Anti-phosphorylcholine Antibodies in Cardiovascular Disease

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the industrialized countries and a growing concern for the developing world. The underlying cause of almost all CVD is atherosclerosis, a process that is characterized by a low-grade inflammation in the artery walls. This inflammation is believed to be initiated by components of low density lipoproteins which, upon oxidation, display pro-inflammatory self-neo epitopes such as malonyldialdehyde (MDA) and phosphorylcholine (PC). Antibodies directed against the PC-epitope (anti-PC) have been researched for several decades but the relatively recent realization that these antibodies also recognize oxidized phospholipids has revolutionized the field and opened up a whole new avenue of investigation. Anti-PC has been shown to aid the clearance of apoptotic cells and prevent the formation of foam cells by clearing oxidized low density lipoprotein. Murine studies with PC-vaccination have shown strong beneficial effects on experimental atherosclerosis in vivo and human epidemiology has consistently linked anti-PC insufficiency to CVD. All humans have detectable anti-PC IgM in serum, though the concentration varies greatly between individuals. Our group has previously shown that people with low serum/plasma levels of anti-PC IgM have increased risk of CVD and the subgroup analysis had indicated that this association was particular strong with regard to the incidence of stroke. In paper I, we tested this hypothesis in a stroke material from northern Sweden. A significant association between low plasma level of anti-PC IgM at baseline and incident stroke was seen for the whole group at anti-PC levels below the 30th percentile (OR 1.62; CI 1.11 to 2.35). Analyses of gender-specific associations indicated fairly strong associations for females, especially at the lowest 30th percentile (OR 2.65; CI 1.41 to 4.95). However, no association was noted for men. Paper II focused on the properties of different anti-PC antibody classes/subclasses. We report that anti-PC IgM, IgA and IgG1 (but not IgG2) were negatively associated with IMT-progression, which is a surrogate marker for atherosclerosis development. Examination of binding profiles revealed that the protective isotypes (IgM, IgA and IgG1) have a different fine-specificity than the non-protective IgG2. Analysis of serum samples taken four years apart in study participants showed that anti-PC IgM titers, essentially, do not change over time. In this paper, we also demonstrate that anti-PC IgM inhibits LPC-induced cell death in vitro and propose that this is yet another protective mechanism. For paper III, I had successfully designed a PC-specific probe to identify, isolate and characterize PC-reactive B cells from ten healthy human donors. We found that all ten had mounted somatically mutated antibodies towards PC utilizing a broad variety of immunoglobulin-genes. PC-reactive B cells were primarily found in the IgM memory subset though significant numbers were also detected among naïve, IgG and CD27CD43 B cells. From the isolated B cells, we derived several human monoclonal antibodies (mAbs) with proven PC specificity. In conclusion, the future of CVD treatment lies with immunomodulation and the premise of this thesis represents one avenue of research in the quest for novel diagnostic tools and improved treatment options. My work has focused on understanding the properties and molecular ontogeny of human anti-PC antibodies. Though this thesis represents one important step in the direction of clinical application, much more research is needed before we will see use of anti-PC mAbs or PC-vaccination in the treatment of patients. LIST OF PUBLICATIONS RELATED PUBLICATIONS Ajeganova S, Fiskesund R, de Faire U, Hafström I, Frostegård J. Effect of biological therapy on levels of atheroprotective antibodies against phosphorylcholine and apolipoproteins in rheumatoid arthritis a one year study. Clin Exp Rheumatol. 2011 Nov-Dec;29(6):942-50. Liu A, Ming JY, Fiskesund R, Ninio E, Karabina SA, Bergmark C, Frostegård AG, Frostegård J. Induction of dendritic cell-mediated T cell activation by modified but not native LDL in humans and inhibition by Annexin A5: heat shock proteins as possible T-cell antigens. Manuscript to be published I. Fiskesund R, Stegmayr B, Hallmans G, Vikstrom M, Weinehall L, de Faire U and Frostegard J. "Low levels of antibodies against phosphorylcholine predict development of stroke in a populationbased study from northern Sweden." Stroke. 2010 Apr; 41(4): 607-612. II. Fiskesund R, Su J, Bulatovic I, Vikström M, de Faire U, Frostegard J. IgM phosphorylcholine antibodies inhibit cell death and constitute a strong protection marker for atherosclerosis development, particularly in combination with auto-antibodies against modified LDL. Results in Immunology. 2012. 2(0): 13-18. III. Fiskesund R, Steen J, Amara K, Murray F, Szwajda A, Liu A, Douagi I, Malmström V and Frostegård J. Naturally occurring human phosphorycholine antibodies predominantly products of affinity-matured B cells in the adult. Manuscript to be published. LIST OF ABBREVATIONS PC Phosphorylcholine Anti-PC Antibody against phosphorylcholine CVD Cardiovascular Disease Ig Immunoglobulin LDL Low density lipoprotein oxLDL Oxidized low density lipoprotein TCR T cell receptor BCR B cell receptor AID Activation induced deaminase MDA Malonyldialdehyde PTC Phosphatidylcholine PAF Platelet activating factor LPC Lysophosphatidylcholine ScR Scavenger receptor APC Antigen presenting cell DC Dendritic cell NK cell Natural Killer cell NKT cell Natural Killer T cell SMC Smooth muscle cell PAMP Pathogen associated molecular pattern PRR Pattern recognition receptor TLR Toll-like receptor LPS Lipopolysacharide CRP C-reactive protein mAb monoclonal antibody ELISA Enzyme linked immunosorbent assay IMT Intima-media thicknness FACS Flowcytometry activated cell sorting CDR Complementarity determining region Lp-PLA2 Lipoprotein associated phospholipase A2