Dose-Dependent Induction of an Idiotypic Cascade by Anti-Glycosaminoglycan Monoclonal Antibody in apoE-/- Mice: Association with Atheroprotection - PubMed (original) (raw)

Dose-Dependent Induction of an Idiotypic Cascade by Anti-Glycosaminoglycan Monoclonal Antibody in apoE-/- Mice: Association with Atheroprotection

Roger Sarduy et al. Front Immunol. 2017.

Abstract

Atherosclerosis, the underlying pathology of most cardiovascular diseases, is triggered by the retention of apolipoprotein B (apoB)-containing lipoproteins in the arterial wall through electrostatic interactions with glycosaminoglycan (GAG) side chains of proteoglycans. Previously, we reported the antiatherogenic properties of the chimeric monoclonal antibody (mAb) chP3R99-LALA, which binds sulfated GAGs, inhibits low-density lipoprotein (LDL)-chondroitin sulfate (CS) association, and abrogates LDL oxidation and foam cell formation. In preventive and therapeutic settings, apoE-deficient (apoE-/-) mice immunized with 50 μg of this mAb showed reduced atherosclerotic lesions related with the induction of autologous anti-GAG antibodies. Knowing that age and sex are major non-modifiable risk factors in the development of atherosclerosis, the present study aimed to assess the influence of these variables on the capacity of chP3R99-LALA mAb to generate an anti-CS antibody response. Also, we aimed at defining the impact of the dose of chP3R99-LALA on the anti-CS antibody induction and the atheroprotective effect of this mAb in apoE-/- mice. Neither age nor sex had an impact in the IgG anti-CS antibody response induced by s.c. immunization with this mAb. Moreover, chP3R99-LALA mAb reduced atherosclerotic lesions to a similar extent in both young male and female apoE-/- mice fed a hypercholesterolemic diet and, in middle-aged female apoE-/- mice, with spontaneous lesions. On the other hand, increasing the dose of chP3R99-LALA (200 vs. 50 μg) elicited an anti-idiotype antibody cascade characterized by higher levels of anti-idiotype (Ab2), anti-anti-idiotype (Ab3), and anti-CS antibody responses. Moreover, this dose increment resulted in a striking reduction of aortic atherosclerotic lesions in immunized mice.

Keywords: antibodies; atheroprotection; atherosclerosis; glycosaminoglycans; idiotypic cascade; monoclonal antibody.

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Figures

Figure 1

Figure 1

Effect of age and gender on the induction of anti-chondroitin sulfate (CS) antibody response in apoE−/− mice immunized with chP3R99-LALA monoclonal antibody (mAb). Mice fed a chow diet received four s.c. injections of 50 μg of chP3R99-LALA mAb at weekly intervals and two additional immunizations biweekly. Sera were obtained prior to the first antibody injection and after the fourth, fifth and sixth immunization of (A) adolescent (6-week olds, n = 5), (B) young adult (16-week olds, n = 5), (C) middle-aged/old (56- to 76-week olds, n = 3) male, and (D) middle-aged (35-week olds, n = 9) female apoE−/− mice. To measure the kinetics of the response against CS, sera from immunized mice (diluted 1:400) were added to enzyme-linked immunoadsorbent assay plates coated with 10 μg/mL of CS and the reaction was developed with peroxidase-conjugated goat anti-mouse IgG. The levels of anti-CS antibodies are expressed as optical density (OD) values. Results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey post hoc test.

Figure 2

Figure 2

Effect of immunization with chP3R99-LALA monoclonal antibody (mAb) on atherosclerosis development in apoE−/− mice of both genders. (A) Experimental design: arrows represent immunization time points with 50 μg of chP3R99-LALA mAb or the isotype-matched control hR3 mAb. Blue and red bars indicate treatment and high-fat high-cholesterol diet period, respectively. (B) Representative en face Oil-Red-O stained aortas from each experimental group. (C) Mean percentage of aortic lesion areas in male and female apoE−/− mice treated with chP3R99-LALA (n = 11 or 8) or hR3 (n = 8 or 4). Results are mean ± SEM. *P < 0.05, Student’s _t_-test.

Figure 3

Figure 3

Antibody response to chondroitin sulfate (CS) in the sera of apoE−/− mice immunized with different doses of chP3R99-LALA monoclonal antibody (mAb). A 6-week-old mice fed with chow diet received four weekly s.c. injections of (A) 50 μg (n = 9) or (B) 200 μg (n = 10) of chP3R99-LALA mAb. Sera obtained before starting immunizations and 1 week after the third and fourth antibody injection (diluted 1:400) were added to enzyme-linked immunoadsorbent assay plates coated with 10 μg/mL of CS and the reaction was developed with peroxidase-conjugated goat anti-mouse IgG. The levels of IgG are expressed as OD values. Results are mean ± SEM. *P < 0.05, ***P < 0.001, one-way ANOVA followed by Tukey post hoc test.

Figure 4

Figure 4

Anti-idiotype (A,B) and anti-anti-idiotype (C,D) antibody responses induced in apoE−/− mice by immunization with doses of 50 μg (A,C) and 200 μg (B,D) of chP3R99-LALA monoclonal antibody (mAb). The anti-idiotype (A,B) and anti-anti-idiotype (C,D) antibody responses were measured by enzyme-linked immunoadsorbent assay, adding mice sera (diluted 1:1,000) to chP3R99-LALA mAb- or ch1E10 Ab2 mAb-coated plates, respectively (white bars). hR3 mAb-coated plates were used to measure the anti-isotype response (black bars). The reaction was developed with peroxidase-conjugated goat anti-mouse IgG. Sera were obtained before the first antibody injection and 1 week after the third and fourth immunization. The serological antibody responses are expressed as OD values. Results are mean ± SEM. *P < 0.05, ***P < 0.001, Student’s _t_-test when the levels of anti-chP3R99-LALA or anti-ch1E10 mAb serological antibody responses were compared with those against hR3 mAb.

Figure 5

Figure 5

Antiatherosclerotic effect induced by chP3R99-LALA monoclonal antibody (mAb) immunization at different doses in apoE−/− mice. (A) Experimental design: arrows represent immunization points according to the experimental procedure with 50 μg of chP3R99-LALA mAb (n = 11), 200 μg of chP3R99-LALA mAb (n = 14), or 200 μg of hR3 mAb (n = 8). (B) Representative en face Oil-Red-O-stained aortas from the different groups. (C) Mean percentage of aortic lesion area in apoE−/− mice treated with 50 or 200 μg of chP3R99-LALA or hR3. Results are mean ± SEM. *P < 0.05, ***P < 0.001, one-way ANOVA followed by Tukey post hoc test.

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