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1Department of Cardiovascular Medicine and 2Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
Submitted 28 October 2002 ; accepted in final form 24 March 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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knockout mice; Fc receptor
receptor KO mice
(7), respectively, have
resulted in the reduction of lesions. We
(22) have previously reported
that blockade of c-fms, a receptor for macrophage colony stimulating
factor, caused marked suppression of atherogenesis in apo E-deficient mice,
where macrophage differentiation was impaired. These results suggest that
immunomodulation may be used to treat or prevent atherosclerosis. Therapy with immunoglobulin has been investigated in a wide range of immune-mediated disorders (6, 13, 27). The mode of action of immunoglobulin is still unclear and may involve both Fc and V region-dependent mechanisms: blockage of Fc receptors on macrophages and effector cells, anti-inflammatory effects by attenuation of complement-mediated damage, regulation of the production of cytokines, or inhibition of lymphocyte proliferation. Several of these mechanisms might be beneficial in atherosclerosis (23). Here, we evaluated whether administration of immunoglobulin could affect the development of atherosclerosis, and the antiatherosclerotic mechanisms conferred by immunoglobulin were also investigated. For this purpose, 6-wk-old apo E-deficient mice were fed a high-fat diet and injected with 1 g·kg–1·day–1 of intact immunoglobulin or F(ab')2 fragments intraperitoneally on alternate days. The fatty streaks and fibrofatty plaques were markedly reduced by the immunoglobulin treatment, but F(ab')2 fragments failed to reduce atherosclerosis. The antiatherosclerotic effects of immunoglobulin may be related not only to suppression of the cytotoxic activity of atherogenic T cells from atherosclerotic mice but to a reduction of the macrophage accumulation in the lesions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The apo E-deficient 129ola x C57BL/6 hybrid mice were generous gifts of Dr. Edward M. Rubin (University of California, Berkeley, CA). These mice were mated with C57BL/6 mice to produce F1 hybrids. The F1 apo E+/– mice were then backcrossed to C57BL/6 mice for 10 generations. Mice homogeneous for the apo E-null allele on a C57BL/6 background were subsequently generated. Male mice were subjected to subsequent experiments. The mice were kept in a temperature-controlled facility on a 14:10-h light-dark cycle with free access to food and water. After being weaned at 4 wk of age, mice were fed a normal chow diet (CMF, Oriental Yeast) until 6 wk of age, when the animals were switched to a high-fat diet containing 20% fat and 0.3% cholesterol as previously described (Fig. 1) (22).
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We performed animal experiments in accordance with the Declaration of Helsinki, and these were approved by our institutional ethics committee for animal experiments.
Immunoglobulin Treatment
Protocol I. In 6-wk-old male apo E-deficient mice, intact immunoglobulin (Venoglobulin-IH, Mitsubishi Pharma; a polyethylene glycol-treated human immunoglobulin) was administered intraperitoneally on alternate days at a dose of 1 g · kg–1 · day–1 for 8 wk (fatty streak stage: intact immunoglobulin, n = 5; controls, n = 6) or for 12 and 16 wk (fibrofatty plaque stage: intact immunoglobulin, n = 4 for 12 wk and n = 5 for 16 wk; controls, n = 4 for 12 wk and n = 6 for 16 wk) (Fig. 1). Littermate controls were injected with 1 g · kg–1 · day–1 of human serum albumin (HSA) intraperitoneally.
Protocol II. To determine the mechanisms and importance of the Fc portion of immunoglobulin on the development of atherosclerosis, mice were intraperitoneally injected on alternate days at a dose of 1 g · kg–1 · day–1 with either intact immunoglobulin or F(ab')2 fragments of human immunoglobulin (Gamma-Venin, Aventis; a polyethylene glycol-treated human immunoglobulin) for 8 wk [intact immunoglobulin, n = 6; F(ab')2 fragments, n = 6; controls, n = 6] or 16 wk [intact immunoglobulin, n = 5; F(ab')2 fragments, n = 5; controls, n = 5]. As shown in previous studies (15, 23, 27, 32), immunoglobulin antigenicity between humans and mice did not appear to be a problem. The molecular weight of F(ab')2 fragments is not completely equal to that of intact immunoglobulin at the same dosage. In addition, both intact immunoglobulin and F(ab')2 fragments have the same molecular structure in the Fab region of immunoglobulin except that the F(ab')2 fragments have no Fc region.
Tissue Processing
Mice were killed by bleeding with puncture of the right ventricle. The blood was collected and allowed to clot. After the serum was separated, lipid profiles were analyzed, as described in Serum Lipid Measurement. The vasculature was perfused with sterile PBS. The root of the aorta was dissected under a macroscope and frozen in OCT embedding medium for serial cryosectioning covering 1.0 mm of the root. The first section was harvested when the first cusp became visible in the lumen of the aorta. Four sections of 10 µm thickness were harvested per slide, and thus 20 slides per mouse were prepared. All sections were immersed for 2 min in 60% isopropanol, stained for 15 min in a saturated oil red-O solution at 37°C, counterstained with hematoxylin, and then mounted under coverslips with glycerol gelatin.
Quantitation of Atherosclerotic Lesions
The oil red-O-stained sections were analyzed at a magnification of x10, as previously described (22, 23, 26). The image was captured directly from the RGB camera attached to a light microscope and displayed on a microcomputer to quantify the cross-sectional surface area of the lesion and the cross-sectional surface area of the vessel. The fraction area of the lesion was calculated by dividing the whole vessel area including the lumen, intima, media, and adventitia, as previously described (23). For each animal, 20 sections, i.e., every fourth section, were examined, and the mean of the fraction area was calculated and expressed as a percentage.
Immunohistochemistry
Aortic root cryosections from mice treated with immunoglobulin,
F(ab')2 fragments, or HSA for 8 wk were also processed for
immunohistochemistry as described previously with minor modifications
(14,
27). In brief, anti-macrophage
(anti-M
, M3/84, 1:400, PharMingen), anti-CD4 (GK1.5, 1:50, PharMingen),
anti-CD8 (53–6.7, 1:50, PharMingen), anti-I-Ab
(25–9-17, 1:25, PharMingen), and anti-ICAM-1 (M-19, 1:100, Santa Cruz
Biotechnology) antibodies were applied to acetone-fixed cryosections. After
being washed, the sections were then exposed to a second antibody (horseradish
peroxidase-conjugated antibodies), and the antibody binding was visualized
with diaminobenzidine. Sections were counterstained with 1% methyl green. The
percentage of positively stained cells per infiltrating cells in the lesions
was calculated for each antibody as previously described
(14,
23). That is, lesions of the
aortic root were analyzed. Data were obtained by dividing the number of
positively stained cells by all methyl green-stained cells inside the internal
elastic lamina. Three to five random microscopic fields were analyzed at
x200.
Serum Lipid Measurement
Serum was separated by centrifugation and stored at –80°C. Serum total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and triglyceride (TG) levels were measured with assay kits (Wako) according to the manufacturer's instructions.
Cell Culture
Spleens from mice treated with immunoglobulin, F(ab')2 fragments, or HSA for 16 wk in protocol II were harvested, and single-cell suspensions were obtained by passing through a stainless steel mesh screen. Cells were suspended in RPMI-1640 supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% nonessential amino acids, 5 x 10–5 M 2-mercaptoethanol, and a penicillin-streptomycin mixture.
F-2 cells, a murine endothelial cell line established from an ultraviolet-induced tumor, and rat aorta smooth muscle cells (SMCs) were maintained in 10% fetal calf serum-supplemented DMEM at 37°C in 5% CO2.
Cytotoxicity Assays
Splenocytes from mice treated with immunoglobulin, F(ab')2
fragments, or HSA for 16 wk in protocol II were used as effector
cells. SMCs and F-2 cells (2 x 104 cells/well) plated in
96-microwell plates were labeled with 37 kBq/well sodium chromate
(51Cr, Amersham International) for 1 h. After being labeled, target
cells were washed with PBS three times, and splenocytes were incubated at the
effector-target ratios of 50:1, 100:1, and 200:1 for 4 h. The supernatant was
collected, and the radioactivity of 51Cr release into the
supernatant was measured by a gamma counter. The percentage of cytotoxicity
was calculated using the following formula
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Statistical Analysis
Values are expressed as means ± SD. Statistical analysis of the data was determined by t-test or by one-way ANOVA, followed by the Fisher protected least-significant-difference test. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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As shown in Table 1, treatment with immunoglobulin did not significantly modify the serum lipid profiles in protocols I or II. The HDL-C-to-TC ratio was also similar in all groups of mice. In addition, immunoglobulin did not reduce or increase body weights.
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Effects of Immunoglobulin on Fatty Streak and Fibrofatty Plaque Formation
Apo E-deficient mice were injected with immunoglobulin or HSA and kept on a cholesterol-rich diet for 8 wk to induce fatty streak formation and for 12 and 16 wk to form fibrofatty plaques, respectively. The surface area covered by fatty streak and fibrofatty plaque lesions was quantified in oil red-O-stained samples, and specimens from immunoglobulin-treated mice were compared with HSA-treated controls. Controls developed extensive lesions in the root of the aorta in both stages (Fig. 2). In mice treated with immunoglobulin, as shown in Table 2, the fraction area of lesion was markedly reduced (4.2 ± 2.0% vs. 13.6 ± 4.8% in controls, P < 0.01 for 8-wk treatment; 7.1 ± 2.9% vs 18.3 ± 4.0% in controls, P < 0.01 for 12-wk treatment; 8.1 ± 2.7% vs. 21.5 ± 3.9% in controls, P < 0.01 for 16-wk treatment).
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Effects of the Fc Portion of Immunoglobulin on Atherosclerosis
In protocol II, mice were kept on the high-fat diet for 8 or 16 wk to permit the formation of atherosclerotic lesions, and immunoglobulin, F(ab')2 fragments, or HSA injection were given for 8 or 16 wk. Control treatment resulted in extensive fatty streak formation and advanced fibrofatty plaques in HSA-treated mice (Fig. 3). In mice treated with immunoglobulin, the fractions of the cross-sectional area covered by lesions were markedly reduced (4.6 ± 3.1% vs. 12.9 ± 4.4% in controls, P < 0.01 for 8-wk treatment; 9.6 ± 3.7% vs. 24.4 ± 6.8% in controls, P < 0.01 for 16-wk treatment). In contrast, F(ab')2 fragment treatment did not reduce the lesion areas significantly (11.2 ± 3.7% for 8-wk treatment, 22.8 ± 5.7% for 16-wk treatment, P = not significant vs. HSA each; Table 2).
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Effects of the Fc Portion of Immunoglobulin on Inflammatory Cell Infiltration in Fatty Streak Lesions
The inflammatory cell infiltrations were assessed immunohistologically by
the number of M, CD4+, CD8+, and
I-Ab+ cells divided by the number of methyl
green-stained cells. The percentage of M-positive cells was
significantly reduced in the immunoglobulin-treated group compared with the
control group (Fig. 4 and
Table 3). Treatment with
F(ab')2 fragments did not suppress the frequency of M
compared with the control group. ICAM-1 was expressed on the intimal cells
just below plaques in the control groups. Treatment with intact
immunoglobulin, but not with F(ab')2 fragments, reduced the
expression of ICAM-1 in the lesions.
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Fc Portion of Immunoglobulin Suppresses the Cytotoxic Activity of Splenocytes from Atherosclerotic Mice
The cytotoxic activities of splenocytes against SMCs and F-2 cells from atherosclerotic mice were examined. At an effector-to-target ratio of 200:1, the cytotoxic activities in immunoglobulin-treated mice were significantly suppressed compared with those in HSA-treated mice (Fig. 5). F(ab')2 fragment treatment did not suppress the cytotoxic activities of splenocytes compared with control mice. Accordingly, the antiatherosclerotic effect of immunoglobulin may be due, at least in part, to the inhibition of cytotoxic activity of T cell-enriched splenocytes from atherosclerotic mice.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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accumulation in lesions was present in mice treated with intact immunoglobulin
but not in mice treated with F(ab')2 fragments. Because the
significant role of M in the development of early atherosclerosis has
already been demonstrated (3,
29,
34), and we
(22) have already reported
that administration of an antagonistic rat monoclonal antibody against murine
c-fms could prevent accumulation of macrophages in the aortic intima
and thereby protect atherogenesis in apo E-deficient mice, suppression of one
antigen-presenting cell, M, may lead to a reduction in the severity of
atherosclerosis. Also, reduced ICAM-1 expression in the lesions was
demonstrated by the intact immunoglobulin treatment. Because ICAM-1 expression
is regulated through nuclear factor (NF)-
B, the results suggest that
the treatment may attenuate proinflammatory NF-B signaling in the
artery wall. Our study clearly indicates that the Fc portion of immunoglobulin
plays an important role in the antiatherosclerotic action and hence in the
reduction of the disease. CD4+ T cells play an important role in atherosclerosis (7, 11, 30), and transfer of T cell-enriched splenocytes from atherosclerotic mice aggravates atherosclerosis in immunodeficient apo E KO mice (34). In the present study, the cytotoxicity assay indicated that the cytotoxic activities of splenocytes against endothelial cells and SMCs in immunoglobulin-treated mice were significantly suppressed compared with those in HSA-treated mice. F(ab')2 fragment treatment did not suppress the cytotoxic activities of splenocytes compared with the control mice. Therefore, the antiatherosclerotic effect of immunoglobulin may be due, at least in part, to the inhibition of cytotoxic activity of T cell-enriched splenocytes from atherosclerotic mice, and this action may be mediated via the Fc portion of immunoglobulin.
Fc receptors act as trigger molecules for inflammatory, allergic,
endocytotic, and inhibitory activities of immune effector cells
(4). It has been demonstrated
that human monocyte-derived macrophages uptake pathogens [e.g., oxidized
low-density lipoprotein-containing immune complexes (oxLDL-ICs)] and lead not
only to transformation of macrophages into foam cells but also to macrophage
activation and release of cytokines. Both processes depend on the engagement
of Fc receptor I by oxLDL-ICs
(9,
19). Most recently, it has
been shown that Fc receptors play a pivotal role in experimental neointimal
vascular hyperplasia via the immunoreceptor tyrosine-based activation
motif/immunoreceptor tyrosine-based inhibition motif
(16,
33). We and others have
reported that F(ab')2 fragments did not ameliorate
experimental myocarditis in rats
(27) and that the
anti-inflammatory activity of immunoglobulin was mediated through the
inhibitory Fc receptor (13,
25). In the present study,
treatment with intact immunoglobulin, but not with F(ab')2
fragments, markedly suppressed the progression of atherosclerosis. In a
previous in vitro study, we
(27) have demonstrated that
intact immunoglobulin, but not F(ab')2 fragments,
downregulated CD32 (Fc receptor II) expression in U937 cells. Our study
adds additional information on the precise mode of antiatherosclerotic action
of immunoglobulin, that is, via the Fc portion. It may be reasonably
hypothesized that injected immunoglobulin may block the Fc receptor of immune
effector cells from binding to oxLDL-ICs, resulting in inhibiting T cell
expansion and macrophage activation. Taken together, it appears that the Fc
portion of immunoglobulin may play a role in the pathogenesis of
antiatherosclerosis.
In addition to the immunomodulatory effects, several functions of immunoglobulin have been proposed. Microorganisms have recently been implicated in the pathogenesis of atherosclerosis (5), and there is strong evidence for an association between infections, such as Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus, and atherosclerosis in humans (20). Immunoglobulin preparations contain many different antibody and autoantibody specificities that can neutralize a large number of pathogens and autopathogens (10, 12). Antibodies directed against several soluble and membrane molecules have also been identified in immunoglobulin preparations (2, 17). It is suggested that expression of cytokines may be blocked not only by the anticytokine antibodies included in immunoglobulin but also by the anti-inflammatory action mediated through the inhibitory Fc receptor (1, 31). The possibility of antipathogen and anticytokine antibody effects in the current study is probably very low because F(ab')2 fragments, the antigen-binding portion of immunoglobulin, did not suppress the disease progression, which, theoretically, may possess anticytokine and antipathogen antibodies. Although experiments directly examining the effects of the Fc portion of immunoglobulin itself upon suppression of atherosclerotic lesions were not performed in the present study, it was clearly demonstrated that the antiatherosclerotic effects of immunoglobulin may due to Fc receptor-mediated anti-inflammatory and immunomodulating actions, because intact immunoglobulin, but not F(ab')2 fragments, suppressed the atherosclerosis.
In conclusion, the present study provides evidence that intact immunoglobulin therapy markedly suppresses atherosclerosis due to Fc receptor-mediated anti-inflammatory action. The effects were not associated with a reduction of high serum lipid levels. The antiatherosclerotic effects of immunoglobulin may be related to the suppression of cytotoxic activity of atherogenic T cells and a reduction of macrophage accumulation in the lesions. The findings of the present study may yield important insights into an immunological approach in the treatment and future clinical use of this therapy in human atherosclerosis.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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  K. Sumiyoshi, H. Mokuno, T. Iesaki, K. Shimada, T. Miyazaki, A. Kume, T. Kiyanagi, K. Kuremoto, Y. Watanabe, N. Tada, et al. Deletion of the Fc receptors {gamma} chain preserves endothelial function affected by hypercholesterolaemia in mice fed on a high-fat diet Cardiovasc Res, December 1, 2008; 80(3): 463 - 470. [Abstract] [Full Text] [PDF]
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 C. Kishimoto A Novel Approach to the Suppression of Atherosclerosis by Fc{gamma} Receptor Blockade Circ. Res., November 24, 2006; 99(11): 1154 - 1155. [Full Text] [PDF]
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P. Hernandez-Vargas, G. Ortiz-Munoz, O. Lopez-Franco, Y. Suzuki, J. Gallego-Delgado, G. Sanjuan, A. Lazaro, V. Lopez-Parra, L. Ortega, J. Egido, et al. Fc{gamma} Receptor Deficiency Confers Protection Against Atherosclerosis in Apolipoprotein E Knockout Mice Circ. Res., November 24, 2006; 99(11): 1188 - 1196. [Abstract] [Full Text] [PDF]
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