ABSTRACT
In humans and animal models of atherosclerosis, antibodies against oxidized LDL have been associated with atherosclerotic lesion development. It has been suggested that IgM anti-oxLDL antibodies are anti-atherogenic, whereas IgG anti-oxLDL antibodies are pro-atherogenic. In this study, we examined the relation between IgM and IgG antibody levels and atherosclerosis severity in APOE(-/-)CD40L(-/-) mice, which are deficient for IgG and develop moderate advanced atherosclerosis, and compared results with mice developing severe (APOE(-/-)) or no atherosclerosis (C57Bl/6). Mice were followed in time for anti-oxLDL antibodies while on high-fat diet or normal chow. Anti-oxLDL antibody levels were determined by ELISA. Results revealed that 24-week-old APOE(-/-)CD40L(-/-) mice had enhanced IgM anti-oxLDL antibody levels when compared with wild-type mice, but similar levels to those of APOE(-/-) mice. As expected, IgG anti-oxLDL antibody levels were almost absent in APOE(-/-)CD40L(-/-) mice. The transition from early to advanced lesions in APOE(-/-) mice was reflected by elevated IgM anti-oxLDL antibody levels. IgM anti-oxLDL levels did not further increase during progression to more advanced lesions. No relation was found between IgG anti-oxLDL levels and atherosclerosis severity. In conclusion, the severity of advanced atherosclerosis in mice is not reflected by IgM and/or IgG anti-oxLDL antibody levels. Furthermore, less advanced atherosclerotic lesion development in APOE(-/-)CD40L(-/-) mice does not seem to be the result of higher levels of protective IgM anti-oxLDL antibodies. Therefore, our study does not support the idea that the previously observed inconsistency in the relation between anti-oxLDL and atherosclerosis severity is due to differences in antibody isotypes.
Subject(s)
Atherosclerosis/diagnosis , Autoantibodies/blood , Lipoproteins, LDL/immunology , Animals , Apolipoproteins E/genetics , Atherosclerosis/immunology , Biomarkers/blood , CD40 Ligand/genetics , Disease Models, Animal , Disease Progression , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.
Subject(s)
Atherosclerosis/immunology , CD4-Positive T-Lymphocytes/immunology , CD40 Ligand/blood , Receptors, Interleukin-2/immunology , Animals , Aorta, Thoracic/pathology , Atherosclerosis/blood , Atherosclerosis/pathology , Bone Marrow/immunology , Bone Marrow Transplantation/immunology , CD40 Ligand/immunology , Disease Models, Animal , Disease Progression , Female , Flow Cytometry , Immunohistochemistry , Lymphocyte Count , Male , Mice , Mice, Inbred C57BL , Phenotype , T-Lymphocytes, Regulatory/immunologyABSTRACT
Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four different sequences of the PR3 cDNA were amplified by PCR: two clones containing the pro-peptide of PR3 with or without a His-tag (rproPR3-his and rproPR3, respectively) and two clones without the pro-peptide and with or without a His-tag (rPR3-his and rPR3, respectively). The PR3 sequences were cloned behind the polyhedrin promoter and the honeybee melittin signal peptide enabling secretion of rPR3. Plasmids were transposed into the genome of baculovirus, and wild types as well as PR3-containing virus genomes were transfected into Sf21 insect cells. All four rPR3 variants were secreted into the medium and were recognized by anti-neutrophil PR3 rabbit serum and by at least two anti-PR3 monoclonal antibodies. Mature forms of PR3 were recognized by almost all patient sera, whereas the pro-forms of PR3 were recognized by 14 of 18 PR3-ANCA sera tested. On SDS-PAGE, the four rPR3 forms migrated at approximately 32 kDa. RPR3-his and rproPR3-his could be purified by means of this His-tag. In conclusion, especially the mature rPR3s are well recognized by PR3-ANCA sera. The presence of a C-terminal His-tag facilitated purification of His-tagged rPR3. Thus, rPR3 expressed in insect cells can be used as a tool for diagnostic tests as well as for epitope mapping studies.
