CXCL13 is the major determinant for B cell recruitment to the CSF during neuroinflammation

Markus C Kowarik 0 Sabine Cepok 0 Johann Sellner 0 Verena Grummel 0 Martin S Weber 0 Thomas Korn 0 Achim Berthele 0 Bernhard Hemmer 0 0 Department of Neurology, Klinikum rechts der Isar, Technische Universitat Munchen , Ismaninger Str. 2281675, Munich , Germany Background: The chemokines and cytokines CXCL13, CXCL12, CCL19, CCL21, BAFF and APRIL are believed to play a role in the recruitment of B cells to the central nervous system (CNS) compartment during neuroinflammation. To determine which chemokines/cytokines show the strongest association with a humoral immune response in the cerebrospinal fluid (CSF), we measured their concentrations in the CSF and correlated them with immune cell subsets and antibody levels. Methods: Cytokine/chemokine concentrations were measured in CSF and serum by ELISA in patients with noninflammatory neurological diseases (NIND, n = 20), clinically isolated syndrome (CIS, n = 30), multiple sclerosis (MS, n = 20), Lyme neuroborreliosis (LNB, n = 8) and patients with other inflammatory neurological diseases (OIND, n = 30). Albumin, IgG, IgA and IgM were measured by nephelometry. CSF immune cell subsets were determined by seven-color flow cytometry. Results: CXCL13 was significantly elevated in the CSF of all patient groups with inflammatory diseases. BAFF levels were significantly increased in patients with LNB and OIND. CXCL12 was significantly elevated in patients with LNB. B cells and plasmablasts were significantly elevated in the CSF of all patients with inflammatory diseases. CXCL13 showed the most consistent correlation with CSF B cells, plasmablasts and intrathecal Ig synthesis. Conclusions: CXCL13 seems to be the major determinant for B cell recruitment to the CNS compartment in different neuroinflammatory diseases. Thus, elevated CSF CXCL13 levels rather reflect a strong humoral immune response in the CNS compartment than being specific for a particular disease entity. - Background B cells play an essential role in the humoral immune response in neuroinflammation and serve as antigen presenting cells for T cells. Recruitment, clonal selection and expansion of B cells require a specialized milieu of secondary lymphoid organ chemokines and cytokines [1]. Chemokines, such as CCL19 (MIP-3, Macrophage Inflammatory Protein-3), CCL21 (SLC, secondary lymphoid-tissue chemokine), CXCL12 (SDF-1, stromal cellderived factor-1) and CXCL13 (BCA-1, B cell attracting chemokine-1), are known to influence migration of B cells [2]. These chemokines are constitutively expressed in lymphoid organs and regulate the migration and compartmentalization of lymphocytes and antigen presenting cells [3,4]. Some of the chemokines are present in the cerebrospinal fluid (CSF) and central nervous system (CNS), especially during neuroinflammation. CXCL13 is produced by stromal cells, binds to the CXCR5 receptor and regulates homing of B cells and subsets of T cells to lymphoid follicles [57]. In addition, CXCL13 seems to play a role in the formation of ectopic lymphoid tissues within the CNS in chronic inflammatory CNS diseases [8,9]. Elevated CXCL13 levels were found in the CSF of patients with MS, neuroborreliosis and other inflammatory neurological diseases [4,1013]. Furthermore, CXCL13 levels correlated with B and T cell numbers in the CSF and intrathecal immunoglobulin production [10]. CXCL13 has recently been suggested as a prognostic marker for multiple sclerosis and clinically isolated syndrome (CIS) [1214]. Based on the observation that high CXCL13 levels are found in the CSF of patients with acute neuroborreliosis, CXCL13 was proposed as a specific diagnostic marker and a key regulator for B cells in acute Lyme neuroborreliosis [11,1517]. CXCL12 binds to the receptor CXCR4 [18] and acts as a potent chemoattractant for B cells, plasma cells, T cells and monocytes [4,10]. The chemokine plays an important role in germinal center organization. CXCL12 is also expressed in the normal brain and is crucial for neuronal guidance [10,19]. Higher CSF levels of CXCL12 were found in inflammatory CNS diseases. CXCL12 was also found in MS lesions but CSF levels were only slightly elevated in these patients [1,10]. CCL19 and CCL21 bind to the CCR7 receptor, which is present on activated B cells, naive and central memory T cells, and dendritic cells. Both chemokines strongly guide subsets of T cells, B cells and mature dendritic cells into secondary lymphatic organs [1,20,21]. Additionally, CCL19 levels have been shown to be increased in the CSF of patients with inflammatory neurological diseases and MS [1,22]. CCL19 mRNA was shown to be overexpressed in MS lesions [4,22]. The cytokines BAFF (B cell activating factor) and APRIL (A Proliferation Inducing Ligand) are members of the TNF (Tumor Necrosis Factor) family and are both expressed by monocytes, macrophages, dendritic cells, astrocytes and, at lower levels, by T cells [23]. APRIL binds to the receptors BCMA (B cell maturation), TACI (transmembrane activator and CAML interacting protein) and syndecan-1 (CD138), BAFF receptors comprise BAFF-R, TACI and BCMA. These receptors are mainly expressed on B cells and to a lower extent on T cells [23,24]. APRIL and BAFF are key factors for the development and survival of B cells; furthermore, BAFF acts as a potent B cell activator [25]. It has been shown that BAFF mRNA is up-regulated in MS lesions and secreted by astrocytes upon stimulation [25]. APRIL protein expression was up-regulated in astrocytes in MS patients whereas APRIL protein levels were not elevated in the CSF [2628]. Taken together, these data suggest a possible role for each of these chemokines and cytokines in neuroinflammation. So far, most cytokines and chemokines were studied in small cohorts of patients often focusing on a single specific neuroinflammatory disease. To obtain a more complete picture of these cytokines and chemokines, we performed a study in a set of 108 patients with non-inflammatory neurological diseases (NIND), clinically isolated syndrome (CIS), multiple sclerosis (MS), Lyme neuroborreliosis (LNB) and patients with other inflammatory neurological diseases (OIND). The objective of the present study was to determine which cytokines and chemokines are most strongly associated with the occurrence of B cells, plasmablasts and the secretion of antibodies in the CSF compartment, irrespective of the specific disease. In parallel to measurements of chemokine and cytokine concentrations by ELISA, seven-color flow cytometry was performed in 107 patients to dissect the major immune cell subsets in the CSF. Methods Patients Patients were recruited at the Department of Neurology of the Technische Universitt Mnchen. All CSF samples were primarily obtained for routine diagnostic work-up, patients consented to the scientific use of their biosamples. Furthermore, the ethics committee of the Technische Universitt Mnchen approved the scientific use of CSF biosamples. A total of 20 patients wi (...truncated)