Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients.

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Genetic background of anticyclic citrullinated peptide autoantibody production in Hungarian patients with rheumatoid arthritis.

Polymorphisms of the peptidylarginine deiminase 4 (PADI4) gene encoding for the isoenzyme that converts arginyl into citrullyl residues have been shown to contribute to susceptibility to rheumatoid arthritis (RA), depending on the population studied. We aimed at determining whether PADI4 single nucleotide polymorphisms (SNPs) are associated with RA in a Hungarian population. The relationship between anticyclic citrullinated peptide (anti-CCP) production and HLA-DRB1 alleles encoding the shared epitope (SE) was also investigated. DNA samples were obtained from RA (n = 261) patients and from control donors (n = 120). HLA-DRB1 genotyping was carried out by polymerase chain reaction (PCR) with sequence-specific priming. PAD4_92 G/C and PAD4_104 T/C SNPs were genotyped using real-time PCR allele discrimination. Autoantibodies against CCP were detected by ELISA. All healthy controls tested anti-CCP negative, whereas 171 (66%) RA patients were anti-CCP positive. No significant difference in allele or genotype frequencies were found between RA patients and controls for any of the PADI4 SNPs. Anti-CCP seropositivity was unrelated to these two SNPs. No association was found between any of the PADI4 SNPs and HLA-DRB1 subtypes. Presence of the HLA-RB1 SE alleles was significantly associated with anti-CCP seropositivity; HLA-DRB1*0401 and HLA-DRB1*1001 carriers showed the strongest association. In conclusion, our data suggest that polymorphisms of the PADI4 gene are not associated with rheumatoid arthritis and are unlikely to be responsible for the presence of anti-CCP autoantibodies in a white Hungarian population. HLA-DRB1 SE alleles, however, may significantly contribute to the genetic determination of anti-CCP production in Hungarian patients with RA.

Redox status and expression of chemokines in the rat lungs on exposure to asbestos and asbestos substituents.

OBJECTIVES: Activity of cytoplasmatic superoxide dismutase, glutathione peroxidase,- and reductase, gamma-glutamyl transpeptidase and expression of macrophage chemoattractant protein-1 and macrophage inhibitory protein-1alpha was determined in primary culture of rat alveolar macrophages and type II pneumocytes after exposure to stone-wool, wollastonite and crocidolite (blue asbestos). METHODS: The activity of redox enzymes was examined by RANDOX kits, chemokines were studied by ELISA. RESULTS: The UICC crocidolite (positive control) decreased the activity of all redox enzymes and increased the expression of chemokines, whereas the two asbestos substituents did not alter the activity of redox enzymes either in the alveolar macrophages or pneumocytes. Stone-wool and wollastonite moderately increased the expression of chemokines in both cells. CONCLUSIONS: The redox status and production of chemokines changed in the opposite direction, presumably owing to stronger toxic effect of asbestos. These data suggest that stone-wool and wollastonite, as potential substituents for asbestos, are less toxic than the human carcinogenic and fibrogenic crocidolite.

The effect of stone-wool on rat lungs and on the primary culture of rat alveolar macrophages and type II pneumocytes.

The effect of stone-wool has been studied in both in vivo long term sequential and in vitro methods in male Sprague-Dawley rats. Stone-wool was administered by single intratracheal instillation and the lungs were examined after 1, 3 and 6 months of exposure by morphological methods. UICC crocidolite was applied as a positive control. In addition, the effects of both fibres were examined in primary cultures of alveolar macrophages (AM) and type II pneumocytes (T2) by morphological, biochemical and immunological methods. By the end of 6 months stone-wool induced moderate pulmonary interstitial inflammation and fibrosis without progression, whereas crocidolite induced progressive interstitial inflammation and fibrosis as a function of time. Although stone-wool inhibited phagocytosis, it did not induce serious membrane damage to the cells examined and did not destroy their ultrastructure. It significantly reduced the activity of Cu,Zn/superoxide dismutase (SOD) and alkaline phosphatase (AP) in alveolar macrophages and significantly decreased the activity of AP and gamma-glutamyl transpeptidase (GGT) in type II pneumocytes. Crocidolite, on the other hand, decreased the activity of all enzymes (glutathione peroxidase, GSH-Px; glutathione reductase, GSH-Rd) of glutathione metabolism as well as alkaline phosphatase in alveolar macrophages. It decreased the activity of all enzymes in type II pneumocytes, except for Cu,Zn/SOD. On exposure to stone-wool, the production of inflammatory proteins, macrophage chemoattractant protein-1 (MCP-1) and macrophage inhibitory protein-1alpha (MIP-1alpha) increased in both cultured cells but did not reach the level induced by crocidolite. Our results suggested that stone-wool is less toxic than crocidolite. Whether it is carcinogenic or not, is still an open question.

The effect of asbestos and stone-wool fibres on some chemokines and redox system of pulmonary alveolar macrophages and pneumocytes type II.

The in vitro effect of stone-wool has been studied in primary cultures of pulmonary alveolar macrophages (AM) and type II pneumocytes (T2) by morphological, biochemical and immunological methods. UICC crocidolite was applied as a positive control. Although stone-wool brought about frustrated phagocytosis, it did not induce serious membrane damage, whereas crocidolite gave rise to very severe membrane alterations. Stone-wool significantly reduced the activity of Cu,Zn/superoxide dismutase (SOD) in alveolar macrophages and significantly decreased the activity of gamma-glutamyl transpeptidase (GGT) in pneumocytes type II. Crocidolite, on the other hand, decreased the activity of all enzymes (glutathione peroxidase - GSH-Px, glutathione reductase - GSH-Rd) of glutathione metabolism in alveolar macrophages. It decreased the activity of all enzymes in pneumocytes type II except for Cu,Zn/SOD. After exposure to stone-wool, the production of inflammatory proteins, macrophage chemoattractant protein-1 (MCP-1) and macrophage inhibitory protein-1alpha (MIP-1alpha) increased in both cultured cells but did not reach the level induced by crocidolite. Although this study provided a useful insight in the toxicity of the stone-wool, we can not draw the conclusions how the intact pulmonary tissue may respond on the exposure to these fibres, exclusively based on the in vitro tests.

Indoor air pollutants and immune biomarkers among Hungarian asthmatic children.

The authors examined the relationship between immune biomarkers and indoor air pollution cross-sectionally in school children 9-11 yr of age who had immunologically related respiratory diseases and who resided in Hungarian cities. Nitrogen dioxide, formaldehyde, benzene, xylene, and toluene were measured passively indoors prior to the collection of venous blood samples for blood counts and identification of immune biomarkers. House dust mite allergen was also measured. Numerous immune biomarkers were significantly elevated in these sensitive children, compared with normal children, and several biomarker alterations in these children were related to high concentrations of air pollutants in the home. The strongest and most significant associations were seen between high indoor nitrogen dioxide concentrations and increased white blood cells, monocytes, red blood cells, and immunoglobulin G (IgG), as well as decreased immunoglobulin M (IgM) and Klebsiella pneumoniae-specific IgM. Bacterial-specific IgGs were related significantly to formaldehyde concentrations. These findings suggest the important role of indoor air pollutants in immune reactions.

A synthetic corticosteroid, dexamethasone regulates generation of soluble form of interleukin-6 receptor of human lymphocytes, in vitro.

In contrast to most of the soluble cytokine receptor antagonists properties, the soluble IL-6 receptor (sIL-6R) occurring in various body fluids of healthy persons and patients with various diseases is an agonist. The enhancing effect is due to its ability to form complex with IL-6 and to bind to gp130 making constitutively IL-6 receptor negative cells responsive for IL-6. The generation as well as the functional role of soluble IL-6 receptor is poorly understood. Earlier, we found that the sIL-6R levels in sera of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were higher than those of the control group measured by ELISA sandwich technology. In the present study we detected different levels of sIL-6R in the supernatants of lymphocyte cultures of healthy persons and patients with RA as well as SLE. Moreover, we found, that in vitro dexamethasone treatment stimulated generation of sIL-6R in both healthy persons and in active SLE, while it strongly suppressed production of sIL-6R in both RA groups. At mRNA level, we found that in SLE both the IL-6R mRNA encoding the membrane spanning and alternatively spliced (soluble) variants increased. Surprisingly, the strong decrease of sIL6R protein in RA was not found at mRNA level.