Rayya J Alsalameh1, Rachael C Casey2, Jürgen Mollenhauer2, Joachim R Kalden3, Gerd R Burmester4, Saifeddin M Alsalameh3,5. 1. Institute of Clinical Microbiology and Hospital Hygiene, University Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany. 2. Department of Biochemistry, Rush University Medical Center, Chicago, Illinois, USA. 3. Department of Medicine III, Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nuremberg, Erlangen, Germany. 4. Department of Internal Medicine, Rheumatology and Clinical Immunology, Charite Centre for Internal Medicine and Dermatology, Charite-Universitätsmedizin Berlin, Berlin, Germany. 5. Division of Rheumatology and Immunology, Department of Medicine, Tawam Hospital, Al Ain, Abu Dhabi, UAE.
Abstract
OBJECTIVE: To analyze proliferation and pro-inflammatory cytokine production of peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis (RA) patients following stimulation with a purified chondrocyte membrane-associated autoantigen (CH65). METHODS: CH65 was highly purified from bovine chondrocyte membranes by solubilization and ion exchange chromatography. PBMC of RA patients (n = 37; 28 seropositive, nine seronegative) and non-arthritic donors (n = 20) were isolated by ficoll centrifugation and used in cell proliferation assays. The levels of interleukin (IL)-1, tumo necrosis factor (TNF) and IL-6 produced after stimulation with CH65 were determined by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using Mann-Whitney U-test and Spearman rank test and the software SPSS 13.0TM (SPSS Inc.; Chicago, IL, USA). RESULTS: Peripheral blood mononuclear cells exhibited a strong proliferative response to purified CH65 in approximately 50% of the RA patients (seropositive > seronegative), with a maximum reactivity at 0.15 or 0.30 μg/mL culture medium. In contrast, PBMC from normal donors did not show a proliferative response to CH65 at any dose. The proliferative response in RA patients peaked at days 7-9 and returned to control levels at day 13, indicating an antigen-driven process. CH65-stimulated RA PBMC produced moderate to high amounts of IL-1, TNF and IL-6. This was comparable to the response after exposure to isolated whole chondrocyte membranes or purified collagen type II. CONCLUSION: These results demonstrate a significant cellular immune response to CH65 protein in RA patients. Given the high similarity between bovine and human CH65, the results suggest a pathogenetic involvement of this molecule as a cartilage-specific potential target autoantigen in RA.
OBJECTIVE: To analyze proliferation and pro-inflammatory cytokine production of peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis (RA) patients following stimulation with a purified chondrocyte membrane-associated autoantigen (CH65). METHODS: CH65 was highly purified from bovine chondrocyte membranes by solubilization and ion exchange chromatography. PBMC of RApatients (n = 37; 28 seropositive, nine seronegative) and non-arthritic donors (n = 20) were isolated by ficoll centrifugation and used in cell proliferation assays. The levels of interleukin (IL)-1, tumo necrosis factor (TNF) and IL-6 produced after stimulation with CH65 were determined by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using Mann-Whitney U-test and Spearman rank test and the software SPSS 13.0TM (SPSS Inc.; Chicago, IL, USA). RESULTS: Peripheral blood mononuclear cells exhibited a strong proliferative response to purified CH65 in approximately 50% of the RApatients (seropositive > seronegative), with a maximum reactivity at 0.15 or 0.30 μg/mL culture medium. In contrast, PBMC from normal donors did not show a proliferative response to CH65 at any dose. The proliferative response in RApatients peaked at days 7-9 and returned to control levels at day 13, indicating an antigen-driven process. CH65-stimulated RA PBMC produced moderate to high amounts of IL-1, TNF and IL-6. This was comparable to the response after exposure to isolated whole chondrocyte membranes or purified collagen type II. CONCLUSION: These results demonstrate a significant cellular immune response to CH65 protein in RApatients. Given the high similarity between bovine and human CH65, the results suggest a pathogenetic involvement of this molecule as a cartilage-specific potential target autoantigen in RA.
Authors: Anna Osiecka-Iwan; Anna Hyc; Dorota M Radomska-Lesniewska; Adrian Rymarczyk; Piotr Skopinski Journal: Cent Eur J Immunol Date: 2018-06-30 Impact factor: 2.085