Interference of the CD30–CD30L Pathway Reduces Atherosclerosis Development
Objective—Costimulatory molecules tightly control immune responses by providing positive signals that promote T-cell activation or by transducing inhibitory signals that limit T-cell responses. CD30 and CD30L are members of the tumor necrosis factor receptor superfamily and are involved in the activation and proliferation of T and B cells, which have been implicated in the initiation and progression of atherosclerosis. In the present study, we thus aimed to determine the role of the CD30–CD30L pathway in the development of atherosclerosis.
Methods and Results—Western-type diet–fed low-density lipoprotein receptor–deficient mice were treated with an anti-CD30L antibody for 8 weeks, which resulted in a reduction of atherosclerotic lesion formation in the aortic root by 35%. Reduced numbers of adventitial CD3+ T cells were found in anti-CD30L–treated mice, whereas no differences were observed in collagen and macrophage content of the atherosclerotic lesions. B-cell and mast cell responses were also not affected on anti-CD30L treatment. Interestingly, splenocyte proliferation was reduced by 53%, whereas T-cell numbers were concomitantly reduced in anti-CD30L–treated mice compared with control mice. These data thus indicate that the CD30–CD30L pathway solely exerts its function via inhibition of T-cell responses.
Conclusion—In the present study, we are the first to show that interruption of the CD30–CD30L pathway reduced initial atherosclerosis development by modulating T-cell function.
Atherosclerosis is considered a chronic autoimmune-like disease resulting from endothelial damage and subsequent cholesterol accumulation in the arterial wall.1,2 Within the atherosclerotic lesion, a chronic inflammation manifests by a continuous infiltration of immune cells. Antigen-presenting cells, such as dendritic cells and macrophages, present antigens such as oxidized low-density lipoprotein (LDL)-cholesterol to T cells, resulting in their activation. As a result, both T cells and macrophages secrete cytokines, and more immune cells are attracted to the site of inflammation, which aggravates atherosclerotic lesion development.
T-cell activation is tightly controlled by a complex network of costimulatory molecules, which can either provide positive or negative signals. Two large families of costimulatory molecules are known: the B7-CD28 superfamily, which includes CD28/CD80/CD86 and programmed death-1 (PD-1)/PD-L1/2, and the tumor necrosis factor (TNF)-TNF receptor superfamily, which includes OX40/OX40L and CD40/CD40L. Numerous studies have shown the crucial role of costimulatory molecules in the pathogenesis of atherosclerosis.3–5 Previously, our laboratories showed that interruption of the OX40–OX40L interaction using an OX40L-blocking antibody leads to a reduction in the initiation of atherosclerosis.4 Signaling of CD40–CD40L has been shown to affect advanced atherosclerosis, because lesions of CD154−/− apolipoprotein E–deficient mice contained fewer lipids and showed increased collagen levels and reduced numbers of immune cells, such as T cells and macrophages, compared with apolipoprotein E–deficient mice.3 In addition, Gotsman et al5 showed that the negative costimulatory pathway PD-1/PD-L1/2 downregulates proatherogenic T-cell responses and atherosclerosis, because PD-L1/2 LDL receptor (LDLr) double-knockout mice developed significantly larger atherosclerotic lesions compared with LDLr-deficient (LDLr−/−) mice.5
CD30 (TNFRSF8) and CD30L (TNFSF8, CD153) also belong to the TNF-TNF receptor superfamily. Whereas CD30 and CD30L are both present on activated B and T cells, CD30L is also expressed on other cell types, such as mature dendritic cells, macrophages, and mast cells (MCs). Triggering via CD30–CD30L has been shown to induce the activation and proliferation of T cells.6,7 Furthermore, the CD30–CD30L pathway has been implicated as a major player in secondary humoral immune responses. CD30−/− mice have impaired follicular germinal center responses and reduced secondary antibody responses.8,9 In addition, CD30L transgenic mice show increased numbers and activity of splenic germinal centers and have elevated serum antibody levels, such as IgG2b and IgE.10
The in vivo role of the interaction between CD30 and CD30L can be investigated using anti-CD30L antibodies, which interrupt the CD30–CD30L pathway. CD30 deficiency or treatment with a CD30L blocking antibody (RM153) significantly reduced airway inflammation in a murine asthma model,11 whereas Blazar et al12 showed that anti-CD30L prolongs survival of mice in graft versus host disease. Furthermore, administration of anti-CD30L completely suppressed the development of spontaneous/type I diabetes mellitus in NOD mice.13
Although macrophages bearing CD30 have been identified in ruptured plaques of patients with coronary artery disease,14 the involvement of the CD30–CD30L pathway in the development of atherosclerosis has not been investigated. In the present study, we therefore investigated the role of the CD30–CD30L pathway in the initiation of atherosclerosis by treatment of LDLr−/− mice with a CD30L blocking antibody.
Materials and Methods
Female LDLr−/− mice, 10 to 12 weeks old, were obtained from Jackson Laboratories. The animals were kept under standard laboratory conditions and were fed a normal chow diet or a Western-type diet containing 0.25% cholesterol and 15% cocoa butter (Special Diet Services, Witham, Essex, United Kingdom). Diet and water were provided ad libitum. All animal work was approved by the regulatory authority of Leiden University and carried out in compliance with the Dutch government guidelines.
CD30L Expression During Atherosclerosis
After 2 weeks of Western-type diet, atherosclerosis was induced in LDLr−/− mice by collar placement (2-mm long, diameter 0.3 mm) around both carotid arteries and continuous Western-type diet feeding.15 Mice were euthanized at 0, 2, 4, 6, 8, and 10 weeks after collar placement, and tissues were harvested after in situ perfusion using PBS. Carotid arteries and spleens (n=4–6 per time point) were isolated, and mRNA was extracted using the guanidium isothiocyanate method and reverse-transcribed (RevertAid M-MuLV reverse transcriptase). Quantitative gene expression analysis was performed on a 7500 Fast Real-Time polymerase chain reaction system (Applied Biosystems, CA) using SYBR green technology. The following primer pairs were used: 5′-CCAAGAAGTCATGGGCCTACCTCCAA-3′ and 5′-GCAAACGATGAAGTACAAGCCA-GGGAA-3′ for CD30L, 5′-GAGCTCTTGTTG-GTTGGGAA-3′ and 5′-CGAACATCTG-TGAAGGCAAA-3′ for CD4, and 5′- GTTGGGGCAGTTGTAGGAAG-3′ and 5′-TGTGAAGCCAGAGGACAGTG-3′ for CD8. The following primers were used as endogenous references: 5′-ggacccgagaagacctcctt-3′ and 5′ gcacatcactcagaatttca-atgg-3′ for acidic ribosomal phosphoprotein PO (36B4) and 5′-ttgctcgagatgtcatgaagga-3′ and 5′ agcaggtcagcaaagaactt-atag-3′ for hypoxanthine phosphoribosyltransferase. Protein levels of CD30L were determined in blood of LDLr−/− mice fed a Western-type diet (n=5) or a chow diet (n=5) for 0, 4, and 8 weeks. Red blood cells were removed from blood using erythrocyte lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L NaHCO3, 0.1 mmol/L EDTA, pH 7.3). Cells were stained with CD4 and CD30L, and positive cells were determined with flow cytometry. All antibodies were purchased from eBioscience (Vienna). Fluorescence-activated cell sorter analysis was performed on a FACSCantoII analyzer (Beckton Dickinson, Mountain View, CA). Data were analyzed using FACSDiva software (Beckton Dickinson).
Functionality of the Anti-CD30L Antibody Under Hypercholesterolemic Conditions
To determine the effect of anti-CD30L on splenocyte proliferation, splenocytes from Western-type diet–fed mice (n=3) were cultured for 24 hours in triplicate in a 96-well round-bottom plate (2 × 105 cells/well) in RPMI 1640 supplemented with l-Glutamine, 100 U/mL streptomycin/penicillin, and 10% FCS. Splenocytes were cultured in the absence or presence of anti-CD3 and anti-CD28 (2 μg/mL) with anti-CD30L (0.01–10 μg/mL). Proliferation was measured by the addition of 3H-thymidine (0.5 μCi/well; Amersham Biosciences, The Netherlands) 16 hours before cell lysis. The amount of 3H-thymidine incorporation was measured using a liquid scintillation analyzer (Tri-Carb 2900R). Responses are expressed as stimulation index: ratio of mean counts per minute of triplicate cultures with anti-CD3/CD28 stimulation to triplicate cultures without stimulation.
Atherosclerosis was induced in LDLr−/− mice by feeding a Western-type diet for 8 weeks. Mice were treated intraperitoneally with 250 µg anti-mouse CD30L (RM153; n=12) or sterile PBS (n=12) twice a week. Anti-mouse CD30L was kindly provided by Hideo Yagita and prepared as previously described.16 At week 8, mice were euthanized and tissues were harvested after in situ perfusion using PBS. Tissues for histology were fixed in Zinc Formal-Fixx (Shandon Inc, Pittsburg, PA). Tissues were frozen in nitrogen and stored at −80°C until further use.
Serum Cholesterol Levels
During the experiments, mice were weighed and blood samples were obtained by tail vein bleeding. The total cholesterol levels in serum were determined at weeks 0, 2, 4, 6, and 8 after start of the Western-type diet feeding. The concentrations of serum cholesterol were determined using enzymatic colorimetric procedures (Roche/Hitachi, Mannheim, Germany). Precipath (Roche/Hitachi) was used as an internal standard.
Histological Analysis and Morphometry
Cryosections of the aortic root (10 µm) were made and stained with Oil-red-O. To determine the number of adventitial T cells, a CD3 staining was performed using anti-mouse CD3 (1:100, SP7; Immunologic, The Netherlands). Lesion collagen content was determined with Masson trichrome staining. Furthermore, corresponding sections on separate slides were stained immunohistochemically with an antibody directed against a macrophage-specific antigen (Moma-2, monoclonal rat IgG2b, diluted 1:1000). Goat anti-rat IgG alkaline phosphatase conjugate (dilution 1:100) was used as a secondary antibody and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as enzyme substrates. MCs were visualized by staining with toluidine blue (Sigma-Aldrich) according to the manufacturer’s protocol. MC numbers and the extent of MC degranulation were assessed manually. The necrotic core was defined as the acellular, debris-rich plaque area as percentage of total plaque area. In addition, the aortic arch and its main branch points were excised (4 µm), fixed, and embedded in paraffin. Longitudinal sections of the aortic arch were analyzed for lesion extent with hematoxylin and eosin staining. Spleen sections were stained with hematoxylin and eosin. Morphology was studied using a Leica DM-RE microscope and LeicaQwin software (Leica imaging systems, Cambridge, United Kingdom).
At euthanization, blood, spleen, and mediastinal lymph nodes were isolated (n=5 per group). Single-cell suspensions were obtained by squeezing the organs through a 70-µm cell strainer. Red blood cells were removed from blood and splenocytes using erythrocyte lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L NaHCO3, 0.1 mmol/L EDTA, pH 7.3). Cells were stained with CD4, CD8, and CD19 to detect T cells and B cells. For intracellular staining, cells were fixed and permeabilized according to the manufacturer’s protocol (eBioscience, Vienna). Subsequently, the cells were stained for the transcription factors T-bet, GATA-3, RORyt, or Foxp3 and the cytokines interferon-γ, interleukin (IL)-4, IL-5, IL-10, and IL-17. Fluorescence-activated cell sorter analysis was performed as described above.
Spleen Cell Proliferation
At euthanization, splenocytes (n=5 per group) were cultured for 72 hours in quintuplicate in a 96-well round-bottom plate (2 × 105 cells/well) in RPMI 1640 supplemented with l-glutamine, 100 U/mL streptomycin/penicillin, and 10% FCS. As a positive control, cells were stimulated with anti-CD3 and anti-CD28 (2 μg/mL). Proliferation was measured as described above.
Cytokine Determination in Serum and Supernatant of Splenocytes
To detect IL-8 in serum, an ELISA was performed according to the manufacturer’s protocol (Biosource). Serum samples were 1:1 diluted in assay diluent, and absorbance was detected at 450 nm. To detect IL-4 and IL-5 in the serum, an ELISA was performed according to the manufacturer’s protocol (eBioscience, Vienna). Serum samples were 1:1 diluted in assay diluent, and absorbance was detected at 450 nm.
Serum Antibody Detection
IgM, IgG1, IgG2a, and IgG2b levels against oxidized LDL were detected in serum using antibodies recognizing mouse IgM, IgG1, IgG2a, and IgG2b and horseradish peroxidase–labeled goat anti-rat Ig (BD Pharmingen). Oxidized LDL (5 µg/mL) was dissolved in NaHCO3/Na2CO3 buffer (pH 9.6) and was coated overnight onto a flat-bottom 96-well high-binding plate (Corning, NY). Serum samples were 1:1 diluted in PBS, and absorbance was detected at 450 nm. Total IgE in serum was determined by a mouse IgE quantitative ELISA according to the manufacturer’s protocol (Bethyl Laboratories, Montgomery, TX).
All data are expressed as mean±SEM. An unpaired 2-tailed Student t test was used to compare normally distributed data between 2 groups of animals. Probability values of P<0.05 were considered significant.
CD30L Is Upregulated in the Initial Stages of Atherosclerosis
Because we aimed to interrupt the CD30–CD30L pathway via blockade of CD30L, we first monitored the expression of CD30L in atherosclerotic lesions and the lymphoid tissue. Although it is known that costimulatory molecules such as OX404 and PD-117 are upregulated in atherosclerotic lesions, it is unclear whether CD30L is regulated as well. LDLr−/− mice were fed a Western-type diet, and slightly constrictive perivascular collars were placed around the carotid arteries, which leads to the development of shear stress–induced atherosclerotic lesions at the proximal site of the collars.15 At 0 to 10 weeks after collar placement, RNA was isolated from the atherosclerotic lesions. As shown in Figure 1A, induction of atherosclerotic lesion development is reflected by an increase in CD68 expression over time (P<0.01). CD30L was also induced during lesion development and showed the highest expression after 2 weeks of Western-type diet feeding (P<0.05), coinciding not only with the influx of CD68+ macrophages but also with the influx of various immune cells, such as CD4+ and CD8+ T cells (Figure 1B). Parallel to the data on the lesion, CD30L mRNA levels in the spleen of LDLr−/− mice significantly increased during Western-type diet feeding (P<0.05; Figure 1C). In addition, we determined protein levels of CD30L and, as shown in Figure 1D and 1E, CD4+ T cells expressing CD30L increase in blood of Western-type diet–fed mice (P<0.05).
Blockage of CD30L Reduces Proliferation of Splenocytes from Western-Type Diet–Fed Mice
To determine whether interruption of the CD30–CD30L pathway impairs T-cell function of LDLr−/− mice fed a Western-type diet for 8 weeks ex vivo, we stimulated splenocytes with anti-CD3/CD28 in the presence or absence of RM153, a blocking CD30L antibody. This antibody was generated by Shimozato et al16 and potently inhibits the binding of CD30 to CD30L and the proliferation of αCD3/CD28 activated T cells. As shown in Figure I in the online-only Data Supplement, blockage of the CD30–CD30L pathway by using RM153 dose-dependently reduced splenocyte proliferation of Western-type diet–fed LDLr−/− mice (P<0.01).
Impaired T-Cell Numbers and Function in Anti-CD30L–Treated Mice
RM153 has been shown to reduce murine autoimmune diabetes mellitus,13 prolong survival of mice in a graft versus host disease model,12 and together with OX40L blockade reduced autoimmune disease in Foxp3-deficient mice18 when administered intraperitoneally twice a week during the experiments (200–500 μg/dose). To study the effect of CD30–CD30L interruption on T cells in vivo, we treated LDLr−/− mice with 250 μg of RM153 twice a week, while the mice were fed a Western-type diet for 8 weeks. The relative number of CD4+ T cells (Figure 2A) in spleen and mediastinal heart lymph nodes of anti-CD30L–treated mice was reduced compared with control mice (P<0.05), whereas CD8+ T cell numbers were not affected by the anti-CD30L treatment (Figure 2B). Furthermore, the differentiation of naive CD4+ T cells into T helper (Th) 1, Th2, Th17, or regulatory T cells was unaffected by anti-CD30L treatment (Figure 2C). To determine the proliferative capacity of T cells from anti-CD30L–treated mice in comparison with control mice, splenocytes from both groups were cultured for 72 hours in the presence of αCD3/αCD28 stimulation. A significant 53% decrease in splenocyte proliferation was observed in mice treated with anti-CD30L (stimulation index of 27.0 ± 2.5) compared with control mice (stimulation index of 58.1 ± 6.1, P<0.01; Figure 2D). As shown in Figure 2E, we demonstrate that CD4+ T cells are the main effector cells, because CD4+ T cells isolated from anti-CD30L–treated mice (n=5) showed a 52% reduction in αCD3/αCD28-mediated proliferation in comparison with CD4+ T cells isolated from control mice (n=5; P<0.05), whereas CD8+ T-cell proliferation was unaffected (Figure II in the online-only Data Supplement). In addition, we determined cytokine secretion by these CD4+ T cells with flow cytometry; no significant differences in cytokine profiles between control and anti-CD30L–treated mice were observed (Figure 2F).
Interference in the CD30–CD30L Pathway Reduces the Development of Atherosclerosis
To determine whether the anti-CD30L–mediated reduction in T-cell responses affects atherosclerosis development, we determined atherosclerotic lesion sizes upon treatment with anti-CD30L. Figure 3A shows representative cross-sections of lesions in the 3-valve area of the aortic root. We observed a significant 35% reduction in the aortic root lesion size in anti-CD30L–treated mice (4.3 × 105±0.3 × 105 μm2) compared with control mice (6.5 × 105±0.5 × 105 μm2; P<0.001). Treatment with rat IgG (isotype control for RM153) did not alter atherosclerotic lesion size in comparison with PBS treatment (Figure III in the online-only Data Supplement). In addition, lesion formation in the aortic arch was reduced in anti-CD30L–treated mice compared with control mice (P=0.09; Figure IV in the online-only Data Supplement). During the experiment, anti-CD30L treatment did not affect body weight and total plasma cholesterol levels (Figure V in the online-only Data Supplement). In line with reduced T-cell percentages and splenocyte proliferation after anti-CD30L treatment, we observed a significant 31% reduction in the number of CD3+ T cells within the adventitia of anti-CD30L–treated mice (71.9 ± 5.3 T cells/section) compared with control mice (104.9 ± 14.1 T cells/section, P<0.05; Figure 3B). This reduction in adventitial T cells in anti-CD30L–treated mice was not a consequence of impaired migration and adhesion of CD4 and CD8 T cells (Figure VI in the online-only Data Supplement). In addition, we determined T-cell subsets and cytokine expression locally in the plaque with quantitative polymerase chain reaction. In line with our previous findings, we did not find any differences in T-cell subsets and their cytokines (Figure VII in the online-only Data Supplement).
Anti-CD30L Treatment Does Not Affect Lesion Composition
With respect to the composition of the lesion (Figure 4), no differences were found in lesion collagen content (18.6 ± 1.3% versus 19.3 ± 1.3%) and macrophage content (46.2 ± 3.6% versus 49.7 ± 2.7%) between anti-CD30L–treated mice and control mice, respectively. In addition, no differences in necrotic cores were observed between anti-CD30L–treated mice (49.9 ± 3.7%) and control mice (54.8 ± 3.2%).
Humoral Responses in Anti-CD30L–Treated Mice Are Not Affected
The CD30–CD30L pathway is described to be involved in germinal center responses and secondary antibody responses.8,9 However, in our study, both the percentage of B cells in blood and spleen (Figure 5A) and the levels of oxidized LDL–specific IgM, IgG1, IgG2a, and IgG2b in serum did not differ between control mice and anti-CD30L–treated mice (Figure 5B). Furthermore, we did not observe any differences in spleen morphology in anti-CD30L–treated mice compared with control mice (Figure 5C).
Anti-CD30L Treatment Does Not Inhibit MCs
Treatment with anti-CD30L significantly reduced serum IgE levels in a murine asthma model,11 and several other studies reported decreased IgE levels after CD30–CD30L pathway interruption.8,9 In our study, a trend toward lowered serum IgE was observed in anti-CD30L–treated mice (363 ± 90 ng/mL) compared with control mice (609 ± 132 ng/mL; Figure 6A). Because IgE is a common MC activator and MCs can aggravate atherosclerosis,19 we analyzed the number of adventitial MCs. However, the numbers of activated MCs and total number of MCs (Figure 6B) in the aortic root remained unaffected by anti-CD30L treatment (activated: 13.8 ± 1.9 MC/mm2 and total: 29.4 ± 2.4 MC/mm2 versus controls; activated: 12.5 ± 1.6 MC/mm2 and total: 28.1 ± 3.0 MC/mm2). In addition, the percentage of activated MCs did not differ (control: 44.2 ± 2.8% versus anti-CD30L: 45.1 ± 4.5%; Figure 6C). Interestingly, CD30–CD30L signaling can induce degranulation-independent MC activation via the secretion of IL-8.20 However, anti-CD30L treatment also did not influence serum levels of KC, the mouse analog of IL-8 (Figure 6D).
Optimal T-cell activation is regulated by costimulatory signals, and modulation of these signals provides a very promising therapeutic strategy to improve the outcome of autoimmune diseases. T cells play an important role in atherosclerosis, and whereas the CD30–CD30L pathway has been implicated in various autoimmune diseases, such as asthma,11 graft-versus-host disease,12 and type I diabetes mellitus,13 no studies describe a role for the CD30–CD30L axis in atherosclerosis.
In the present study, we found that CD30L expression within the atherosclerotic lesion highly correlated with CD4 and CD8 T-cell infiltration and that CD30L expression in the spleen was upregulated after 8 and 12 weeks of Western-type diet feeding. In addition, CD4+ T cells expressing CD30L are increased in Western-type diet–fed mice, suggesting a proatherogenic role of the CD30–CD30L pathway. We therefore chose to intervene with the CD30–CD30L pathway during the development of atherosclerosis as a therapeutic approach. Interruption of CD30–CD30L by using the CD30L blocking antibody RM153 reduced atherosclerosis development in LDLr−/− mice by 35% and coincided with a 31% reduction in adventitial T-cell numbers. CD30L signaling is reported to enhance proliferation of T cells,16 and blocking CD30L therefore diminishes proliferation of T cells as shown by several studies.11,13,21 We showed that anti-CD30L inhibited the proliferation of splenocytes from Western-type diet–fed mice ex vivo. Interruption of the CD30–CD30L interaction also potently reduced T-cell numbers in vivo, as we found reduced percentages of CD4+ T cells in the spleen and lymph nodes of anti-CD30L–treated mice compared with control mice. Furthermore, a 56% reduction in splenocyte proliferation was observed after anti-CD30L treatment, which was particularly a result of reduced CD4+ T-cell proliferation. In addition, we show that anti-CD30L does not interfere with the migration and adhesion capacity of T cells. The role of T cells in atherosclerosis has been established already in several studies.22–24 CD4+ T cells can be subdivided into several subclasses: Th1, Th2, Th17, and regulatory T cells. However, no differences were found in T-cell subsets after anti-CD30L treatment, which indicates that interruption of the CD30–CD30L pathway under hypercholesterolemic conditions impairs T-cell numbers and function but does not influence their differentiation. In line with our findings, Chakrabarty et al13 showed that anti-CD30L (RM153) treatment reduced T-cell proliferation in response to islet antigens and markedly inhibited the development of spontaneous diabetes mellitus in NOD mice. Furthermore, they showed that anti-CD30L inhibited the incidence of diabetes mellitus in NOD-SCID mice after diabetogenic T-cell transfer.13
Signaling via CD30–CD30L may also affect humoral responses. Mice deficient in CD30 mice show reduced levels of several immunoglobulins, such as IgG1, IgG2c, and IgE.8 CD30L Tg mice show increased numbers and activity of splenic germinal centers and elevated basal serum concentrations of IgG2a, IgG2b, and IgE.10 In addition, Shanebeck et al9 showed that mouse splenic B cells stimulated via CD30L induced increased amounts of several immunoglobulins, such as IgG1 and IgE. However, under hypercholesterolemic conditions, we did not find any significant difference in immunoglobulin production or spleen morphology in anti-CD30L–treated mice compared with control mice.
In a murine asthma model, CD30 deficiency or treatment with anti-CD30L significantly reduced airway inflammation, splenocyte proliferation, Th2 responses, and serum IgE levels.11 Whereas in the present study we also observed a reduction in splenocyte proliferation and a trend toward reduced serum IgE, we did not observe reduced Th2 responses as shown by GATA-3–expressing CD4+ cells and serum IL-4 and IL-5 levels (Figure VIII in the online-only Data Supplement). IgE may induce activation of MCs, which are correlated with the incidence of plaque rupture and erosion25 and also play a crucial role in plaque progression and destabilization in vivo.19 Furthermore, MCs are the predominant CD30L-expressing cells in Hodgkin disease, which are involved in tumorigenesis and tumor progression.26 However, despite a reduction in serum IgE, anti-CD30L–treated mice did not have reduced numbers of MCs or activated MCs as shown by adventitial MCs and IL-8 release.
In conclusion, we are the first to demonstrate that anti-CD30L treatment inhibits plaque development in LDLr−/− mice, independent of plasma cholesterol levels and lesional macrophage and collagen content. Given the profound inhibition of anti-CD30L treatment on T-cell proliferation and activation, we propose that anti-CD30L treatment, at least partly, exerts its protective effects by modulating this process. These data thus identify anti-CD30L treatment as a novel therapeutic modality in the inhibition of atherosclerotic lesion development and the prevention of acute cardiovascular syndromes.
Sources of Funding
This work was supported by Netherlands Heart Foundation grants 2008B048 and 2007T039 and by grant 016.86.046 (Veni) from the Netherlands Organization for Scientific Research (NWO).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300509/-/DC1.
- Received May 3, 2012.
- Accepted October 9, 2012.
- © 2012 American Heart Association, Inc.
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