Th2-predominant inflammation and blockade of IFN-γ signaling induce aneurysms in allografted aortas (original) (raw)
Allografts in WT and GRKO recipients haveIFN-γ– or IL-4–predominant cytokine environments, respectively. The aortic allograft model essentially allows the induction of a focal inflammatory response in discrete aortic segments. In WT combinations, this inflammation results in an intimal hyperplastic lesion without aneurysm formation (18). To test the role of specific cytokine subsets in the pathogenesis of AAA in this model, we induced either IFN-γ– or IL-4–dominant responses in transplanted BALB/c (B/c, H-2d) aortic segments in total allo-mismatched 129SvEv (129Sv, H-2b) hosts. Recipients were either WT, which resulted in predominant IFN-γ production; or congenitally deficient in GRKO, which resulted in IL-4–predominant cytokine expression. Aortic allografts harvested one week after transplantation, at the peak of inflammatory cell infiltration (18), exhibited significantly greater IL-4 mRNA expression in the GRKO hosts compared with WT recipients (Figure 1, A and B). Other Th2 cytokine mRNAs (IL-10 and IL-15) were modestly increased in allografts in GRKO hosts compared with WT recipients (Figure 1A). Western blots (Figure 1C) showed the same marked skewing in IL-4 protein production in allografts recovered from GRKO versus WT recipients. Although intragraft IFN-γ levels are comparable in WT and GRKO hosts, GRKO inflammatory cells cannot respond to IFN-γ signals. Consequently, IL-4 largely governs responses to inflammation in GRKO hosts, whereas IFN-γ responses direct inflammation in WT hosts.
Aortic allografts from WT or GRKO recipients were harvested 1 week after transplantation and were analyzed for cytokine expression by RNase protection assay (RPA) (A and B) and Western blot for IL-4 (C). (A) Gel image of RPA. (B) Histogram of the optical densities from RPA data normalized to GAPDH and averaged for 6 or 7 grafts. (C) Protein extracts (25 μg/lane) of the aortic grafts of WT or GRKO recipients analyzed by Western blot for IL-4.
Allografts in GRKO hosts develop aortic aneurysms. Lack of IFN-γ receptor in the host resulted in profound aneurysm formation, in some cases leading to spontaneous rupture. Figure 2 shows the gross appearance (A and B), representative echoaortograms (C and D), and the histologic appearance (E and F, same magnification) of transverse sections of aortic allografts from WT (A, C, and E) or GRKO (B, D, and F) recipients 12 weeks after transplantation. The aortic diameter of the allografts of GRKO hosts was approximately three times larger than that of WT recipients. Aneurysmal allografts in GRKO hosts also showed markedly fragmented and attenuated elastic laminae in the aortic media (Figure 2H) not seen in the WT recipient allografts (Figure 2G).
Aortic allografts from WT (A, C, E, and G) or GRKO (B, D, F, and H) recipients 12 weeks after transplantation. (A and B) Representative gross appearance of aortas. (C and D) Echoaortograms. (E and F) Histologic appearance of transverse sections. (G and H) Elastic van Gieson staining (EVG). Analysis of 9–10 aortic transplants from different donors yielded similar results. Control (WT) experiments, shown in panels A, C, E, and G, did not reveal measurable aneurysm formation or elastic tissue degradation. Arrowheads indicate elastic lamellae.
IL-4 blockade prevents aortic aneurysm formation in allografts in GRKO recipients. To examine specifically whether IL-4 blockade prevents aneurysm development, we treated GRKO recipients of B/c aortic allografts with anti–IL-4 monoclonal antibody (11B11). GRKO hosts received 2 mg of anti–IL-4 antibody by intraperitoneal injection 1 day before and 1 day after surgery, followed by 1 mg antibody per week thereafter (n = 3). We also transplanted B/c aortas into mice congenitally double deficient in IL-4 and IFN-γ receptor (DKO, n = 6) and harvested the grafts 12 weeks after transplantation. Aneurysms did not develop in aortic allografts from GRKO recipients receiving anti–IL-4 antibody (Figure 3A) or in allografts from DKO hosts (Figure 3B). Moreover, elastic van Gieson staining showed minimal elastic tissue fragmentation in grafts from GRKO hosts treated with 11B11 (Figure 3C) and no sign of elastolysis in grafts from DK O recipients (Figure 3D).
Aortic allografts from GRKO recipients treated with anti–IL-4 antibody (A and C) or from DKO recipients (B and D) harvested at 12 weeks after transplantation. (A and B) Representative histologic appearance of transverse sections. (C and D) Elastic van Gieson staining. (E) Analysis of aortic diameter from the isograft (Iso) (n = 5), WT recipient allografts (n = 9), GRKO recipient allografts (n = 10), allografts in GRKO recipients with anti–IL-4 antibody (11B11) treatment (n = 3), and DKO (n = 6) recipient allografts 12 weeks after transplantation. *P < 0.01 vs. GRKO; **P < 0.0001 vs. GRKO.
Echoaortograms performed 12 weeks after transplantation and just before harvest showed in situ graft diameters of 0.8 ± 0.1 mm in isografts (n = 5); 1.0 ± 0.2 mm in WT hosts (n = 9; P = NS vs. isografts); 2.9 ± 0.8 mm in GRKO hosts (n = 10; P < 0.0001 vs. WT); 1.3 ± 0.1 mm in GRKO hosts receiving anti–IL-4 treatment (n = 3; P = 0.005 vs. GRKO); and 1.0 ± 0.2 mm in DKO hosts (n = 6; P < 0.0001 vs. GRKO). Our results indicate that the presence of IL-4 predominance induces aortic aneurysm formation (Figure 3E) and that IL-4 blockade prevents aneurysm formation.
Aneurysm formation does not result from increased blood pressure. To examine whether IL-4 elaboration or blockade affected systemic blood pressure (and thereby influenced AAA formation), we measured the recipient systolic arterial pressure 12 weeks after transplantation by a tail cuff. Blood pressure in all transplant groups was similar: 106 ± 18 mmHg in WT; 101 ± 13 mmHg in GRKO; and 96 ± 11 mmHg in DKO (P = NS).
Increased mRNA expression of elastolytic MMPs mRNA correlates with allograft aneurysm formation in GRKO recipients. The ECM protein elastin consists of highly cross-linked, hydrophobic tropoelastin monomers that confer resilience to the elastic fibers. The hydrophobicity and extensive cross-linking of tropoelastin result in an insoluble fiber that resists proteolysis. Under normal conditions, elastin has minimal turnover (19). Nevertheless, destruction of elastic fibers characterizes certain pathological situations such as AAAs (20).
The ability of certain MMPs, e.g., MMP-2, -3, -9, and -12, to degrade elastin may have particular relevance to AAA formation, as human AAAs exhibit abundant MMP-2 and MMP -9 expression (21, 22) and, in some cases, excessive MMP-1 and MMP-3 (4–6). In experimental models, targeted disruption of MMP-9 results in decreased elastin fiber degradation after elastase perfusion in mouse aortas and suppresses the subsequent development of aortic aneurysms (23). The other elastolytic MMPs such as matrilysin (MMP-7) (24) and macrophage metalloelastase (MMP-12) (25, 26) may play even greater roles in very large aneurysms.
In addition to the MMPs, AAAs contain excessive serine proteases such as plasmin, as well as elevated plasminogen activators (PAs) (i.e., urokinase PA, uPA; and tissue PA, tPA), compared with normal aortic tissue (4, 5). Cysteine proteinases such as cathepsin-L, -S, and -K or serine proteinases such as neutrophil elastases have elastinolytic potential (27) and may also contribute to AAA formation.
To identify which elastinolytic or collagenolytic enzymes contribute to elastin degradation and AAA formation in grafts in GRKO mouse hosts, we performed LightCycler-based real-time PCR using cDNA prepared from total RNA extracted from aortic allografts one week after transplant at the peak of inflammatory cell infiltration (18). We examined MMP-1 through MMP-24, TIMP-1 through TIMP-4, cathepsin-L, -S, and -K, neutrophil elastase, uPA, and tPA. MMP-2, -3, -9, -12, -13, -15, and -19 mRNA expression all increased significantly in allografts from GRKO recipients with parallel increases in TIMP-1, TIMP-2, and TIMP-3 compared with grafts in either WT or DKO hosts (n = 6 for each; Figure 4). Allografts from DKO had significantly lower MMP-10 mRNA levels than allografts from GRKO recipients; expression of mRNA encoding the other proteases did not differ between groups (data not shown).
Metalloproteinase expression during AAA development. The MMP mRNA levels of aortic allografts from WT (white bars), GRKO (black bars), or DKO (gray bars) recipients (n = 6, each). Total RNA was extracted from the aortic allografts and first-strand cDNA generated by reverse transcription was subjected to real-time quantitative PCR with the LightCycler. Data represent the mean ± SEM of six determinations of the percentage of mRNA copies relative to untreated growing cells. *P < 0.05; **P < 0.01; ***P < 0.001.
Elevated enzymatic activities for MMP-12 in allografts in GRKO hosts. To test formally whether the elevated MMP mRNA resulted in increased functional enzymatic activity, we performed Western blot analysis, elastase colorimetric assay, and zymography. Western blot analysis demonstrated markedly increased expression of MMP-12 in allografts from GRKO hosts compared with allografts from WT and DKO recipients (Figure 5A). At one week, aortic allografts in GRKO recipients had significantly greater elastase activity (6.61 ± 1.60 μU/g; n = 6) relative to WT hosts (1.75 ± 0.41 μU/g; n = 6; P < 0.0001) and DKO recipients (0.61 ± 0.15 μU/g; n = 6; P < 0.0001). Immunoprecipitation with anti–MMP-12 antibody of the proteins recovered from allografts in GRKO hosts removed the majority of the elastolytic activity with residual elastase colorimetric assay activity of 0.04 ± 0.05 μU/g (P < 0.0001 vs. nonimmunoprecipitated proteins from aortic grafts in GRKO hosts; n = 6; Figure 5B). Gelatin zymography (Figure 5C) and casein zymography (Figure 5D) corroborated these results; increased activities corresponded to MMP-2, MMP-9, and MMP-12 proteases, as indicated. The same amounts of proteins loaded in the gels suggests much higher MMP-12 activities than MMP-9 in GRKO recipient allografts.
Western blot for MMP-12, elastase colorimetric assay, and gelatin and casein zymography. (A) Representative gel image of Western blot analysis. Protein extracts (20 μg/lane) of the aortic grafts of WT (n = 6), GRKO (n = 6), or DKO (n = 6) recipients analyzed by Western blot for MMP-12. The gel images represent qualitatively similar results. (B) The elastase colorimetric assay shows significantly greater elastase activity in the proteins extracted from allografts in GRKO hosts (n = 6) compared with proteins extracted from allografts in WT (n = 6) or DKO (n = 6) hosts. After anti–MMP-12 immunoprecipitation (IP) (n = 6), the proteins from allografts from GRKO recipients had significantly reduced elastase activity, which indicates that the majority of elastolytic activity in those allografts derives from MMP-12. Bar shows mean ± SEM; *P < 0.0001. (C and D) Representative gel images of gelatin zymogram (C) and casein zymogram (D). Protein extracts (20 μg/lane) of the aortic grafts of WT (n = 6), GRKO (n = 6), or DKO (n = 6) recipients analyzed by (C) gelatin- or (D) casein-zymogram for MMPs. The gel images represent qualitatively similar results. We could detect only 92 kDa and 72 kDa active bands from GRKO recipient allografts in the gelatin zymogram (C) and only 20 kDa active band from GRKO recipient allografts in the casein zymogram (D).
The majority of the graft-infiltrating cells during aneurysm formation are macrophages. A number of cell types including endothelial cells, medial smooth muscle cells, and adventitial connective tissue cells might contribute to the local pool of MMPs in vascular tissues (28). Monocyte-derived macrophages in particular can express abundant active MMPs (4, 5, 29), especially MMP-9 and MMP-12 in diseased tissues. MMP activity depends on control at the transcriptional level by cytokines, on activation of the proenzyme forms of the MMPs, and on the local concentrations of TIMPs (28, 30). We observed medial elastinolysis in the allografts in GRKO hosts within 1–4 weeks of transplantation (18), when the majority of the graft-infiltrating cells consist of CD11b+ macrophages (Figure 6, B and D), with a lesser contribution of CD4+ T cells (Figure 6, A and C) and CD8+ T cells (data not shown). Immunoreactive MMP-12 (Figure 6, F and H) colocalized predominantly with CD11b+ cells (Figure 6, E and G), which suggests that infiltrating macrophages provide much of the MMP-12 in developing AAA.
Representative immunohistochemistry of CD4+ (A and C) and CD11b+ (B and D) cells in allografts from GRKO recipients harvested 4 weeks after transplantation (n = 6). Boxed area in A or B (original magnification, ×100) is enlarged in C or D (original magnification, ×400), respectively. The majority of the graft infiltrating cells, especially adjacent to degraded elastic lamellae, consisted of CD11b+ macrophages. Expression of MMP-12 (F and H) colocalized with CD11b+ cells (E and G) in allografts from GRKO recipients harvested 4 weeks after transplantation (n = 6).
IL-4 augments and IFN-γ diminishes MMP-12 mRNA expression in macrophages. To examine whether IL-4 and IFN-γ can directly modulate MMP-12 expression in macrophages, we cultured bone marrow–derived macrophages from WT and GRKO mice in the presence of IL-4, IFN-γ, and/or anti–IL-4 antibody. Both conventional PCR (23 cycles) with subsequent gel electrophoresis (Figure 7A) and real-time quantification of MMP-12 gene copy number using LightCycler (Figure 7B) were performed on cDNA from total RNA extracted after 18 hours of macrophage culture. IL-4 increased MMP-12 mRNA expression, and anti–IL-4 antibody abolished this effect. Moreover, IFN-γ decreased IL-4–induced MMP-12 mRNA expression in WT macrophages but did not inhibit MMP-12 expression in GRKO macrophages (data not shown). MMP-9 mRNA expression exhibited the same responses to IFN-γ and IL-4 as MMP-12 (data not shown).
Conventional RT-PCR (A) and real-time PCR (B) for MMP-12 from WT bone marrow×derived macrophages incubated 18 hours with IL-4 (10 ng/ml), IFN-γ (500 U/ml), and/or anti×IL-4 antibody (5 μg/ml, 11B11). IL-4 augmented and IFN-γ inhibited MMP-12 mRNA expression of the WT macrophages. In addition, 11B11 inhibited IL-4×induced augmentation of MMP-12 mRNA expression by the WT macrophages. Bar shows mean ± SEM (n = 6, each). *P < 0.0001.