Antonella Capozzi

Advanced glycation end products of human β2 glycoprotein I modulate the maturation and function of DCs

In chronic disorders related to endothelial cell dysfunction, plasma β₂ glycoprotein I (β₂GPI) plays a role as a target antigen of pathogenetic autoimmune responses. However, information is still lacking to clarify why β₂GPI triggers autoimmunity. It is possible that posttranslational modification of the protein, such as nonenzymatic glycosylation, leads to the formation of advanced glycation end products (AGEs). The aim of our study was to explore whether glucose-modified β₂GPI is able to interact and activate monocyte-derived immature dendritic cells (iDCs) from healthy human donors. SDS-PAGE and spectrofluorometric analyses indicated that β₂GPI incubated with glucose was sugar modified, and that this modification likely consisted of AGE formation, resulting in AGE-β₂GPI. AGE-β₂GPI caused phenotypical and functional maturation of iDCs involving the activation of p38 MAPK, ERK, and NF-κB. It also induced on DCs a significant up-regulation of RAGE, the receptor for AGEs. Evidence for RAGE involvement comes from blocking experiments with an anti-RAGE mAb, confocal analysis, and coimmunoprecipitation experiments. AGE-β₂GPI-stimulated DCs had increased allostimulatory ability and primed naive T lymphocytes toward a Th2 polarization. These findings might explain in part the interactive role of β₂GPI, AGEs, and DCs in chronic disorders related to endothelial cell dysfunction.

Autoantibodies specific to a peptide of β2-glycoprotein I cross-react with TLR4, inducing a proinflammatory phenotype in endothelial cells and monocytes

β(2)-glycoprotein I (β(2)GPI) is the major antigenic target for antiphospholipid Abs. Anti-β(2)GPI Abs are a heterogeneous population of Igs targeting all domains of the molecule. Abs specific to β(2)GPI domain I are strongly associated with thrombosis and obstetric complications. In the present study, we sought to understand the possible pathogenic mechanism for this subset of anti-β(2)GPI Abs, investigating their potential cross-reactivity with other self-proteins involved in inflammatory or coagulant events. We compared the amino acid sequence of the β(2)GPI domain I with human proteins in a protein databank and identified a peptide sharing 88% identity with an epitope of human TLR4. A high percentage of patients with antiphospholipid syndrome (41%) and systemic lupus erythematosus (50%) presented serum IgG specific to this peptide. Anti-β(2)GPI peptide Abs binding the TLR4 were able to induce NF-κB activation in HEK293 cells that were stably transfected with the TLR4 gene. Anti-β(2)GPI peptide Abs induced activation of TLR4 and triggered interleukin-1 receptor-associated kinase phosphorylation and NF-κB translocation, promoting VCAM expression on endothelial cells and TNF-α release by monocytes. In conclusion, our observations suggest a novel pathogenic mechanism in the TLR4 stimulation by anti-β(2)GPI peptide Abs that links adaptive immune responses with innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Autoantibodies to the adenosine triphosphate synthase play a pathogenetic role in Alzheimer's disease

It has become evident that an autoimmune component could play a role in Alzheimers disease (AD) onset and/or progression. The aim of this study was to identify neuronal antigenic targets specifically recognized by serum autoantibodies and to investigate their cellular effects and their possible pathogenetic role. We identified, by an immunoproteomic approach using mouse brain proteins, the adenosine triphosphate (ATP) synthase β subunit as a new autoantigen in AD. Using an ELISA assay we found that serum anti-ATP synthase autoantibodies were present in 38% of patients with AD, but in no age-matched healthy subjects or in patients with Parkinsons disease or atherosclerosis. Analytical cytology studies, using SH-SY5Y neuroblastoma cell line, showed that ATP synthase autoantibodies were capable of inducing the inhibition of ATP synthesis, alterations of mitochondrial homeostasis and cell death by apoptosis. These findings suggest that autoantibodies specific to ATP synthase can exert a pathogenetic role via a mechanism that brings into play the impairment of the extracellular ATP homeostasis and the alteration of mitochondrial function triggering cell death by apoptosis.

The mosaic of "seronegative" antiphospholipid syndrome.

In the clinical practice it is possible to find patients with clinical signs suggestive of antiphospholipid syndrome (APS), who are persistently negative for the laboratory criteria of APS, that is, anti-cardiolipin antibodies (aCL), anti-β 2-GPI antibodies and lupus anticoagulant. Therefore, it was proposed for these cases the term of seronegative APS (SN-APS). In order to detect autoantibodies with different methodological approaches, sera from 24 patients with SN-APS were analysed for anti-phospholipid antibodies using TLC immunostaining, for anti-vimentin/cardiolipin antibodies by enzyme-linked immunosorbent assay (ELISA), and for anti-annexin V and anti-prothrombin antibodies by ELISA and dot blot. Control groups of our study were 25 patients with APS, 18 with systemic lupus erythematosus (SLE), and 32 healthy controls. Results revealed that 13/24 (54.2%) SN-APS sera were positive for aCL (9 of whom were also positive for lysobisphosphatidic acid) by TLC immunostaining, 11/24 (45.8%) for anti-vimentin/cardiolipin antibodies, 3/24 (12.5%) for anti-prothrombin antibodies, and 1/24 (4.2%) for anti-annexin V antibodies. These findings suggest that in sera from patients with SN-APS, antibodies may be detected using “new” antigenic targets (mainly vimentin/cardiolipin) or methodological approaches different from traditional techniques (mainly TLC immunostaining). Thus, SN-APS represents a mosaic, in which antibodies against different antigenic targets may be detected.

Thin-layer chromatography immunostaining in detecting anti-phospholipid antibodies in seronegative anti-phospholipid syndrome

In clinical practice it is possible to find patients with clinical signs suggestive of anti‐phospholipid syndrome (APS) who are persistently negative for the routinely used anti‐phospholipid antibodies (aPL). Therefore, the term proposed for these cases was seronegative APS (SN‐APS). We investigated the clinical usefulness of thin‐layer chromatography (TLC) immunostaining in detecting serum aPL in patients presenting clinical features of SN‐APS. Sera from 36 patients with SN‐APS, 19 patients with APS, 18 patients with systemic lupus erythematosus (SLE), 20 anti‐hepatitis C virus (HCV)‐positive subjects and 32 healthy controls were examined for aPL using TLC immunostaining. Anti‐β2‐glycoprotein‐I, anti‐annexin II, anti‐annexin V and anti‐prothrombin antibodies were tested by enzyme‐linked immunosorbent assays (ELISA). Eahy926, a human‐derived endothelial cell line, was incubated with immunoglobulin (Ig)G fraction from SN‐APS patients and analysis of phospho‐interleukin (IL)‐1 receptor‐associated kinase (IRAK) and phospho‐nuclear factor (NF)‐κB was performed by Western blot, vascular cell adhesion molecule 1 (VCAM‐1) expression by cytofluorimetric analysis and supernatants tissue factor (TF) levels by ELISA. TLC immunostaining showed aPL in 58·3% of SN‐APS patients: anti‐cardiolipin in 47·2%, anti‐lyso(bis)phosphatidic acid in 41·7% and anti‐phosphatidylethanolamine in 30·5%. Six of 36 patients showed anti‐annexin II. Incubation of Eahy926 cells with IgG from SN‐APS induced IRAK phosphorylation, NF‐κB activation, VCAM‐1 surface expression and TF cell release. TLC immunostaining could identify the presence of aPL in patients with SN‐APS. Moreover, the results suggest the proinflammatory and procoagulant effects in vitro of these antibodies.

New antigenic targets and methodological approaches for refining laboratory diagnosis of antiphospholipid syndrome.

Antiphospholipid antibodies (aPLs) are a heterogeneous group of antibodies directed against phospholipids or protein/phospholipid complexes. Currently, aPLs are assessed using either “solid-phase” assays that identify anticardiolipin antibodies and anti-β2-glycoprotein I antibodies or “liquid-phase” assay that identifies lupus anticoagulant. However, in the last few years, “new” antigenic targets and methodological approaches have been employed for refining laboratory diagnosis of antiphospholipid syndrome (APS). In this review the potential diagnostic value of antibodies to domains of β2-GPI, prothrombin/phosphatidylserine, vimentin/cardiolipin, protein S, protein C, annexin A2, annexin A5, and phospholipid antigens is discussed. Moreover, new technical approaches, including chemiluminescence, multiline dot assay, and thin layer chromatography (TLC) immunostaining, which utilize different supports for detection of aPL, have been developed. A special focus has been dedicated on “seronegative” APS, that is, those patients with a clinical profile suggestive of APS (thromboses, recurrent miscarriages, or foetal loss), who are persistently negative for the routinely used aPL. Recent findings suggest that, in sera from patients with SN-APS, antibodies may be detected using “new” antigenic targets (mainly vimentin/cardiolipin) or methodological approaches different from traditional techniques (TLC immunostaining). Thus, APS represents a mosaic, in which antibodies against different antigenic targets may be detected thanks to the continuously evolving new technologies.

Subclinical Atherosclerosis in Systemic Lupus Erythematosus and Antiphospholipid Syndrome Focus on β2GPI-Specific T Cell Response

Objective— Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. &bgr;2 glycoprotein I (&bgr;2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, &bgr;2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to &bgr;2GPI and its relationship with atherosclerotic process. Approach and Results— Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte &bgr;2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to &bgr;2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte &bgr;2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte &bgr;2GPI and TF with IMT or FMD was detected. &bgr;2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to &bgr;2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm. Conclusions— A significant percentage of patients with SLE and PAPS show a &bgr;2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies

OBJECTIVES Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). METHODS For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. RESULTS Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients. CONCLUSION These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.

Subclinical Atherosclerosis in Systemic Lupus Erythematosus and Antiphospholipid Syndrome

Objective— Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. &bgr;2 glycoprotein I (&bgr;2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, &bgr;2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to &bgr;2GPI and its relationship with atherosclerotic process. Approach and Results— Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte &bgr;2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to &bgr;2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte &bgr;2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte &bgr;2GPI and TF with IMT or FMD was detected. &bgr;2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to &bgr;2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm. Conclusions— A significant percentage of patients with SLE and PAPS show a &bgr;2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

Streptococcal-vimentin cross-reactive antibodies induce microvascular cardiac endothelial proinflammatory phenotype in rheumatic heart disease

Rheumatic heart disease (RHD) is characterized by the presence of anti‐streptococcal group A antibodies and anti‐endothelial cell antibodies (AECA). Molecular mimicry between streptococcal antigens and self proteins is a hallmark of the pathogenesis of rheumatic fever. We aimed to identify, in RHD patients, autoantibodies specific to endothelial autoantigens cross‐reactive with streptococcal proteins and to evaluate their role in inducing endothelial damage. We used an immunoproteomic approach with endothelial cell‐surface membrane proteins in order to identify autoantigens recognized by AECA of 140 RHD patients. Cross‐reactivity of purified antibodies with streptococcal proteins was analysed. Homologous peptides recognized by serum cross‐reactive antibodies were found through comparing the amino acid sequence of streptococcal antigens with human antigens. To investigate interleukin (IL)‐1R‐associated kinase (IRAK1) and nuclear factor‐κB (NF‐κB) activation, we performed a Western blot analysis of whole extracts proteins from unstimulated or stimulated human microvascular cardiac endothelial cells (HMVEC‐C). Adhesion molecule expression and release of proinflammatory cytokines and growth factors were studied by multiplex bead based immunoassay kits. We observed anti‐vimentin antibodies in sera from 49% RHD AECA‐positive patients. Cross‐reactivity of purified anti‐vimentin antibodies with heat shock protein (HSP)70 and streptopain streptococcal proteins was shown. Comparing the amino acid sequence of streptococcal HSP70 and streptopain with human vimentin, we found two homologous peptides recognized by serum cross‐reactive antibodies. These antibodies were able to stimulate HMVEC‐C inducing IRAK and NF‐κB activation, adhesion molecule expression and release of proinflammatory cytokines and growth factors. In conclusion, streptococcal–vimentin cross‐reactive antibodies were able to activate microvascular cardiac endothelium by amplifying the inflammatory response in RHD.