelve Il1rn–/–-Il1r1+/+ and 20 Il1r1–/–-Il1rn–/– littermates were then reared to 200 days. Aortic root lesions were found in 10 of 12 Il1rn–/–/Il1r1+/+ and 0 of 20 Il1rn–/–/Il1r1–/– mice (P<0.001, 1-tailed Fisher’s exact test). A small group of Il1rn–/–-Il1r1+/– mice from this cross was also initially retained pending genotyping. When reared to 200 days, 4 of 7 of these mice were found to be significantly affected (compared with the double-null group, P<0.002, Fisher’s exact test). Two had grade 3 lesions.
Time Course of Onset of Aortitis in Il1rn–/– Sf3 Mice
To gain insight into the early pathology of arteritis, mice used in the present study were all killed while apparently healthy and graded as described in Methods. Figure 1 illustrates the status of infiltration as a function of age in 23 Sf3 Il1rn–/– mice (median age 105 days) and 8 wild-type controls (median age 172 days). In the wild-type group, 2 of 8 cases of grade 1 infiltration were observed. In the Il1rn–/– cohort, 19 of 23 scored grade 1 or above. The Il1rn-deficient animals were significantly more likely to score 1 than the wild type (P<0.01, 1-tailed Fischer exact test). The 2 small IFN-+ infiltrates in 24-day-old and 108-day-old wild-type mice also appeared to contain CD4+ cells (Figures 2e through 2g); however, because we saw no evidence of destructive aortitis in wild-type animals, we chose to reject a grade 1 score as a reliable indication of developing aortitis. We have found no evidence for a minimum age of onset of aortitis (the earliest histologically confirmed case was at 31 days), whereas all Il1rn–/– mice in the present study were affected at 200 days. Assuming irreversible onset in Il1rn–/– mice and age-independent rate of onset, we found that the data best fit a half-life of onset (grade >1) of 52 days. The susceptibility and rate of onset of aortitis grade >1 were not detectably different between 17 male and 11 female Sf3. Inflammatory infiltrate in Il1rn–/– mice typically was observed near the opening of the coronary arteries, where turbulence is high. Examples are shown in Figures 2a through 2d of IFN-–immunostained frozen sections of aortic roots that were unaffected or had grade 1, grade 2, and grade 3 infiltration. We note that lethal disease appeared to be retarded in the Sf3 Il1rn–/– mice when reared under barrier conditions compared with the original outbred colony in quarantine.6
General Vascular Activation Does Not Precede Inflammation in Il1rn–/– Mice
We detected no expression of the endothelial activation marker E-selectin (Figure 3a) in unaffected Il1rn–/– mice, whereas CD31+ endothelial cells were readily detectable in adjacent sections (Figure 3b). In affected animals, E-selectin (Figures 3c and 3d) was expressed strongly and focally and appeared to correlate with sites of apparent destructive activity, such as the margin of damaged elastin (Figure 3e).
Destruction of Elastic Laminae
Both GCA and TA feature localized transmural infiltration of the vessel by leukocytes from the microvasculature of the tunica adventitia.9,10,11,13 Our observations (such as Figures 2c and 4 a) indicate the same route of infiltration in Il1rn–/– mice. In Figures 4a, 4b, and 4c, adjacent sections of tissue from a severely affected Il1rn–/– mouse show the root of a coronary artery (indicated by "C"). For comparison, Figure 4d shows a grazing section of a coronary artery of a wild-type mouse. Here, it was possible to trace the ribbons of healthy elastin from the aortic sinus into the neck of the vessel. The elastin was coarsely structured and strongly stained (Figure 4e). Figures 4f and 4h, by contrast, show typical thin, diffuse, disorganized elastin in a severely inflamed coronary artery from an Il1rn–/– mouse but a heavily thickened fibrous (red) layer. Figures 4i and 4k show low-resolution images of connective tissue and macrophages, respectively, at the base of a severely affected aortic valve. An area of intense infiltration (arrow in Figure 4k) coincided with ragged elastin (arrow in Figure 4i). At high resolution (Figure 4j), ribbons of elastin appeared to have been severed. The absence of organized elastin from the infiltrated section was confirmed by ultraviolet autofluorescence (Figure 4l) of the same section as Figure 4k. This showed dim, amorphous fluorescence, clearly different from the bright, localized ribbons of elastin seen in a healthy aortic root (Figures 4n and 4o).
Chondrometaplasia was also observed in established fibrotic lesions. In several cases (5 of 23 affected animals, aged 125 to 209 days), nodules that resembled cartilage (and that were stained with toluidine blue) were located at the base of the aortic valve (Figure 4m) or in the media of the aorta.
Involvement of Activated T Cells
The inset in Figure 4k, which shows macrophage-specific staining, is an approximately equivalent area to those shown in Figures 5a through 5e. IL-4+ cells (Th2) were detectable (Figure 5b) although not abundant at heavily inflamed sites. In sections from 2 Sf3 mice and 1 BALB/c Il1rn–/– mouse, the ratio of IL-4+ to IFN-+ cells was estimated to be <0.1, 0.2, and 0.1, respectively.
CD4+ cells (Figure 5a) are similarly distributed and are colocalized with dense staining for IFN- (Figure 5c). We therefore stained for IFN- and CD4 on the same section (Figure 5m) taken from another mouse. Coincidence of black (IFN-) and red (CD4) staining indicated the presence of activated Th1 cells. (Figures 5k and 5l show the antigens separately.) Localized expression of the chemokine CCL2 (Figure 5f) was detectable at sites of CD4+ cell infiltration (Figure 5g), where there were also indications of localized E-selectin expression (Figure 5h) in the CD31+ vascular network (Figure 5i). CCL2 was not detected in wild-type mice.
Dendritic Cells
Dendritic cells are intensely MHC II+ in situ. Stimulated endothelial cells and macrophages are also MHC II+, although expression by dendritic cells is expected to be much more intense. MHC II+ cells in wild-type mice (Figure 5e) did not consistently line vessels (and so were not endothelial) and were too few, compared with F4/80+ cells, to be identified with macrophages (Figure 4k). Also, some MHC II+ cells had dendritic morphology (see the indicated cells in Figure 5e). Ramification yielded a large stained area with anti-MHC II. CD205 has a strong specificity toward dendritic cells and identified a similar group of cells to anti-MHC II (Figure 5d). Fainter CD205+ cells are probably macrophages. As in human vessels,14 dendritic cells (visualized as MHC II+ cells) tended to form a ring around the media in the wild-type artery (Figure 5j) and occupied the fibrous tissue of the valve itself. Anti-CD205 antibodies detected no cells in wild-type aortic roots, possibly because CD205 is only upregulated to detectable levels during maturation. Thus, although dendritic cells appear to be recruited to affected tissue (Figures 4a and 5 e) versus wild-type tissue (Figure 5j), they were not obviously codistributed with the majority of effector T cells (Figure 5c).
Discussion
If IL-1 and IL-1; are the only agonists for IL-1R1, and IL-1ra has no other role but to block that interaction, then deficiency of the receptor15 should suppress the Il1rn–/– phenotype completely. Irikura et al16 have shown that the induced phenotypic features and general malaise observed in C57BL/6 Il1rn–/– animals are suppressed by overexpression of IL-1ra or by elimination of IL-1R1. We show that a single functional copy of the IL-1R1 gene (Il1r1) is essential for the pathology of IL-1ra deficiency, which implicates IL-1 as the mediator of the disease process.
Small aortic root infiltrates of CD4+ and IFN-+ cells, which could have been Th1 or NK-T cells, were observed in 2 young wild-type mice. We interpret these as normal responses to subclinical events. In an Il1rn–/– mouse, such events might instigate chronic disease, because a stimulus that causes IL-1 to be released in an Il1rn–/– mouse would be expected to produce an enhanced or prolonged proinflammatory response compared with the wild type. If IL-1ra were absent, then perhaps IL-1 that is released from the vasculature in response to constitutive physiological stimuli might be sufficient to cause general activation of the adventitial microvasculature, without involving an autoimmune response. However, we did not detect vascular activation (as E-selectin expression) in preaffected susceptible mice. It thus appears that strong, large-scale vascular activation is part of the inflammatory process and not an immediate consequence of IL-1ra deficiency.
In TA and GCA, the elastic laminae are subject to wholesale fragmentation and digestion by macrophages. Krettek et al17 have shown that de novo synthesis of disorganized elastin by vascular smooth muscle cells and lesional macrophages is simultaneous with laminar elastin destruction in human vascular disease. In Il1rn–/– mice, we have already noted invasion of the media by macrophages and destruction of the elastic laminae.6 We show here that synthesis of disorganized elastin as small fibers and fibrosis, which causes general tissue thickening, appears to occur very close to sites of elastin destruction.
The presence of an active IFN-–rich, CD4+, Th1-like population within arteritic lesions, in conjunction with macrophages and dendritic cells, is highly suggestive of an autoimmunity-driven pathology: in humans, MHC haplotype association with GCA18 and TA,19,20 strongly biased T-cell receptor usage in affected GCA arteries,21,22 and adoptive transfer of disease with CD4+ T cells in the GCA graft model11 all suggest autoimmunity that centrally involves T cells. However, Shimizu and colleagues23 have recently shown that Th2 rather than Th1 cells drive elastinolysis in aortic allografts, and a similar role is possible for the IL-4+ cells that are also present in Il1rn–/– arteritic lesions. In GCA and TA, dense accumulations of activated macrophages are involved in continuous mutual interaction between macrophages and CD4+ T cells.11,13 Iwakura24 recently reviewed the evidence for the involvement of IL-1 in T-cell activation in arthritis in Il1rn–/– mice and has presented a model in which T-cell receptor engagement with antigen presenting cells (probably dendritic cells) increases IL-1R expression on the T cell. Trapped dendritic cells have already been implicated in GCA.11,14 Maturation of dendritic cells can be triggered by a combination of IL-1 and CD40L,25 which could provide a direct link between IL-1 hyperactivity and the onset of a putative autoimmune state. Chemokine secretion would be expected to mediate the recruitment of more macrophages and effector T cells, as well as immobilize dendritic cells,22 and we show evidence of CCL2 in the mouse lesions.
Because of the apparent similarity in gross pathology and immunohistology, we suggest that Il1rn–/– mice might prove to be a tractable spontaneous experimental model for GCA and TA. In the future, manipulation of the leukocyte populations in Il1rn–/– mice may yield useful information about the possible nature of vascular autoantigens that could be beneficial to the therapy of GCA and TA.
Acknowledgments
This study was supported by grant RGA014661 from the British Heart Foundation. We thank Orla Gallagher for technical assistance and advice and the staff of the Field Laboratories for expert animal husbandry.
References
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Beasley D, McGuiggin ME, Dinarello CA. Human vascular smooth muscle cells produce an intracellular form of interleukin-1 receptor antagonist. Am J Physiol. 1995; 269: C961–C968.
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Nicklin MJH, Hughes DE, Barton JL, Ure JM, Duff. Arterial inflammation in mice lacking the interleukin-1 receptor antagonist gene. J. Exp. Med. 2000; 191: 303–311.
Shepherd J, Little MC, Nicklin MJH. Psoriasis-like cutaneous inflammation in mice lacking interleukin-1 receptor antagonist. J Invest Dermatol. 2004; 122: 665–669.
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Nordborg E, Nordborg C. Giant cell arteritis: epidemiological clues to its pathogenesis and an update on its treatment. Rheumatology. 2003; 42: 413–421.
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Ma-Krupa W, Jeon S, Spoerl S, Tedder TF, Goronzy JJ, Weyand CM. Activation of arterial wall dendritic cells and breakdown of self-tolerance in giant cell arteritis. J Exp Med. 2003; 199: 173–183.
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Inder SJ, Bobryshev YV, Cherian SM, Lord RS, Masuda K, Yutani C. Accumulation of lymphocytes, dendritic cells, and granulocytes in the aortic wall affected by Takayasu’s disease. Angiology. 2000; 51: 565–579.
Ma-Krupa W, Dewan M, Jeon M-S, Kurtin PJ, Younge BR, Goronzy JJ, Weyand CM. Trapping of misdirected dendritic cells in granulomatous lesions of giant cell arteritis. Am J Pathol. 2002; 161: C1815–C1823.
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Krettek A, Sukhova GK, Libby P. Elastogenesis in human arterial disease. Arterioscler Thromb Vasc Biol. 2003; 23: 582–587.
Gonzalez-Gay MA. Genetic epidemiology in giant cell arteritis polymyalgia rheumatica. Arthritis Res. 2001; 3: 154–157.
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Abstract
Background— In mice that lack interleukin-1 receptor antagonist (IL-1ra), transmural inflammation of the elastic arteries develops at sites of turbulent flow. We described late histopathology previously. Here, we investigate the cellular events in nonlethal arteritis at the aortic root and compare them with Takayasu’s arteritis and giant cell arteritis.
Methods and Results— IL-1ra–deficient mice were inbred from the original stocks and from BALB/c backcrosses. Disease was ascertained histologically and immunohistologically postmortem at the aortic root. Onset appeared to be stochastic and was not detectably age dependent; in our local Sf3 strain, the half-time of onset was 52 days. Loss of the type I IL-1 receptor suppressed the arteritis. Microvascular activation, as determined by absence of strong E-selectin expression, was absent from preaffected vessels. In mildly affected cases, infiltration was adventitial. In severely affected animals, infiltrates appeared to be active in destroying elastin, but resynthesis of disorganized elastin occurred at closely adjacent sites. Infiltrates consisted predominantly of macrophages but were rich in CD4+–interferon-+ cells, which are likely to represent Th1 cells. Dendritic cells accumulated in lesional areas.
Conclusions— The arteritic phenotype of IL-1ra deficiency is mediated by the interleukin-1 receptor and involves effector Th1 cells. The destructive pattern and many of the cellular features of arteritis in IL-1ra–deficient mice resemble the human elastic-vessel arteritides, for which these mice may be a useful animal model.
Key Words: immunohistochemistry ; immunology ; inflammation ; interleukins ; receptors
Introduction
The interleukin-1 (IL-1) receptor antagonist protein (IL-1ra; gene symbol Il1rn) is a homolog and an endogenous competitive antagonist of the proinflammatory cytokines IL-1 and IL-1; (collectively called IL-1) for binding to their single known functional (type I) IL-1 receptor (IL-1R1; see review in Dinarello1), which is widely distributed. IL-1ra is produced at a low basal level by many tissues and has been reported to be inducibly expressed by vascular smooth muscle and endothelial cells.2,3 It is most actively expressed, along with IL-1, by monocytes and macrophages that are subjected to a proinflammatory stimulus in vitro or in vivo.4 Cells respond strongly to partial occupancy of receptors by IL-1, despite an often large local excess of competing IL-1ra.5
IL-1ra deficiency in laboratory mice creates 3 apparently spontaneous inflammatory diseases, with strain selectivity. Humoral autoimmunity appears limited or absent. We have demonstrated transmural inflammation at sites of turbulence in the elastic arteries from mice derived from outbred 129xMF1 Il1rn–/– mice6 and BALB/c mice (Harlan)7 and localized psoriasiform cutaneous inflammation in Il1rn–/– BALB/c,7 whereas Horai and colleagues have demonstrated arthritis in the same strain, which we have confirmed.7,8
Chronic inflammation of the elastic arteries is seen in 2 rare but medically serious human disorders: giant cell or temporal arteritis (GCA)9 and Takayasu’s arteritis (TA).10 GCA is a disease of age, whereas TA affects young adults. GCA tends to focus on the upper extracranial branches of the aorta, whereas TA affects the thoracic and brachial branches. Both affect the aorta in a significant minority of cases. Both GCA and TA are regarded as medical emergencies because they can result in ischemia to vital organs, particularly the retina, brain, and myocardium. TA and GCA are treated with steroids but can be refractory and usually relapse when the drug is withdrawn. To avoid the side effects of chronic systemic use of steroids, steroid-sparing immunosuppression is often indicated, which carries other obvious risks. Consequently, new therapeutic strategies should be sought; however, the rarity of the diseases is an impediment to clinical trials, and animal models are likely to be valuable. In one currently available model, arteries from patients affected with GCA can be grafted into severe combined immunodeficient (SCID) mice, which allows manipulation of the transferred leukocyte populations.11 The other type of model can be induced by persistent infection, particularly by Herpesviridae in mice, but remains dependent on viral replication.12 We suggest that apparently spontaneous arteritis in Il1rn–/– mice might also be considered. We have already reported that there are pathological features of the mouse disease6 that are shared with TA13 and GCA9: lesions are sited in the high-pressure arterial system; inflammation extends throughout the vessel wall but is longitudinally delimited; and there is dense macrophage accumulation, elastin destruction (which sometimes leads to aneurysms), and smooth muscle proliferation into the vessel intima that causes vessel obstruction. Acute illness in Il1rn–/– mice also tends to be caused by vessel rupture or by ischemia resulting from vessel stenosis. At the cellular level, we also showed that the destruction appears to be the result of activated macrophages associated with T cells, although giant multinuclear cells are absent.
To identify some of the processes in the etiology of the arteritis in Il1rn–/–-deficient mice, we now report an immunohistological analysis of disease lesions in apparently healthy mice. We selected the aortic root as an easily identifiable and frequently affected site. We show genetically that arteritis is mediated by IL-1R1 and hence by IL-1.
Methods
Mice
BALB/c and C57BL/6 stocks were from Harlan (United Kingdom). The Sf3 line was originally derived from a cell line from a 129P2/Hsd mouse and the outbred line MF1 (Harlan)6 and has been sib-sib inbred. The line was rederived into specific pathogen-free conditions at generation 5. All Sf3 mice described here are descended from 1 inbred pair from the tenth generation (expected residual heterozygosity of 12%). Both alleles of Il1rn are retained in the stock. Wild-type and Il1rn–/– animals are from the same or closely related litters. The null allele of Il1rn was backcrossed onto BALB/c to N5 before inbreeding to generate Il1rn–/– and Il1rn+/+ littermates. All mice were housed behind a positive-pressure barrier; technical staff showered before entering the facility and wore full-body overalls. All material that came into the facility was sterilized. Mice were sampled periodically and found to be seronegative for specific pathogens, described previously.6 Mice were reared under UK Home Office Project and Personal Licenses and were usually killed by cervical dislocation. The work received approval from the appropriate ethics review panel of the University of Sheffield.
Sections and Inflammation Scoring
Frozen sections (6 μm) were prepared by standard methods and stored at –70°C. Alternatively, tissue was fixed for >24 hours in 3.7% neutral phosphate-buffered formaldehyde or zinc-Tris fixative or dissected from formaldehyde-fixed cadavers and processed to paraffin. At least 1 section per 120 μm was stained with hematoxylin and eosin to reveal sites of inflammation in the arterial wall. In time-course samples, in which frozen sections were examined, the degree of inflammation was graded by estimating the number of interferon (IFN)-+ cells present in the lesion on the most densely inflamed single 6-μm section. Grades were assigned thus: 0 (<10 cells), 1 (10 to 100 cells), 2 (100 to 1000 cells), and 3 (>1000 cells).
With the exception of some major histocompatibility complex II (MHC II) and F4/80 staining, immunohistological visualization of antigens was done, by necessity, on frozen sections. Primary monoclonal antibodies were detected with biotinylated secondary antibodies and then revealed with peroxidase or phosphatase-coupled avidin-biotin complexes (ABC reagent kits, Vector Laboratories). The peroxidase substrate was diaminobenzidine, and the phosphatase substrate was Vector Red (Vector Laboratories). All washes and incubations were performed with PBS/0.1% saponin; otherwise, standard methods were used in all cases. A list of the antibodies that we used in the study is available from the authors on request. Connective tissue remodeling and degradation was detected by elastin and van Gieson staining by standard procedures.
Results
Epistasis Between Il1rn and Il1r1
We bred together receptor-deficient (Il1rn–/– C57BL/6) and Il1rn–/– Sf3 mice. Progeny were crossed for 2 further generations against the Sf3 line. Double heterozygotes (Il1r1+/–-Il1rn+/–) were inbred, and Il1r1+/–- Il1rn–/– progeny were inbred again. Twelve Il1rn–/–-Il1r1+/+ and 20 Il1r1–/–-Il1rn–/– littermates were then reared to 200 days. Aortic root lesions were found in 10 of 12 Il1rn–/–/Il1r1+/+ and 0 of 20 Il1rn–/–/Il1r1–/– mice (P<0.001, 1-tailed Fisher’s exact test). A small group of Il1rn–/–-Il1r1+/– mice from this cross was also initially retained pending genotyping. When reared to 200 days, 4 of 7 of these mice were found to be significantly affected (compared with the double-null group, P<0.002, Fisher’s exact test). Two had grade 3 lesions.
Time Course of Onset of Aortitis in Il1rn–/– Sf3 Mice
To gain insight into the early pathology of arteritis, mice used in the present study were all killed while apparently healthy and graded as described in Methods. Figure 1 illustrates the status of infiltration as a function of age in 23 Sf3 Il1rn–/– mice (median age 105 days) and 8 wild-type controls (median age 172 days). In the wild-type group, 2 of 8 cases of grade 1 infiltration were observed. In the Il1rn–/– cohort, 19 of 23 scored grade 1 or above. The Il1rn-deficient animals were significantly more likely to score 1 than the wild type (P<0.01, 1-tailed Fischer exact test). The 2 small IFN-+ infiltrates in 24-day-old and 108-day-old wild-type mice also appeared to contain CD4+ cells (Figures 2e through 2g); however, because we saw no evidence of destructive aortitis in wild-type animals, we chose to reject a grade 1 score as a reliable indication of developing aortitis. We have found no evidence for a minimum age of onset of aortitis (the earliest histologically confirmed case was at 31 days), whereas all Il1rn–/– mice in the present study were affected at 200 days. Assuming irreversible onset in Il1rn–/– mice and age-independent rate of onset, we found that the data best fit a half-life of onset (grade >1) of 52 days. The susceptibility and rate of onset of aortitis grade >1 were not detectably different between 17 male and 11 female Sf3. Inflammatory infiltrate in Il1rn–/– mice typically was observed near the opening of the coronary arteries, where turbulence is high. Examples are shown in Figures 2a through 2d of IFN-–immunostained frozen sections of aortic roots that were unaffected or had grade 1, grade 2, and grade 3 infiltration. We note that lethal disease appeared to be retarded in the Sf3 Il1rn–/– mice when reared under barrier conditions compared with the original outbred colony in quarantine.6
General Vascular Activation Does Not Precede Inflammation in Il1rn–/– Mice
We detected no expression of the endothelial activation marker E-selectin (Figure 3a) in unaffected Il1rn–/– mice, whereas CD31+ endothelial cells were readily detectable in adjacent sections (Figure 3b). In affected animals, E-selectin (Figures 3c and 3d) was expressed strongly and focally and appeared to correlate with sites of apparent destructive activity, such as the margin of damaged elastin (Figure 3e).
Destruction of Elastic Laminae
Both GCA and TA feature localized transmural infiltration of the vessel by leukocytes from the microvasculature of the tunica adventitia.9,10,11,13 Our observations (such as Figures 2c and 4 a) indicate the same route of infiltration in Il1rn–/– mice. In Figures 4a, 4b, and 4c, adjacent sections of tissue from a severely affected Il1rn–/– mouse show the root of a coronary artery (indicated by "C"). For comparison, Figure 4d shows a grazing section of a coronary artery of a wild-type mouse. Here, it was possible to trace the ribbons of healthy elastin from the aortic sinus into the neck of the vessel. The elastin was coarsely structured and strongly stained (Figure 4e). Figures 4f and 4h, by contrast, show typical thin, diffuse, disorganized elastin in a severely inflamed coronary artery from an Il1rn–/– mouse but a heavily thickened fibrous (red) layer. Figures 4i and 4k show low-resolution images of connective tissue and macrophages, respectively, at the base of a severely affected aortic valve. An area of intense infiltration (arrow in Figure 4k) coincided with ragged elastin (arrow in Figure 4i). At high resolution (Figure 4j), ribbons of elastin appeared to have been severed. The absence of organized elastin from the infiltrated section was confirmed by ultraviolet autofluorescence (Figure 4l) of the same section as Figure 4k. This showed dim, amorphous fluorescence, clearly different from the bright, localized ribbons of elastin seen in a healthy aortic root (Figures 4n and 4o).
Chondrometaplasia was also observed in established fibrotic lesions. In several cases (5 of 23 affected animals, aged 125 to 209 days), nodules that resembled cartilage (and that were stained with toluidine blue) were located at the base of the aortic valve (Figure 4m) or in the media of the aorta.
Involvement of Activated T Cells
The inset in Figure 4k, which shows macrophage-specific staining, is an approximately equivalent area to those shown in Figures 5a through 5e. IL-4+ cells (Th2) were detectable (Figure 5b) although not abundant at heavily inflamed sites. In sections from 2 Sf3 mice and 1 BALB/c Il1rn–/– mouse, the ratio of IL-4+ to IFN-+ cells was estimated to be <0.1, 0.2, and 0.1, respectively.
CD4+ cells (Figure 5a) are similarly distributed and are colocalized with dense staining for IFN- (Figure 5c). We therefore stained for IFN- and CD4 on the same section (Figure 5m) taken from another mouse. Coincidence of black (IFN-) and red (CD4) staining indicated the presence of activated Th1 cells. (Figures 5k and 5l show the antigens separately.) Localized expression of the chemokine CCL2 (Figure 5f) was detectable at sites of CD4+ cell infiltration (Figure 5g), where there were also indications of localized E-selectin expression (Figure 5h) in the CD31+ vascular network (Figure 5i). CCL2 was not detected in wild-type mice.
Dendritic Cells
Dendritic cells are intensely MHC II+ in situ. Stimulated endothelial cells and macrophages are also MHC II+, although expression by dendritic cells is expected to be much more intense. MHC II+ cells in wild-type mice (Figure 5e) did not consistently line vessels (and so were not endothelial) and were too few, compared with F4/80+ cells, to be identified with macrophages (Figure 4k). Also, some MHC II+ cells had dendritic morphology (see the indicated cells in Figure 5e). Ramification yielded a large stained area with anti-MHC II. CD205 has a strong specificity toward dendritic cells and identified a similar group of cells to anti-MHC II (Figure 5d). Fainter CD205+ cells are probably macrophages. As in human vessels,14 dendritic cells (visualized as MHC II+ cells) tended to form a ring around the media in the wild-type artery (Figure 5j) and occupied the fibrous tissue of the valve itself. Anti-CD205 antibodies detected no cells in wild-type aortic roots, possibly because CD205 is only upregulated to detectable levels during maturation. Thus, although dendritic cells appear to be recruited to affected tissue (Figures 4a and 5 e) versus wild-type tissue (Figure 5j), they were not obviously codistributed with the majority of effector T cells (Figure 5c).
Discussion
If IL-1 and IL-1; are the only agonists for IL-1R1, and IL-1ra has no other role but to block that interaction, then deficiency of the receptor15 should suppress the Il1rn–/– phenotype completely. Irikura et al16 have shown that the induced phenotypic features and general malaise observed in C57BL/6 Il1rn–/– animals are suppressed by overexpression of IL-1ra or by elimination of IL-1R1. We show that a single functional copy of the IL-1R1 gene (Il1r1) is essential for the pathology of IL-1ra deficiency, which implicates IL-1 as the mediator of the disease process.
Small aortic root infiltrates of CD4+ and IFN-+ cells, which could have been Th1 or NK-T cells, were observed in 2 young wild-type mice. We interpret these as normal responses to subclinical events. In an Il1rn–/– mouse, such events might instigate chronic disease, because a stimulus that causes IL-1 to be released in an Il1rn–/– mouse would be expected to produce an enhanced or prolonged proinflammatory response compared with the wild type. If IL-1ra were absent, then perhaps IL-1 that is released from the vasculature in response to constitutive physiological stimuli might be sufficient to cause general activation of the adventitial microvasculature, without involving an autoimmune response. However, we did not detect vascular activation (as E-selectin expression) in preaffected susceptible mice. It thus appears that strong, large-scale vascular activation is part of the inflammatory process and not an immediate consequence of IL-1ra deficiency.
In TA and GCA, the elastic laminae are subject to wholesale fragmentation and digestion by macrophages. Krettek et al17 have shown that de novo synthesis of disorganized elastin by vascular smooth muscle cells and lesional macrophages is simultaneous with laminar elastin destruction in human vascular disease. In Il1rn–/– mice, we have already noted invasion of the media by macrophages and destruction of the elastic laminae.6 We show here that synthesis of disorganized elastin as small fibers and fibrosis, which causes general tissue thickening, appears to occur very close to sites of elastin destruction.
The presence of an active IFN-–rich, CD4+, Th1-like population within arteritic lesions, in conjunction with macrophages and dendritic cells, is highly suggestive of an autoimmunity-driven pathology: in humans, MHC haplotype association with GCA18 and TA,19,20 strongly biased T-cell receptor usage in affected GCA arteries,21,22 and adoptive transfer of disease with CD4+ T cells in the GCA graft model11 all suggest autoimmunity that centrally involves T cells. However, Shimizu and colleagues23 have recently shown that Th2 rather than Th1 cells drive elastinolysis in aortic allografts, and a similar role is possible for the IL-4+ cells that are also present in Il1rn–/– arteritic lesions. In GCA and TA, dense accumulations of activated macrophages are involved in continuous mutual interaction between macrophages and CD4+ T cells.11,13 Iwakura24 recently reviewed the evidence for the involvement of IL-1 in T-cell activation in arthritis in Il1rn–/– mice and has presented a model in which T-cell receptor engagement with antigen presenting cells (probably dendritic cells) increases IL-1R expression on the T cell. Trapped dendritic cells have already been implicated in GCA.11,14 Maturation of dendritic cells can be triggered by a combination of IL-1 and CD40L,25 which could provide a direct link between IL-1 hyperactivity and the onset of a putative autoimmune state. Chemokine secretion would be expected to mediate the recruitment of more macrophages and effector T cells, as well as immobilize dendritic cells,22 and we show evidence of CCL2 in the mouse lesions.
Because of the apparent similarity in gross pathology and immunohistology, we suggest that Il1rn–/– mice might prove to be a tractable spontaneous experimental model for GCA and TA. In the future, manipulation of the leukocyte populations in Il1rn–/– mice may yield useful information about the possible nature of vascular autoantigens that could be beneficial to the therapy of GCA and TA.
Acknowledgments
This study was supported by grant RGA014661 from the British Heart Foundation. We thank Orla Gallagher for technical assistance and advice and the staff of the Field Laboratories for expert animal husbandry.
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