Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

Objective. To test the hypothesis that deimination of viral sequences containing Arg-Gly repeats could generate epitopes recognized by anti-citrullinated protein antibodies (ACPAs) that are present in rheumatoid arthritis (RA) sera.Methods. Multiple antigen peptides derived from Epstein-Barr virus (EBV)-encoded Epstein-Barr nuclear antigen 1 (EBNA-1) were synthesized, substituting the arginines with citrulline, and were used to screen RA sera. Anti-cyclic citrullinated peptide antibodies were purified by affinity chromatography and tested on a panel of in vitro deiminated proteins. Their ability to bind in vivo deiminated proteins was evaluated by immunoprecipitation, using EBV-infected cell lines.Results. Antibodies specific for a peptide corresponding to the EBNA-1 35-58 sequence containing citrulline in place of arginine (viral citrullinated peptide [VCP]) were detected in 50% of RA sera and in <5% of normal and disease control sera. In addition, affinitypurified anti-VCP antibodies from RA sera reacted with filaggrin-derived citrullinated peptides, with deiminated fibrinogen, and with deiminated recombinant EBNA-1. Moreover, anti-VCP antibodies immunoprecipitated, from the lysate of calcium ionophore-stimulated lymphoblastoid cell lines, an 80-kd band that was reactive with a monoclonal anti-EBNA-1 antibody and with anti-modified citrulline antibodies.Conclusion. These data indicate that ACPAs react with a viral deiminated protein and suggest that EBV infection may play a role in the induction of these RA-specific antibodies.

In systemic autoimmune diseases, autoantibodies specific for α‐enolase are detected more frequently in patients with active renal involvement. To analyze the properties of anti‐α‐enolase antibodies and the distribution of the enzyme in the cell, mouse monoclonal and polyclonal antibodies were obtained from mice immunized with a glutathione‐S‐transferase‐α‐enolase fusion protein. Anti‐α‐enolase antibodies were purified from patient sera on enolase from human kidney. Using these antibodies, the distribution of α‐enolase in the cell was analyzed in subcellular fractions and in the cell membrane by flow cytometry and immunoprecipitation. Plasminogen binding was studied by an immunoenzymatic assay. We observed that α‐enolase was present in the cytosol and membrane fractions obtained from kidney and U937 cells. By flow cytometry, mouse polyclonal anti‐enolase antibodies, one monoclonal and 7/9 human anti‐enolase antibodies bound the membrane of U937 cells. One monoclonal antibody and mouse polyclonal anti‐enolase antibodies immunoprecipitated a 48‐kDa molecule from surface‐labeled U937 cells and this molecule was recognized by rabbit anti‐enolase antibodies. Both immunization‐induced antibodies and 7/9 autoantibodies from patient sera inhibited the binding of plasminogen to α‐enolase. The results show that α‐enolase, an autoantigen in connective tissue diseases, is a cytoplasmic enzyme which is also expressed on the cell membrane, with which it is strongly associated. Anti‐α‐enolase autoantibodies isolated from patient sera recognize the membrane‐associated form of the enzyme and/or interfere with its receptor function, thus inhibiting the binding of plasminogen. Autoantibodies specific for α‐enolase could play a pathogenic role, either by a cytopathic effect or by interfering with membrane fibrinolytic activity.

These data demonstrate that patients with different systemic autoimmune diseases differ in the expression of activating receptors and co-receptors on CD3-CD56+ NK cells. The down-regulation of receptors and co-receptors in SSc with lung involvement suggests their possible role in this manifestation of the disease.

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