Adrienn Bíró

Studies on the interactions between C-reactive protein and complement proteins

Several studies have investigated the interactions between C‐reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b‐binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme‐linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1‐INH. Native, ligand‐unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea‐treatment or by site‐directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1‐activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.

Relationship of Anti-60 kDa Heat Shock Protein and Anti-Cholesterol Antibodies to Cardiovascular Events

Background—Several recent studies have indicated an association between key inflammatory mediators and atherosclerotic diseases. We evaluated whether high levels of antibodies against heat shock proteins and cholesterol (ACHA) predicted cardiovascular (CV) events. Methods and Results—We used blood samples from the Heart Outcomes Prevention Evaluation (HOPE) study to conduct a nested case-control study of 386 cases with CV events and 386 age- and sex-matched HOPE study controls without events. We explored the relationship between anti-hsp antibodies, ACHA, and subsequent outcomes (incident myocardial infarction, stroke, or CV death) during a mean follow-up of 4.5 years using conditional logistic regression. High levels of anti-hsp65 antibodies (≥90th percentile) predicted CV events (OR, 2.1; 95% CI, 1.2 to 3.9, P =0.01). Anti-hsp60 antibodies did not predict any event type, whereas incident stroke developed significantly less frequently in patients with high ACHA levels. Anti-hsp antibodies and ACHA did not correlate with inflammatory (fibrinogen, C-reactive protein, interleukin-6, intracellular adhesion molecule-1) or infectious markers (C pneumoniae or cytomegalovirus antibodies). Anti-hsp65 antibodies (≥90th percentile) and fibrinogen (highest tertile) had a strong joint effect: patients with high concentrations of both had more CV events (OR, 5.5; 95% CI, 1.8 to 17.5, P =0.004) than patients with low levels of both. A similar joint effect (OR, 2.7; 95% CI, 1.3 to 5.7, P =0.01) was found for high levels of anti-hsp65 and presence of cytomegalovirus antibodies. Conclusions—Serum antibodies to hsp65 were associated with subsequent CV events in this study of high-risk patients, independent of conventional cardiovascular risk factors and other inflammatory markers.

Proteins of the Innate Immune System Crystallize on Carbon Nanotubes but Are Not Activated

The classical pathway of complement is an essential component of the human innate immune system involved in the defense against pathogens as well as in the clearance of altered self-components. Activation of this pathway is triggered by C1, a multimolecular complex comprising a recognition protein C1q associated with a catalytic subunit C1s-C1r-C1r-C1s. We report here the direct observation of organized binding of C1 components C1q and C1s-C1r-C1r-C1s on carbon nanotubes, an ubiquitous component in nanotechnology research. Electron microscopy imaging showed individual multiwalled carbon nanotubes with protein molecules organized along the length of the sidewalls, often over 1 μm long. Less well-organized protein attachment was also observed on double-walled carbon nanotubes. Protein-solubilized nanotubes continued to attract protein molecules after their surface was fully covered. Despite the C1q binding properties, none of the nanotubes activated the C1 complex. We discuss these results on the adsorption mechanisms of macromolecules on carbon nanotubes and the possibility of using carbon nanotubes for structural studies of macromolecules. Importantly, the observations suggest that carbon nanotubes may interfere with the human immune system when entering the bloodstream. Our results raise caution in the applications of carbon nanotubes in biomedicine but may also open possibilities of novel applications concerning the many biochemical processes involving the versatile C1 macromolecule.

The 8.1 ancestral MHC haplotype is strongly associated with colorectal cancer risk

Many recent data indicate that some alleles encoded in the central major histocompatibility complex (MHC) region (Class III) of short arm of chromosome 6 may modify the risk of cancer development. Therefore we determined 4 single nucleotide polymorphisms (SNPs) of this region (TNF‐α −308 G > A, RAGE −429 T > C, HSP70‐2 −1267 A > G, LTA 252 A > G) in genomic DNA samples from 183 Hungarian patients with colorectal cancer and 141 age matched control subjects representing the Hungarian population of the same age and gender. No significant differences were found in either SNP tested. When, however, three‐ or four‐locus haplotypes consisting of known constituents of the so‐called 8.1 ancestral haplotype (8.1AH) were considered, marked differences were observed. Frequency of TNF‐α −308A, RAGE −429C, HSP70‐2 −1267G, LTA 252G (8.1AH) haplotype was significantly (p = 0.006) more frequent (19.1%) among patients than in the controls (7.7%). Age‐ and gender‐adjusted ratio of the 8.1AH carriers vs. non‐carriers to have colorectal cancer was 2.514 (1.130–5.594). This risk was higher in ≤67 years old subjects (4.073 (1.317–12.596)) and in females (3.771 (1.302–10.927). These findings—consistent with similar recent results with ovarian cancer—indicate that carriers of the 8.1AH, encoding for an altered immune response and known to be associated with alterations of several immune functions and autoimmune diseases have an increased risk for some cancer types. These findings may contribute to better understanding how the defense mechanisms against tumors could be enhanced/strengthened.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3β-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Complement Protein C1q Recognizes Enzymatically Modified Low-Density Lipoprotein through Unesterified Fatty Acids Generated by Cholesterol Esterase

We previously reported that enzymatically modified low-density lipoprotein (E-LDL) particles obtained by LDL treatment with trypsin and then cholesterol esterase are recognized by C1q and activate the C1 complex of complement. The objective of this study was to identify the E-LDL component(s) recognized by C1q. In addition to trypsin, plasmin, thrombin, tryptase, and matrix metalloprotease-2 each yielded E-LDL particles with high C1-activating efficiency, and the C1 activation extent was strictly dependent on cholesterol esterase treatment in all cases. When incorporated into vesicles, the lipid fraction of E-LDL, but not of native LDL, triggered C1 activation, and activation correlated with the amount of unesterified cholesterol generated by cholesterol esterase. Whereas treatment of E-LDL particles with human serum albumin reduced their fatty acid content, both cholesterol and unesterified fatty acids were decreased by methyl-beta-cyclodextrin, both treatments resulting in dose-dependent inhibition of the C1-activating ability of the particles. Incorporation of linoleic acid into phosphatidylcholine-containing model vesicles enabled them to interact with the C1q globular domain and to trigger C1 activation, and cholesterol enhanced both processes by facilitating incorporation of the fatty acid into the vesicles. Direct evidence that C1q binds E-LDL through its globular domains was obtained by electron microscopy. This study demonstrates that C1 binding to E-LDL particles involves recognition by the C1q globular domain of the unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed.

Anti-cholesterol antibodies in human sera.

In the last 30 years many research showed that high serum cholesterol level is a great risk for the atherosclerosis. In recent years, it has become clear that the immune system has a major role in atherosclerosis development and progression, and has binding capacity to cholesterol as well. It has been demonstrated in animal experiments, that anti-cholesterol antibodies (ACHA) can prevent cholesterol diet induced atherosclerosis. Our group is looking for the answer, whether ACHA have the same function in animals and in humans, or not. In this review we summarize our studies in human sera. We measured serum ACHA levels in different groups of patients with atherosclerotic diseases in patients with viral infections and in healthy population. In the summary we write about the possible functions of ACHA in the human immune system.

Serum Anti-cholesterol Antibodies in Chronic Hepatitis-C Patients During IFN-α-2b Treatment

Previously we detected more than 3 times higher anti-cholesterol antibody (ACHA) levels in HIV positive patients compared to healthy individuals, however, this level significantly decreased during highly active anti-retroviral therapy (HAART). In our present study we examined whether these findings could also be detected in patients with chronic hepatitis C (CHC). We calculated the correlation between the ACHA levels and the C5b-9 complement activation product. 39 patients with CHC were treated with IFN-alpha-2b (Schering-Plough) 5 MU daily for 6 weeks, followed by 5 MU TIW. Serum levels of ACHA and complement activation products were measured with ELISA. Serum HCV RNA was measured by a highly sensitive branched DNA technique before and 3, 6 and 12 months after the beginning of IFN-alpha-2b therapy. 52 healthy persons served as controls. At the onset of treatment ACHA level was significantly (p = 0.0062) higher in patients (40 (24-69) AU/ml) (median (interquartile range)) than in control sera (26 (20-35) AU/ml). In the 26 responder patients ACHA levels decreased to the normal level during the therapy, but no change was observed in the 13 non-responders. In patients with a sustained response ACHA levels remained low till the end of the 12 months IFN treatment. ACHA levels were significantly (p = 0.0422) higher in the patients with low ( or = 4.0 mmol/l) cholesterol concentrations. The ACHA level before the therapy strongly correlated (r = 0.5499, p = 0.0014) with C5b-9 serum levels. ACHA levels are elevated in CHC, but this elevation is not as high as in HIV. Decrease of viral load by IFN-alpha-2b treatment in the responders results in normalization of ACHA concentration. High ACHA levels in patients with low serum cholesterol concentration suggest that high ACHA levels may contribute to the decrease in cholesterol levels. The correlation between the ACHA and C5b-9 levels indicate, that the ACHA may play a role in the complement activation in CHC.

Anti-cholesterol antibody levels in hereditary angioedema.

Hereditary angioedema (HAE) is a rare disorder caused by the deficiency of the C1‐inhibitor gene (C1INH) and characterized by recurrent bouts of angioedema. Autoimmune disorders frequently occur in HAE. Previously we found, that danazol has an adverse effect on serum lipid profile: reduced high‐density lipoprotein (HDL) and elevated low‐density lipoprotein (LDL) cholesterol levels are associated with long‐term prophylactic use, whereas total cholesterol levels are unchanged. Our aim was to study the anti‐cholesterol antibody (ACHA) production in HAE patients and compare it with those of healthy blood donors, and to investigate the possible associations between ACHA levels and serum lipid profile alterations caused by danazol. Anti‐cholesterol IgG levels were measured by ELISA and their correlation with serum concentrations of total cholesterol, HDL, LDL, triglycerides was determined in HAE patients receiving/not receiving danazol. Serum ACHA levels were significantly higher in HAE patients, compared to healthy blood donors (P < 0.0001). Longterm danazol prophylaxis had no effect on serum ACHA levels in HAE patients. However, we found a significant, negative correlation between ACHA levels and serum total cholesterol (r =−0.4033, P = 0.0200), LDL (r =−0.4565, P = 0.0076) and triglyceride (r =−0.4230, P = 0.0121) levels only in danazol‐treated patients, but not in HAE patients who did not receive long‐term prophylaxis. Patients with HAE have higher baseline ACHA levels compared to healthy subjects, and this might reflect polyclonal B‐cell activation. The latter would be a potential explanation for the lack of an increased incidence of infectious diseases in HAE patients, but might lead to increased autoimmunity.

Enzymatically Modified Low-Density Lipoprotein Is Recognized by C1q and Activates the Classical Complement Pathway

Several studies suggest that the complement system is involved in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of LDL to bind and activate C1, the complex protease that triggers the classical pathway of complement. Unlike native LDL, oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were both recognized by the C1q subunit of C1, but only E-LDL particles, obtained by sequential treatment with a protease and then with cholesterol esterase, had the ability to trigger C1 activation. Further investigations revealed that C1q recognizes a lipid component of E-LDL. Several approaches, including reconstitution of model lipid vesicles, cosedimentation, and electron microscopy analyses, provided evidence that C1 binding to E-LDL particles is mediated by the C1q globular domain, which senses unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed.