Carlos A Núñez-Álvarez1, Diego F Hernández-Ramírez1, Araceli Martinez-Castillo1, Virginia Pascual Ramos1, Javier Cabiedes1, Alicia Ortega2, Antonio R Cabral3. 1. Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico. 2. Department of Biochemistry, School of Medicine, Universidad Nacional Autónoma de México, Mexico City, Mexico; Department of Biochemistry, Instituto Nacional de Perinatología, Mexico City, Mexico. Electronic address: aortega@unam.mx. 3. Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico. Electronic address: arcabral1952@gmail.com.
Abstract
Homozygote genotype V247 of the β2-glycoprotein-I (β2GP-I) gene has been associated with anti-β2GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to β2GP-I247 has been little studied. OBJECTIVE: To evaluate the immune cellular proliferation in response to native and non-native β2GP-I247 valine/leucine phenotype from Mexican patients with PAPS. METHODS: We studied 10 patients with PAPS and 10 healthy control subjects (HC). The polymorphism at position 247 of the β2GP-I gene was determined by PCR-RFLP and the corresponding β2GP-I protein was subsequently purified from normal human plasma by affinity chromatography. PBMC purified from patients and controls were stimulated with β2GP-I under native and in non native (reduced) conditions. We also determined the anti-β2GP-I production in vitro by B cell clones (EBV) generated in cocultures experiments. Differential Scanning Calorimetry (DSC) was studied to determine the structural differences between the β2GP-I247 valine/leucine isoforms. Cytokine profile (IL-2, IL-4, IL-6, TNFα, INFγ) was evaluated in culture supernatants. RESULTS: PAPS and healthy control PBMCs had a higher proliferative response when stimulated with β2GP-I under reduced cultures conditions compared to non-denatured conditions. PBMCs response from PAPS patients was higher. We observed more cell proliferation in response to β2GP-I247 valine/leucine or valine isoforms in non-native conditions. In contrast, this response was not significant against β2GP-I247 leucine. These findings were T CD4+-dependent. Similar results were obtained with B cell clones derived from PAPS patients, which showed more pronounced proliferation in non native conditions and higher against β2GP-I247 valine. No differences were found in anti-β2GP-I production, but high levels of IL-6 in vitro were identified. The structural analysis of both β2GP-I247 isoforms by DSC showed a major conformational change due to a single mutation in the β2GP-I variants. CONCLUSIONS: PAPS PBMCs had a higher cellular response against β2GP-I247 in non-native culture conditions preferentially to the β2GP-I247 valine phenotype. This effect is T CD4+ dependent and appears to be driven by tertiary structural changes adopted by β2GP-I247 polymorphism.
Homozygote genotype V247 of the β2-glycoprotein-I (β2GP-I) gene has been associated with anti-β2GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to β2GP-I247 has been little studied. OBJECTIVE: To evaluate the immune cellular proliferation in response to native and non-native β2GP-I247 valine/leucine phenotype from Mexican patients with PAPS. METHODS: We studied 10 patients with PAPS and 10 healthy control subjects (HC). The polymorphism at position 247 of the β2GP-I gene was determined by PCR-RFLP and the corresponding β2GP-I protein was subsequently purified from normal human plasma by affinity chromatography. PBMC purified from patients and controls were stimulated with β2GP-I under native and in non native (reduced) conditions. We also determined the anti-β2GP-I production in vitro by B cell clones (EBV) generated in cocultures experiments. Differential Scanning Calorimetry (DSC) was studied to determine the structural differences between the β2GP-I247 valine/leucine isoforms. Cytokine profile (IL-2, IL-4, IL-6, TNFα, INFγ) was evaluated in culture supernatants. RESULTS: PAPS and healthy control PBMCs had a higher proliferative response when stimulated with β2GP-I under reduced cultures conditions compared to non-denatured conditions. PBMCs response from PAPS patients was higher. We observed more cell proliferation in response to β2GP-I247 valine/leucine or valine isoforms in non-native conditions. In contrast, this response was not significant against β2GP-I247 leucine. These findings were T CD4+-dependent. Similar results were obtained with B cell clones derived from PAPS patients, which showed more pronounced proliferation in non native conditions and higher against β2GP-I247 valine. No differences were found in anti-β2GP-I production, but high levels of IL-6 in vitro were identified. The structural analysis of both β2GP-I247 isoforms by DSC showed a major conformational change due to a single mutation in the β2GP-I variants. CONCLUSIONS: PAPS PBMCs had a higher cellular response against β2GP-I247 in non-native culture conditions preferentially to the β2GP-I247 valine phenotype. This effect is T CD4+ dependent and appears to be driven by tertiary structural changes adopted by β2GP-I247 polymorphism.