Dysfunctional chemotaxis in Familial Mediterranean Fever
Purpose: Familial Mediterranean fever (FMF) is a heritable autoinflammatory disease characterized by substantial neutrophil influx at sites of serosal and synovial involvement. Inflammatory attacks are usually prevented by prophylaxis with colchicine, a microtubule inhibitor. Pyrin, the FMF protein, colocalizes with microtubules and may impact leukocyte cytoskeletal functions such as adhesion and migration. We therefore sought to further investigate leukocyte migration in FMF. Method: Peripheral blood samples were obtained from 25 treated patients, 10 untreated patients, and 16 controls. Subsets of patients were studied in several assays. Mononuclear cells were stimulated for 24 hours with TLR ligands 2, 4, 5, 7, and 9. Chemokines produced from cultured supernatants and from serum samples were evaluated with the Luminex immunoassay. Analysis of gene expression sequences with the Affymetrix system was utilized in 8-paired patient samples who underwent colchicine withdrawal. Chemotaxis assays included a transwell system and a live imaging protocol with both patient granulocytes and the neutrophil-like PLB cell line transfected with V726A, M680I, and M694V pyrin mutations, stimulated to various chemoattractants, including MIP-1 alpha, IL-8 and fMLP. Results: Microarray analysis revealed a "chemotactic signature" induced by colchicine withdrawal with upregulation of major chemotactic genes including CRK and Integrin beta-1, and downregulation of genes involving chemokine receptors and cell adhesion molecules such as CCR 7 and CXCR3. Moreover, neutrophil activation was increased, as seen with the downregulation of the L-selectin marker, CD 62L. Asymptomatic FMF patients on colchicine showed elevated serum levels of the chemokines, MIP-1 alpha and beta, as compared to controls. In addition, mononuclear cells from treated patients stimulated ex-vivo, suggest a role for TLR4, 5, and 9 in the modulation of chemokines, specifically MIP-1 alpha and beta. Patients off colchicine demonstrated significant hyperresponsive cell movement compared with treated patients and controls, although such movement was found to be random (chemokinetic) and less directed (chemotactic) when visualized. By transwell analysis of the transfected neutrophil-like PLB cell line hypermobility was more apparent with the V726A mutation. Conclusion: A cascade of intracellular events involving chemokine signaling can impact leukocyte migration in FMF. This study has focused on determining the extent to which these effects represent the underlying pathophysiology of FMF, independent of colchicine.
