Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Apolipoprotein E Triggers Complement Activation in Joint Synovial Fluid of Rheumatoid Arthritis Patients by Binding C1q.

We identified apolipoprotein E (ApoE) as one of the proteins that are found in complex with complement component C4d in pooled synovial fluid of rheumatoid arthritis (RA) patients. Immobilized human ApoE activated both the classical and the alternative complement pathways. In contrast, ApoE in solution demonstrated an isoform-dependent inhibition of hemolysis and complement deposition at the level of sC5b-9. Using electron microscopy imaging, we confirmed that ApoE interacts differently with C1q depending on its context; surface-bound ApoE predominantly bound C1q globular heads, whereas ApoE in a solution favored the hinge/stalk region of C1q. As a model for the lipidated state of ApoE in lipoprotein particles, we incorporated ApoE into phosphatidylcholine/phosphatidylethanolamine liposomes and found that the presence of ApoE on liposomes increased deposition of C1q and C4b from serum when analyzed using flow cytometry. In addition, posttranslational modifications associated with RA, such as citrullination and oxidation, reduced C4b deposition, whereas carbamylation enhanced C4b deposition on immobilized ApoE. Posttranslational modification of ApoE did not alter C1q interaction but affected binding of complement inhibitors factor H and C4b-binding protein. This suggests that changed ability of C4b to deposit on modified ApoE may play an important role. Our data show that posttranslational modifications of ApoE alter its interactions with complement. Moreover, ApoE may play different roles in the body depending on its solubility, and in diseased states such as RA, deposited ApoE may induce local complement activation rather than exert its typical role of inhibition.

Activation of Complement by Pigment Epithelium-Derived Factor in Rheumatoid Arthritis.

The aim of this study was to identify molecules that trigger complement activation in rheumatic joints. C4d, the final cleavage product of C4 activation, is found in the diseased joint and can bind covalently to complement-activating molecules. By using a highly specific Ab against a cleavage neoepitope in C4d, several molecules that were specifically bound to C4d were identified from pooled synovial fluid (SF) from four rheumatoid arthritis (RA) patients. One of these molecules, pigment epithelium-derived factor (PEDF), is a broadly expressed multifunctional member of the serine proteinase inhibitor family. Using ELISA, we confirmed the presence of various amounts of complexes between PEDF and C4d in the SF from 30 RA patients, whereas none were detected in SF from control subjects. Correlation analyses suggested that, in arthritis patients, C4d-PEDF complexes found in sera arise from the joints, as well as from other tissues, and levels of the complexes did not differ in sera of RA patients and healthy controls. When immobilized, recombinant PEDF expressed in eukaryotic cells activated the classical complement pathway but not the alternative or lectin pathways. C1q protein was demonstrated to bind immobilized PEDF, and PEDF was shown to bind to immobilized C1q, in particular its head regions, which are known to interact with other activators of the classical pathway. Our results call for further investigation into the role of PEDF in inflammatory processes in the joint, which, in combination with classical complement activation, appears to be part of a (patho-)physiologic response.

Chain length-dependent effects of inulin-type fructan dietary fiber on human systemic immune responses against hepatitis-B.

SCOPE: In vivo studies demonstrating that only specific dietary-fibers contribute to immunity are still inconclusive, as measuring immune effects in healthy humans remains difficult. We applied a relatively inefficacious vaccination-challenge to study chain length-dependent effects of inulin-type fructan (ITF) dietary fibers on human immunity. METHODS AND RESULTS: ITFs with two different 'degree of polymerization-' (DP)-profiles were tested in vitro for effects on PBMC-cytokines and TLR2 activation. In a double-blind placebo-controlled trial, 40 healthy volunteers (18-29 years) were divided into three groups and supplemented from day 1 to day 14 with DP10-60 ITF, DP2-25 ITF (both n = 13), or fructose placebo (n = 14), 8 g/day. On day 7, all volunteers were vaccinated against hepatitis B. Anti-HbsAg-titer development and lymphocyte subsets were studied. In vitro, DP10-60 ITFs stimulated a Th1-like cytokine profile and stimulated TLR2 more strongly than DP2-25 ITFs. In vivo, DP10-60 increased anti-HBsAg titers, Th1-cells, and transitional B-cells. Both ITFs increased CD45ROhi CTLs at day 35, and CD161+ cytokine producing NK-cells at day 21 and 35. CONCLUSION: Support of immunity is determined by the chain length of ITFs. Only long-chain ITFs support immunity against pathogenic hepB-epitopes introduced by vaccination. Our findings demonstrate that specific dietary fibers need to be selected for immunity support.

Toll-like receptor 2 activation by ß2→1-fructans protects barrier function of T84 human intestinal epithelial cells in a chain length-dependent manner.

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that ß2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether ß2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 ß2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by ß2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain ß2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain ß2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with ß2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that ß2→1-fructans are specific ligands for TLR2. To conclude, ß2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of ß2→1-fructan-mediated health effects.