J Neuropathol Exp Neurol Copyright Ó 2014 by the American Association of Neuropathologists, Inc.
LETTER TO Epstein-Barr Virus in the Central Nervous System and Cervical Lymph Node of a Patient With Primary Progressive Multiple Sclerosis Although Epstein-Barr virus (EBV) has strong associations with multiple sclerosis (MS), the mechanisms linking EBV infection to CNS pathology remain elusive (1). Selective EBV deregulation in MS is manifested by elevated antibody titers and T-cell responses to EBV but not to other herpes viruses (1, 2). The absence of EBV in the cerebrospinal fluid of most MS patients and the lack of significant differences in blood EBV load between patients and healthy subjects point to a secluded low-grade active EBV infection (2). Our previous studies suggest that EBV may contribute to MS pathology by establishing a persistent infection in the CNS through infiltrating B cells harboring latent and reactivated virus and by stimulating an immunopathologic response (3, 4). Whereas this model remains controversial (5), no data are available on the EBV status of MS patient lymphoid tissue, where most of the viral life cycle occurs. Using in situ hybridization and immunohistochemistry, we have analyzed for the first time EBV in both postmortem CNS and lymph nodes from a patient with primary progressive MS. A 43-year-old woman with primary progressive MS (10-year disease duration) and severe disability (Expanded Disability Status Scale 7.0) died in 2002 because of an ischemic stroke; she underwent autopsy with a postmortem delay of less than 24 hours at the Pathologic Anatomy Institute, Agostino Gemelli Hospital, Rome, Italy. No therapy or comorbidities were reported. Paraffinembedded tissue blocks from the right cerebral hemisphere, brainstem, and cervical spinal cord, a deep lateral cervical lymph node, and a pulmonary hilar lymph node were analyzed. The use of post-
Vol. 73, No. 7 July 2014 pp. 729Y731
THE
EDITOR
mortem tissue for research purposes was approved by the Ethics Committee of Istituto Superiore di Sanita`. Neuropathologic examination revealed large demyelinated lesions (Figs. 1A, B), with a low level of lymphocytic infiltration in the white matter at all CNS levels and moderate inflammation in the cerebral meninges. Foamy macrophages were present in active lesions in the brain and spinal cord (Fig. 1C). In situ hybridization was used to detect EBV-encoded small RNAs (EBERs), which are abundantly expressed during EBV latent infection. Epstein-Barr virusY encoded small RNAYpositive cells were scattered in localized areas of white matter lesions at all CNS levels examined and in the cerebral meninges (Figs. 1DYG). Immunohistochemistry for the EBV BZLF1 protein expressed during the immediateearly lytic cycle and gp350/220, a glycoprotein of the viral envelope, revealed the presence of lytically infected cells in the cerebral meninges (not shown) and in a perilesional area in the cervical spinal cord white matter (Figs. 1H, I). In the cervical lymph node, there were numerous EBER-positive cells at the edge of most B follicles and in the paracortex (Figs. 1J, K). Using immunohistochemistry, scattered perifollicular cells were found to be immunopositive for EBV nuclear antigen 1 (EBNA1), a protein expressed during all EBV latency programs (III to I); isolated cells expressed EBNA2, which is restricted to the latency III program and essential for EBVmediated B-cell transformation (Figs. 1L, M). Epstein-Barr virus lytically infected cells expressing BZLF1 and, more rarely, gp350/220 were found at the border of some B follicles (Fig. 1N). In the cervical lymph node, 20% to 25% of the follicles contained proliferating CD20-positive B cells, including large B lymphoblasts (Figs. 1O, P); this finding is compatible with EBVinduced B-cell growth/transformation. Numerous CD20-negative immunoblasts were present in the paracortex, suggesting T-cell activation (Figs. 1O, Q). No cells positive for EBER or any of the EBV proteins analyzed and no large immunoblasts were found in the pulmonary hilar lymph node (Figs. 1RYU), similarly to
previously analyzed nonpathologic lymph nodes (3). In conclusion, these findings suggest, for the first time, that both the CNS (3, 4) and its draining lymph nodes may be preferential sites where EBV escapes immune surveillance and abnormally reactivates in MS patients. The biologic implication of this finding is that EBV reactivating in cervical lymph nodes could be a more potent stimulus of an immunopathologic response than virus hidden in the CNS. Therapeutically, eradication of abnormal intracerebral and extracerebral EBV deposits might be an effective means to treat the disease, as suggested by the beneficial effects of monoclonal antibodies depleting B cells (the main EBV reservoir) in MS patients (6). It remains to be established whether EBV deregulation in cervical lymph nodes is a frequent finding in MS or is restricted to patients with primary progressive MS and more aggressive MS clinical courses.
Barbara Serafini, PhD Barbara Rosicarelli, MSc Francesca Aloisi, PhD Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita` Rome, Italy, [email protected]
Egidio Stigliano, PhD Institute of Pathologic Anatomy Policlinico A. Gemelli, Rome, Italy
Conflicts of Interest Disclosures: This study was supported by the Italian Ministry of Health (grant no. 107, Ricerca Finalizzata 2007 to Francesca Aloisi). ACKNOWLEDGMENTS The authors thank Dr Mariangela Cirillo, Policlinico A. Gemelli, Rome, Italy, for expert advice on postmortem tissue characterization and Dr Riccardo Dolcetti, National Cancer Institute, Aviano, Italy, for helpful discussions.
J Neuropathol Exp Neurol Volume 73, Number 7, July 2014
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
729
Letter to the Editor
730
J Neuropathol Exp Neurol Volume 73, Number 7, July 2014
Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 73, Number 7, July 2014
REFERENCES 1. Ascherio A, Munger KL. Epstein-Barr virus infection and multiple sclerosis: A review. J Neuroimmune Pharmacol 2010;5:271Y77 2. Lossius A, Johansen JN, Torkildsen K, et al. Epstein-Barr virus in systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosisVAssociation and causation. Viruses 2012;4:3701Y30
3. Serafini B, Rosicarelli B, Franciotta D, et al. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J Exp Med 2007;204: 2899Y912 4. Angelini DF, Serafini B, Piras E, et al. Increased CD8+ T-cell response to EpsteinBarr virus lytic antigens in the active phase of multiple sclerosis. PLoS Pathog 2013;9(4): e1003220
Letter to the Editor 5. Lassmann H, Niedobitek G, Aloisi F, et al. Epstein-Barr virus in the multiple sclerosis brain: A controversial issueVReport on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain 2011;134:2772Y86 6. Lulu S, Waubant E. Humoral-targeted immunotherapies in multiple sclerosis. Neurotherapeutics 2013;10:34Y43
FIGURE 1. Epstein-Barr virus (EBV) detection in the CNS and lymph node tissues of a patient with primary progressive multiple sclerosis. (AYI) CNS tissue. There is extensive demyelination in the cervical spinal cord (A) and subcortical white matter (B) demonstrated with Luxol fast blue (LFB) staining of formalin-fixed paraffin sections. Perivascular and intraparenchymal foamy macrophages in an active spinal cord lesion are visualized using immunostaining with anti-CD68 monoclonal antibody (KP1; DakoCytomation, Glostrup, Denmark) (C). Epstein-Barr virusYencoded small RNA (EBER)Yspecific in situ hybridization (DakoCytomation) shows EBV latently infected cells (black nuclear staining) in a white matter lesion in the brainstem (D, E; arrows indicate perivascular EBER-positive cells). Scattered EBER-positive cells in the cerebral meninges are also shown in (F) and (G). Epstein-Barr virus lytically infected cells in the cervical spinal cord are visualized with monoclonal antibodies to the EBV immediate-early lytic protein BZLF1 (clone BZ-1; kind gift of Prof J. Middeldorp, VU University Medical Centre, Amsterdam, The Netherlands) (H; nuclear staining) and the EBV envelop glycoprotein gp350/220 (Bioworld Consulting Laboratories, Mt. Airy, MD) (I; red, cytoplasmic staining). (JYQ) Deep cervical lymph node. In paraffin sections of the cervical lymph node, numerous EBER-positive cells are present outside (paracortex) and at the edge of B follicles (J, K); in the inset in K, the arrow indicates a large EBER-positive nucleus. Immunostaining with antibodies to EBV latent proteins (EBNA-1 [clone 1H4; kind gift of Prof G. Niedobitek; Sana Klinikum Lichtenberg/Unfallkrankenhaus, Berlin, Germany] [L, arrows] and EBNA-2 [PE2; Novocastra Laboratories, Newcastle upon Tyne, UK] [M, arrows]) reveals the presence of numerous perifollicular EBNA1-positive and rare EBNA2positive cells in the cervical lymph node (both proteins have a nuclear localization). Using immunofluorescence staining, the nuclei (DAPI stain, blue) of several cells at the edge of lymphoid B follicles are positive for BZLF1 (pink) (N and upper inset) and a few cells are gp350/220-positive (red) (N, lower inset). Double immunofluorescence staining of cervical lymph node sections with antibodies to CD20 (L26; ScyTeK Laboratories, Logan, UT) (green) and the proliferation marker Ki-67 (rabbit polyclonal; Novocastra Laboratories) (red) shows proliferating follicular CD20-positive B cells and perifollicular CD20-negative immunoblasts (O). Large CD20-positive B lymphoblasts at the border of a lymphoid follicle (P and inset) and CD20-negative immunoblasts in the paracortex (Q), including large multinucleated cells (arrows), are shown at high-power magnification. (RYU) Pulmonary hilar lymph node. No EBV-infected cells expressing EBER (R), EBNA1 (S), or EBNA2 (T) and no large immunoblasts (U) are present in the pulmonary hilar lymph node; this lymph node was also negative for BZLF1 and gp350/220 (not shown). Scale bars = (A, B) 500 Km; (C, J) 200 Km; (E, I, S, T, U) 100 Km; (F, G, KYM, O, R) 50 Km; (D, H, N, Q and insets in G, K, P) 20 Km; (insets in E, I, M, N) 10 Km. Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
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LETTER TO Epstein-Barr Virus in the Central Nervous System and Cervical Lymph Node of a Patient With Primary Progressive Multiple Sclerosis Although Epstein-Barr virus (EBV) has strong associations with multiple sclerosis (MS), the mechanisms linking EBV infection to CNS pathology remain elusive (1). Selective EBV deregulation in MS is manifested by elevated antibody titers and T-cell responses to EBV but not to other herpes viruses (1, 2). The absence of EBV in the cerebrospinal fluid of most MS patients and the lack of significant differences in blood EBV load between patients and healthy subjects point to a secluded low-grade active EBV infection (2). Our previous studies suggest that EBV may contribute to MS pathology by establishing a persistent infection in the CNS through infiltrating B cells harboring latent and reactivated virus and by stimulating an immunopathologic response (3, 4). Whereas this model remains controversial (5), no data are available on the EBV status of MS patient lymphoid tissue, where most of the viral life cycle occurs. Using in situ hybridization and immunohistochemistry, we have analyzed for the first time EBV in both postmortem CNS and lymph nodes from a patient with primary progressive MS. A 43-year-old woman with primary progressive MS (10-year disease duration) and severe disability (Expanded Disability Status Scale 7.0) died in 2002 because of an ischemic stroke; she underwent autopsy with a postmortem delay of less than 24 hours at the Pathologic Anatomy Institute, Agostino Gemelli Hospital, Rome, Italy. No therapy or comorbidities were reported. Paraffinembedded tissue blocks from the right cerebral hemisphere, brainstem, and cervical spinal cord, a deep lateral cervical lymph node, and a pulmonary hilar lymph node were analyzed. The use of post-
Vol. 73, No. 7 July 2014 pp. 729Y731
THE
EDITOR
mortem tissue for research purposes was approved by the Ethics Committee of Istituto Superiore di Sanita`. Neuropathologic examination revealed large demyelinated lesions (Figs. 1A, B), with a low level of lymphocytic infiltration in the white matter at all CNS levels and moderate inflammation in the cerebral meninges. Foamy macrophages were present in active lesions in the brain and spinal cord (Fig. 1C). In situ hybridization was used to detect EBV-encoded small RNAs (EBERs), which are abundantly expressed during EBV latent infection. Epstein-Barr virusY encoded small RNAYpositive cells were scattered in localized areas of white matter lesions at all CNS levels examined and in the cerebral meninges (Figs. 1DYG). Immunohistochemistry for the EBV BZLF1 protein expressed during the immediateearly lytic cycle and gp350/220, a glycoprotein of the viral envelope, revealed the presence of lytically infected cells in the cerebral meninges (not shown) and in a perilesional area in the cervical spinal cord white matter (Figs. 1H, I). In the cervical lymph node, there were numerous EBER-positive cells at the edge of most B follicles and in the paracortex (Figs. 1J, K). Using immunohistochemistry, scattered perifollicular cells were found to be immunopositive for EBV nuclear antigen 1 (EBNA1), a protein expressed during all EBV latency programs (III to I); isolated cells expressed EBNA2, which is restricted to the latency III program and essential for EBVmediated B-cell transformation (Figs. 1L, M). Epstein-Barr virus lytically infected cells expressing BZLF1 and, more rarely, gp350/220 were found at the border of some B follicles (Fig. 1N). In the cervical lymph node, 20% to 25% of the follicles contained proliferating CD20-positive B cells, including large B lymphoblasts (Figs. 1O, P); this finding is compatible with EBVinduced B-cell growth/transformation. Numerous CD20-negative immunoblasts were present in the paracortex, suggesting T-cell activation (Figs. 1O, Q). No cells positive for EBER or any of the EBV proteins analyzed and no large immunoblasts were found in the pulmonary hilar lymph node (Figs. 1RYU), similarly to
previously analyzed nonpathologic lymph nodes (3). In conclusion, these findings suggest, for the first time, that both the CNS (3, 4) and its draining lymph nodes may be preferential sites where EBV escapes immune surveillance and abnormally reactivates in MS patients. The biologic implication of this finding is that EBV reactivating in cervical lymph nodes could be a more potent stimulus of an immunopathologic response than virus hidden in the CNS. Therapeutically, eradication of abnormal intracerebral and extracerebral EBV deposits might be an effective means to treat the disease, as suggested by the beneficial effects of monoclonal antibodies depleting B cells (the main EBV reservoir) in MS patients (6). It remains to be established whether EBV deregulation in cervical lymph nodes is a frequent finding in MS or is restricted to patients with primary progressive MS and more aggressive MS clinical courses.
Barbara Serafini, PhD Barbara Rosicarelli, MSc Francesca Aloisi, PhD Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita` Rome, Italy, [email protected]
Egidio Stigliano, PhD Institute of Pathologic Anatomy Policlinico A. Gemelli, Rome, Italy
Conflicts of Interest Disclosures: This study was supported by the Italian Ministry of Health (grant no. 107, Ricerca Finalizzata 2007 to Francesca Aloisi). ACKNOWLEDGMENTS The authors thank Dr Mariangela Cirillo, Policlinico A. Gemelli, Rome, Italy, for expert advice on postmortem tissue characterization and Dr Riccardo Dolcetti, National Cancer Institute, Aviano, Italy, for helpful discussions.
J Neuropathol Exp Neurol Volume 73, Number 7, July 2014
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
729
Letter to the Editor
730
J Neuropathol Exp Neurol Volume 73, Number 7, July 2014
Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 73, Number 7, July 2014
REFERENCES 1. Ascherio A, Munger KL. Epstein-Barr virus infection and multiple sclerosis: A review. J Neuroimmune Pharmacol 2010;5:271Y77 2. Lossius A, Johansen JN, Torkildsen K, et al. Epstein-Barr virus in systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosisVAssociation and causation. Viruses 2012;4:3701Y30
3. Serafini B, Rosicarelli B, Franciotta D, et al. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J Exp Med 2007;204: 2899Y912 4. Angelini DF, Serafini B, Piras E, et al. Increased CD8+ T-cell response to EpsteinBarr virus lytic antigens in the active phase of multiple sclerosis. PLoS Pathog 2013;9(4): e1003220
Letter to the Editor 5. Lassmann H, Niedobitek G, Aloisi F, et al. Epstein-Barr virus in the multiple sclerosis brain: A controversial issueVReport on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain 2011;134:2772Y86 6. Lulu S, Waubant E. Humoral-targeted immunotherapies in multiple sclerosis. Neurotherapeutics 2013;10:34Y43
FIGURE 1. Epstein-Barr virus (EBV) detection in the CNS and lymph node tissues of a patient with primary progressive multiple sclerosis. (AYI) CNS tissue. There is extensive demyelination in the cervical spinal cord (A) and subcortical white matter (B) demonstrated with Luxol fast blue (LFB) staining of formalin-fixed paraffin sections. Perivascular and intraparenchymal foamy macrophages in an active spinal cord lesion are visualized using immunostaining with anti-CD68 monoclonal antibody (KP1; DakoCytomation, Glostrup, Denmark) (C). Epstein-Barr virusYencoded small RNA (EBER)Yspecific in situ hybridization (DakoCytomation) shows EBV latently infected cells (black nuclear staining) in a white matter lesion in the brainstem (D, E; arrows indicate perivascular EBER-positive cells). Scattered EBER-positive cells in the cerebral meninges are also shown in (F) and (G). Epstein-Barr virus lytically infected cells in the cervical spinal cord are visualized with monoclonal antibodies to the EBV immediate-early lytic protein BZLF1 (clone BZ-1; kind gift of Prof J. Middeldorp, VU University Medical Centre, Amsterdam, The Netherlands) (H; nuclear staining) and the EBV envelop glycoprotein gp350/220 (Bioworld Consulting Laboratories, Mt. Airy, MD) (I; red, cytoplasmic staining). (JYQ) Deep cervical lymph node. In paraffin sections of the cervical lymph node, numerous EBER-positive cells are present outside (paracortex) and at the edge of B follicles (J, K); in the inset in K, the arrow indicates a large EBER-positive nucleus. Immunostaining with antibodies to EBV latent proteins (EBNA-1 [clone 1H4; kind gift of Prof G. Niedobitek; Sana Klinikum Lichtenberg/Unfallkrankenhaus, Berlin, Germany] [L, arrows] and EBNA-2 [PE2; Novocastra Laboratories, Newcastle upon Tyne, UK] [M, arrows]) reveals the presence of numerous perifollicular EBNA1-positive and rare EBNA2positive cells in the cervical lymph node (both proteins have a nuclear localization). Using immunofluorescence staining, the nuclei (DAPI stain, blue) of several cells at the edge of lymphoid B follicles are positive for BZLF1 (pink) (N and upper inset) and a few cells are gp350/220-positive (red) (N, lower inset). Double immunofluorescence staining of cervical lymph node sections with antibodies to CD20 (L26; ScyTeK Laboratories, Logan, UT) (green) and the proliferation marker Ki-67 (rabbit polyclonal; Novocastra Laboratories) (red) shows proliferating follicular CD20-positive B cells and perifollicular CD20-negative immunoblasts (O). Large CD20-positive B lymphoblasts at the border of a lymphoid follicle (P and inset) and CD20-negative immunoblasts in the paracortex (Q), including large multinucleated cells (arrows), are shown at high-power magnification. (RYU) Pulmonary hilar lymph node. No EBV-infected cells expressing EBER (R), EBNA1 (S), or EBNA2 (T) and no large immunoblasts (U) are present in the pulmonary hilar lymph node; this lymph node was also negative for BZLF1 and gp350/220 (not shown). Scale bars = (A, B) 500 Km; (C, J) 200 Km; (E, I, S, T, U) 100 Km; (F, G, KYM, O, R) 50 Km; (D, H, N, Q and insets in G, K, P) 20 Km; (insets in E, I, M, N) 10 Km. Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
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