Barbara Rosicarelli

Detection of Ectopic B‐cell Follicles with Germinal Centers in the Meninges of Patients with Secondary Progressive Multiple Sclerosis

Multiple sclerosis (MS) is characterized by synthesis of oligoclonal immunoglobulins and the presence of B‐cell clonal expansions in the central nervous system (CNS). Because ectopic lymphoid tissue generated at sites of chronic inflammation is thought to be important in sustaining immunopathological processes, we have investigated whether structures resembling lymphoid follicles could be identified in the CNS of MS patients. Sections from post‐mortem MS brains and spinal cords were screened using immunohistochemistry for the presence of CD20+ B‐cells, CD3+ T‐cells, CD 138+ plasma cells and CD21+, CD35+ follicular dendritic cells, and for the expression of lymphoid chemokines (CXCL13, CCL21) and peripheral node addressin (PNAd). Lymphoid follicle‐like structures containing B‐cells, T‐cells and plasma cells, and a network of follicular dendritic cells producing CXCL13 were observed in the cerebral meninges of 2 out of 3 patients with secondary progressive MS, but not in relapsing remitting and primary progressive MS. We also show that proliferating B‐cells are present in intrameningeal follicles, a finding which is suggestive of germinal center formation. No follicle‐like structures were detected in parenchymal lesions. The formation of ectopic lymphoid follicles in the meninges of patients with MS could represent a critical step in maintaining humoral autoimmunity and in disease exacerbation.

Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions.

As a result of their close association with the blood-brain barrier, astrocytes play an important role in regulating the homing of different leukocyte subsets to the inflamed central nervous system (CNS). In this study, we investigated whether human astrocytes produce chemokines that promote the migration of myeloid dendritic cells (DCs). By reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, we show that cultured human astrocytes stimulated with interleukin-1β and tumor necrosis factor produce CCL2, CCL3, CCL4, CCL5, CCL20, and CXCL12 that act on immature DCs, but not CCL19 and CCL21, 2 chemokines specific for mature DCs. Compared with controls, supernatants of cytokine-stimulated astrocytes are more effective in promoting the migration of immature monocyte-derived DCs (iMDDCs). Desensitization of CXCR4 (receptor for CXCL12), CCR1-3-5 (shared receptors for CCL3-4-5), and CCR6 (receptor for CCL20) on iMDDC reduces cell migration toward astrocyte supernatants, indicating that astrocytes release biologically relevant amounts of iMDDC-attracting chemokines. By immunohistochemistry, we show that CXCL12 and, to a lesser extent, CCL20 are expressed by reactive astrocytes in multiple sclerosis lesions. These data lend support to the idea that astrocyte-derived chemokines may contribute to immature DC recruitment to the inflamed CNS.

Characterization and Recruitment of Plasmacytoid Dendritic Cells in Synovial Fluid and Tissue of Patients with Chronic Inflammatory Arthritis

Dendritic cells (DCs) are thought to play a key role in driving the immunopathogenic response underlying chronic inflammatory arthritis. In this study, we have examined the presence and phenotype of plasmacytoid DCs (pDCs) in the synovial fluids (SF) of patients with rheumatoid arthritis (RA), psoriatic arthritis (PA), and osteoarthritis (OA) and determined the chemotactic properties of SF from these patients toward pDCs. Flow cytometry analysis showed that the percentage of pDCs, identified as a population of Lin−CD123++ cells, is 4- to 5-fold higher in RA SF and PA SF than in OA SF. The morphological and immunophenotypic characterization of pDCs isolated from PA and RA SF indicates that they are in an immature state, most likely due to inhibitory factors present in RA SF, but are still able to undergo maturation when exposed ex vivo to viral agent or unmethylated DNA. CD123+ and BDCA2+ pDCs were detected by immunohistochemistry in RA synovial tissue in which expression of the IFN-α-inducible protein MxA was also found, suggesting production of type I IFN by maturing pDCs. We also show that CXCR3 and CXCR4 are expressed by both blood-derived pDCs and pDCs isolated from RA and PA SF and that CXCL-10, CXCL-11, and CXCL-12 present in RA and PA SF stimulate chemotaxis of blood-derived pDCs. Altogether, these findings suggest that chemokine-driven recruitment of pDCs from the blood to the inflamed synovium could be important in the regulation of the immune response in chronic inflammatory arthritis.

Epstein-Barr virus latent infection and BAFF expression in B cells in the multiple sclerosis brain: implications for viral persistence and intrathecal B-cell activation.

A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal synthesis of antibodies. Our previous finding that a large fraction of B cells infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the possibility that this virus, because of its ability to establish a latent infection in B cells and interfere with their differentiation, contributes to B-cell dysregulation in MS. The aim of this study was to gain further insight into EBV latency programs and their relationship to B-cell activation in the MS brain. Immunohistochemical analysis of postmortem MS brain samples harboring large EBV deposits revealed that most B cells in white matter lesions, meninges, and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent membrane protein 1and latent membrane protein 2A, 2 EBV-encoded proteins that provide survival and maturation signals to B cells. By combining laser-capture microdissection with preamplification reverse transcription-polymerase chain reaction techniques, EBV latency transcripts (latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS brain samples analyzed. We also found that B cell-activating factor of the tumor necrosis factor family is expressed in EBV-infected B cells in acute MS lesions and ectopic B-cell follicles. These findings support a role for EBV infection in B-cell activation in the MS brain and suggest that B cell-activating factor of the tumor necrosis factor family produced by EBV-infected B cells may contribute to this process resulting in viral persistence and, possibly, disruption of B-cell tolerance.

Epstein‐Barr virus persistence and reactivation in myasthenia gravis thymus

Increasing evidence supports a link between Epstein‐Barr virus (EBV), a ubiquitous B‐lymphotropic human herpesvirus, and common B‐cell–related autoimmune diseases. We sought evidence of EBV infection in thymuses from patients with myasthenia gravis (MG), an autoimmune disease characterized by intrathymic B‐cell activation.

Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

Migration of dendritic cells into the brain in a mouse model of prion disease.

The immune system plays a key role in the dissemination of prion infections from the periphery to the central nervous system (CNS). While follicular dendritic cells are critical for prion replication in lymphoid tissue and subsequent neuroinvasion, myeloid dendritic cells (DCs) have been implicated in both the clearance and propagation of pathological prion protein. Since nothing is known on the ability of DCs to migrate to the CNS during prion diseases, we investigated the immunohistochemical localization of CD205(+) DCs in the brain of C57BL/6 mice intraperitoneally infected with the mouse-adapted KFu strain of Gerstmann-Sträussler-Scheinker syndrome, a human genetic prion disorder. In normal brain, CD205(+) cells were present in the meninges and choroid plexus, whereas in the majority of mice sacrificed between 120 and 300 days post infection, CD205(+) DCs were also detected in the cerebral cortex, subcortical white matter, thalamus and medulla oblongata. These findings demonstrate that DCs can enter the CNS of prion-infected mice, suggesting a possible role for these cells in the pathogenesis of prion disorders.

B-Cell Enrichment and Epstein-Barr Virus Infection in Inflammatory Cortical Lesions in Secondary Progressive Multiple Sclerosis

Abstract Gray matter lesions are thought to play a key role in the progression of disability and cognitive impairment in multiple sclerosis (MS) patients, but whether gray matter damage is caused by inflammationor secondary to axon loss in the white matter, or both, is not clear. In an analysis of postmortem brain samples from 44 cases of secondary progressive MS, 26 cases were characterized by meningeal inflammation with ectopic B-cell follicles and prominent gray matter pathology; subpial cortical lesions containing dense perivascular lymphocytic infiltrates were present in 11 of these cases. Because intracortical immune infiltrates were enriched in B-lineage cells and because we have shown previously that B cells accumulating in the MS brain support an active Epstein-Barr virus (EBV) infection, we investigated evidence of EBV in the infiltrated cortical lesions. Cells expressing EBV-encoded small RNA and plasma cells expressing EBV early lytic proteins (BZLF1, BFRF1) were present in all and most of the intracortical perivascular cuffs examined, respectively. Immunohistochemistry for CD8-positive cells, granzyme B, perforin, and CD107a indicated cytotoxic activity toward EBV-infected plasma cells that was consistently observed in infiltrated cortical lesions, suggesting active immune surveillance. These findings indicate that both meningeal and intraparenchymal inflammation may contribute to cortical damage during MS progression, and that intracortical inflammation may be sustained by an EBV-driven immunopathologic response, similar to findings in white matter lesions and meninges.

Lymphoid chemokines in chronic neuroinflammation

Abstract Lymphoid chemokines play an essential role in the establishment and maintenance of lymphoid tissue microarchitecture and have been implicated in the formation of tertiary (or ectopic) lymphoid tissue in chronic inflammatory conditions. Here, we review recent advances in lymphoid chemokine research in central nervous system inflammation, focusing on multiple sclerosis and the animal model experimental autoimmune encephalomyelitis. We also highlight how the study of lymphoid chemokines, particularly CXCL13, has led to the identification of intrameningeal B-cell follicles in the multiple sclerosis brain paving the way to the discovery that these abnormal structures are highly enriched in Epstein–Barr virus-infected B cells and plasma cells.

Expression of TWEAK and its receptor Fn14 in the multiple sclerosis brain: implications for inflammatory tissue injury.

The expression patterns of tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a pleiotropic cytokine with proinflammatory and cell death-inducing activities, and its receptor, fibroblast growth factor-inducible 14 (Fn14), were examined in postmortem brain tissue samples from patients with multiple sclerosis (MS) and controls. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction demonstrated that both TWEAK and Fn14 were upregulated in the MS compared with control unaffected brain samples. Perivascular and meningeal macrophages and astrocytes and microglia associated with lesions were identified as the main sources of TWEAK in the MS brains. The highest frequency of TWEAK+ cells was found at edges of chronic active white matter lesions and in subpial cortical lesions inMS cases with abundant meningeal inflammation and ectopic B-cell follicles. Neurons and reactive astrocytes expressing Fn14 were mainly localized in the cerebral cortex in highly infiltrated MS brains. Numerous TWEAK-expressing microglia were associated with the extensive loss of myelin and astrocytosis, neuronal damage, and vascular abnormalities in subpial cortical lesions; this suggests that TWEAK could synergize with other cytotoxic factors diffusing from the inflamed meninges to promote cortical injury. Taken together, these findings indicate that the TWEAK/Fn14 pathway contributes to inflammation and tissue injury and is, therefore, a potential therapeutic target in MS.