Serological and genetic evidence for altered complement system functionality in systemic lupus erythematosus: Findings of the GAPAID consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of classswitched autoantibodies targeting nucleic acids

Régulation de la mort cellulaire programmée : vers une conception plus dynamique

La mort cellulaire joue un rôle fondamental dans le maintien de l’intégrité de l’organisme et son dérèglement a été identifié dans de nombreux processus pathologiques. Une activation en cascade de protéases à cystéine - les caspases - conduit au clivage de divers substrats protéiques essentiels à la survie cellulaire et permet l’amplification d’un signal de mort provenant principalement de deux voies : (1) l’une déclenchée par des protéines relarguées par des mitochondries ; (2) l’autre engendrée dans la membrane plasmique par des récepteurs, membres de la famille du TNFR (tumor necrosis factor receptor). Pour chacune de ces voies, les mécanismes d’intégration des signaux s’établissent au niveau d’un complexe multiprotéique, respectivement l’apoptosome et le DISC (death inducing signaling complex). C’est en agissant sur la formation et/ou sur l’activité de ces complexes que la plupart des modulateurs de la mort cellulaire exercent leur action. Par ailleurs, des complexes multiprotéiques localisés dans d’autres lieux stratégiques de la cellule sont également indispensables à l’intégration et/ou à la régulation des signaux de mort cellulaire

An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes

Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death-inducing signaling complex (DISC) that includes Fas, the Fas-associated death domain protein (FADD) and caspase-8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid- and cholesterol-rich membrane rafts in mouse thymocytes as well as the L12.10-Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase-8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas-triggered recruitment of FADD and caspase-8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas-mediated cell death signaling

DCC association with lipid rafts is required for netrin-1-mediated axon guidance.

During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues

Absolute Quantitation of Serum Antibody Reactivity Using the Richards Growth Model for Antigen Microspot Titration

In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen-specific serum antibodies. We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease-specific antigens were treated with different serum dilutions and developed for IgG and IgA binding. By fitting the binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen. By providing universal chemical units, this approach may improve the standardization of serological testing, the quality control of antibodies and the quantitative mapping of the antibody–antigen interaction space

Synergistic stimulation of IgG producing plasma cell differentiation by anti-Ig and CpG ODN in the presence of TAK1 inhibitor.

<p>Purified human blood B cells (10⁵ cells/well) were cultured with anti-Ig (2.5 µg/ml), BAFF (100 ng/ml) and CpG (0.5 µg/ml) as indicated below the figure for 4 days in the presence of IL-2 (50 ng/ml) and IL-10 (50 ng/ml). Numbers of IgG producing plasma cells were determined by ELISPOT assay. Spot numbers in the presence of vehicle (white bars) and spots in the TAK1 inhibitor treated samples (black bars) are shown. Means ±SD of three different experiments.</p

BCR synergizes with TLR9 on a TAK1 dependent manner for cytokine production in primary human B cells.

<p>Secreted IL-6, IL-8, IL-10 and TNFα were measured after 48 h from culture supernatants of vehicle treated (white bars) and TAK1 inhibitor treated (black bars) purified human tonsill (A) and blood (B) B cells. Data represent the mean ±SD of three different experiments with resting tonsill B cells (A), while one experiment with blood B cells (B) is shown. *p<0.05.</p

Phospho-flow analysis of p38 MAPK activation in resting and activated B cells from healthy blood donors and from RA patients.

<p>PBMC (4×10⁶ cells) were left unstimulated (control) or stimulated with anti-Ig (7.5 µg/ml), CpG (4 µg/ml) or both agents for 30 min at 37°C. p38 phosphorylation was tested in the CD19⁺ CD27⁻ naive B cell subset. (A) The activation of p38 MAPK in healthy (square; <i>n</i> = 17) and RA (triangle; <i>n</i> = 13) B cells were analyzed by flow cytometry. (B) The relative activation of p38 MAPK was calculated based on the alteration of phosphorylated MAPKs expression in resting and stimulated naive B cells (stimulated sample MFI/resting sample MFI). Paired Student's t-test was used to assess the differences of phospho-p38 MAPK expression between control and stimulated samples. *<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.0005.</p

TAK1 dependent activation of the MAPK and NFκB pathways in B cells stimulated with combinations of anti-IgG, CPG ODN and BAFF.

<p>Resting tonsill B cells were stimulated with combinations of anti-Ig (2.5 µg/ml), BAFF (100 ng/ml) and CpG (2 µg/ml) for 30 min with or without TAK1 inhibitor ((5Z)-7-Oxozeaenol), and the phosphorylation of various signaling molecules was tested by Western blot.</p