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To prevent and treat experimental arthritis via nasal administration of an altered peptide ligand (APL) from the major arthritogenic epitope in adjuvant-induced arthritis (AIA) and to explore the mechanisms involved. Peptides were administered nasally before and after induction of arthritis. Splenocytes and lymph node cells draining both the site of inflammation and the site of tolerance induction were used for cell transfer and were studied for antigen-specific T cell characteristics. In addition, attempts were made to stop T cell tolerance in vitro, using anticytokine antibodies. Nasal administration of a modulatory APL of the heat-shock protein 60 (Hsp60) 180–188 T cell epitope, alanine 183, had a suppressive effect in AIA that far exceeded that of the wild-type epitope. In addition to its effectiveness in preventing AIA, alanine 183 may be effective in the treatment of ongoing AIA. The protective effect of alanine 183 can be passively transferred using activated splenocytes. Nasal administration of alanine 183 did not lead to detectable T cell proliferation or interleukin-2 (IL-2) production in mandibular lymph node cells, while transforming growth factor β (TGFβ), IL-10, and IL-4 were readily produced. Likewise, after nasally induced tolerance, followed by induction of arthritis, inguinal lymph node cells produced IL-4, TGFβ, and IL-10. After neutralizing in vitro the individual cytokines with anticytokine antibodies, only blocking of IL-10 production led to reversal of tolerance, at the site of tolerance induction and the site of inflammation. Nasal administration of an APL of Hsp60 180–188 induces highly effective protection against AIA through generation of regulatory cells that produce IL-4, TGFβ, and IL-10, whereas the induced tolerance is driven mainly by production of IL-10.

T cell receptor (TCR) peptide immunizations have been demonstrated to protect against experimental autoimmune diseases. These findings have led to clinical trials employing TCR peptides in multiple sclerosis and rheumatoid arthritis patients. Previously, we identified a strongly immunogenic region of the TCR α chain of an arthritogenic T cell clone (AV11 66-80). In this report, we show that rats immunized with AV11 66-80 developed arthritis with clinical symptoms and histology similar to adjuvant arthritis (AA). Transfer of this disease into naive rats using AV11 66-80-specific T cells proved the T cell-mediated character of the disease. The AV11 66-80 arthritic rats developed resistance to Mycobacterium tuberculosis-induced AA, indicating that both forms of arthritis depended on similar regulatory mechanisms. This first demonstration of TCR peptide-induced arthritis, together with an earlier report on a polymorphism in this very same AV11 66-80 region involved in arthritis resistance in mice, suggests a central role of the public epitope AV11 66-80 in the control of autoimmune arthritis. Although TCR peptide immunizations can be exploited to prevent experimental autoimmunity, caution should be taken in the induction of TCR peptide-specific T cells for immunotherapy to avoid adverse effects as shown here.

Adjuvant Arthritis (AA) can be induced by passive transfer of a T cell clone (A2b) derived from arthritic rats, specific for Heat Shock Protein 60, HSP60 176-190. Furthermore, a crucial role for T cells with HSP60 176-190 specificity in AA was shown by induction of tolerance using HSP60 176-190 or by immunization with an altered peptide ligand based on the same sequence. To study clonal expansion of A2b-like T cells during AA and to determine their role in AA induction, we generated a clonotypic antibody, 16C4, specific for the TCR of the A2b T cell clone (TCR AV11S1/BV18). This antibody stained A2b T cells in flow cytometry experiments, induced proliferation of A2b cells when fixed on a solid support, and inhibited antigen-induced A2b proliferation when added in solution. A2b-like T cells were detected in a low frequency in lymphoid organs of arthritic rats. Thus, as in vivo administration of 16C4 did not inhibit AA, cells containing the determinant recognized by 16C4 are possibly not the sole contributors to AA development. Furthermore, epitope specific interventions by antigen administration may be possible even in cases where the epitope specific T cell clonotype is of low frequency.

Zusammenfassung Es wird eine kompetitive Methode „ELISA” (enzyme-linked immunosorbent assay) mit monoklonalen Antikörpern zur Bestimmung von Chloramphenicol-Rückständen (CAP) in Schweinefleisch beschrieben. Für Standardlösungen wurde als Nachweisgrenze 25 ng ml-1 festgestellt (=2.0 ng CAP). Die sehr große Spezifität dieser monoklonalen Antikörper für CAP drückt sich durch die unwichtigen Kreuzreaktionen mit anderen antimikrobiellen Substanzen und mit strukturell verwandten Verbindungen aus. Zur Probenaufarbeitung wurde das gleiche Schnellverfahren benutzt wie bei der Hochleistungs-Flüssigchromatographie. Rückstände > 5 µg kg-1 können im Schweinefleisch leicht bestimmt werden. Bei Zusatzmengen von 10, 15, 25 und 50 µg CAP pro kg sind die Wiederfindungsraten gut. Die Ergebnisse mit der ELISA-Methode wurden mit HPLC bestätigt.

Adjuvant Arthritis (AA) can be induced in Lewis rats by immunisation with mycobacterial antigens. The disease can be passively transferred with T cell clone A2b, which recognises the 180–188 amino acid sequence in mycobacterial heat shock protein 60 (hsp60) and which crossreacts with crude cartilage proteoglycans. We succeeded to induce peripheral tolerance to this AA-associated T cell epitope following nasal administration of a peptide containing this epitope (mycobacterial hsp60 176–190). In rats treated nasally with 176–190 and immunised with mycobacterial hsp60, proliferative responses to 176–190 were reduced. AA was inhibited nasally with 176–190 treated rats and not in rats nasally treated with a control mycobacterial hsp60 peptide (211–225). Moreover, nasal 176–190 led to similar arthritis protective effects in a non-microbially induced experimental arthritis (avridine induced arthritis). In a subsequent study we tried to prevent and to treat AA through nasal administration of mycobacterial hsp60 peptide 180–188 and a peptide analogue of 180–188, 180–188L183->A (Alanine 183), which has been shown to have an increased MHC-binding affinity for rat RT1 B1 and an increased capacity to inhibit the proliferative A2b response in vitro. We found that nasal administration of 180–188 had a moderate arthritis suppressive effect in AA, whereas its analogue peptide Alanine 183, had a strong suppressive effect. This strong arthritis suppressive effect was only partly due to the higher MHC-binding affinity for rat RT1 B1. Furthermore, it was possible to passively transfer nasal Alanine 183 induced disease protection. The present findings may in our view offer novel prospects for immunotherapy through nasal administration of (analogue) peptides, with a mimicry relationship with joint specific cartilage proteoglycan epitopes. [ABSTRACT FROM AUTHOR]

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