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Complement-mediated cytotoxicity of antibodies to the GABAB receptor
Correspondence
Complement-mediated cytotoxicity of antibodies to the GABAB receptor We read with great interest the paper by Lancaster and colleagues,1 which describes a novel autoimmune limbic encephalitis associated with antibodies to the GABAB receptor. In all six tested patients the anti-GABAB antibodies were IgG1 and thus able to activate complement. Nevertheless, in their discussion, the authors consider the role of complement-mediated cytotoxicity in the pathogenesis of this disorder, which was sensitive to immunotherapy in most cases, as questionable. We have described a woman with recurrent acute episodes of respiratory crises, dysautonomic symptoms, and total sleep loss (agrypnia) due to an antibody directed against the GABAB receptor,2 who showed a good clinical response to immunotherapy.2,3 Similar to patients described by Lancaster and colleagues,1 our patient’s purified IgG recognised a band of about 110 kDa on protein extracts of mouse cerebellum, cortex, and brainstem, and immunolabelled cultured Chinese hamster ovary (CHO) cells transfected with human GABAB1 receptors and rat GABAB2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both purified IgG and anti-GABAB1 antibodies from the patient. In our case, in mice the human purified IgG was able to induce a reversible neurological syndrome similar in all respects to that observed in the patient. Moreover, immunohistochemistry on brain sections of mice injected with the patient’s IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on neurons, suggesting a complementmediated cytotoxicity. In our opinion, the clinical response to treatment does not rule out the role of antineural www.thelancet.com/neurology Vol 9 April 2010
complement-fixing autoantibodies in the pathogenesis of autoimmune encephalitis. We have no conflicts of interest.
Anna Paola Batocchi, Giacomo Della Marca, Giovanni Frisullo [email protected] Department of Neurosciences, Catholic University, Rome, Italy 1
2
3
Lancaster E, Lai M, Peng X, et al. Antibodies to the GABAB receptor in limbic encephalitis with seizures: case series and characterisation of the antigen. Lancet Neurol 2010; 9: 67–76. Batocchi AP, Della Marca G, Mirabella M, et al. Relapsing-remitting autoimmune agrypnia. Ann Neurol 2001; 50: 668–71. Frisullo G, Della Marca G, Mirabella M, et al. A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia. Exp Neurol 2007; 204: 808–18.
Authors’ reply We thank Batocchi and colleagues for their comments; however, they seem to have misunderstood us:1 the antibodies of our patients did not recognise a 110 kDa band in denaturing immunoblot. Patients’ antibodies precipitated the B1/B2 subunits of the GABAB receptor, which was confirmed with immunoblots of the immunoprecipitate by use of commercially available specific antibodies, indicating that the target epitope is present in its functional conformation. Additional analyses with cultured neurons showed that the epitope was on the cell surface and that patients had no other antibodies to cell-surface antigens. Our patients’ phenotype resembled that of animals with disrupted GABAB receptors. Therefore, the disorder they had was not only clinically but also immunologically different from that of the patient described by Batocchi and colleagues.2,3 The authors indicate that their patient’s symptoms resulted from antibodies to an intracellular GABAB receptor epitope that competed with a commercial antibody, via complement activation. However, controls to identify the antibody specificity and studies to rule out other antibodies were missing.
For example, reactivity of patient’s IgG with non-transfected cells was not assessed; normal IgG showed some reactivity with transfected Chinese hamster ovary cells, and competition experiments between normal IgG and the commercial antibody were not done.3 Moreover, to model the disorder the authors injected patient’s or control IgG into the cisterna magna of mice at concentrations ranging from 800 to 4000 times the normal levels of IgG in human CSF. Data from these mice showed deposits of IgG mainly inside Purkinje cells—a pattern different from that of their patient’s antibody reactivity, which, according to the authors, spared Purkinje cells2,3—and deposits of complement that did not colocalise with the patient’s IgG (figure 5 in the paper by Frisullo and colleagues3). These findings, along with the well-known property of Purkinje cells to take up proteins from CSF, raises concern about the specificity of the IgG and complement deposits. Furthermore, the symptoms of the mice3 only partially resembled the patient’s syndrome.2 The rapid recovery of the mice is also puzzling in that Purkinje cells are unlikely to recover from supposed complementmediated cytotoxicity and complement activation is unlikely to occur after intracellular penetration of an antibody. More consistent with the reversibility of their patient’s symptoms is that other (undefined) antibodies to cell-surface antigens were involved; these would not activate complement because the data of Batocchi and colleagues2 did not show cell-surface linear deposition of complement. As we suggested,1 the occurrence of antibodies that can fix complement does not demonstrate that this mechanism is operational. JD has received royalties from a patent related to Ma2 autoantibody test and has filed patent applications for NMDA and GABAB receptor autoantibody tests. JD has received funding from Euroimmun for projects unrelated to the study referred to in these letters. All other authors have no conflicts of interest.
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Complement-mediated cytotoxicity of antibodies to the GABAB receptor We read with great interest the paper by Lancaster and colleagues,1 which describes a novel autoimmune limbic encephalitis associated with antibodies to the GABAB receptor. In all six tested patients the anti-GABAB antibodies were IgG1 and thus able to activate complement. Nevertheless, in their discussion, the authors consider the role of complement-mediated cytotoxicity in the pathogenesis of this disorder, which was sensitive to immunotherapy in most cases, as questionable. We have described a woman with recurrent acute episodes of respiratory crises, dysautonomic symptoms, and total sleep loss (agrypnia) due to an antibody directed against the GABAB receptor,2 who showed a good clinical response to immunotherapy.2,3 Similar to patients described by Lancaster and colleagues,1 our patient’s purified IgG recognised a band of about 110 kDa on protein extracts of mouse cerebellum, cortex, and brainstem, and immunolabelled cultured Chinese hamster ovary (CHO) cells transfected with human GABAB1 receptors and rat GABAB2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both purified IgG and anti-GABAB1 antibodies from the patient. In our case, in mice the human purified IgG was able to induce a reversible neurological syndrome similar in all respects to that observed in the patient. Moreover, immunohistochemistry on brain sections of mice injected with the patient’s IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on neurons, suggesting a complementmediated cytotoxicity. In our opinion, the clinical response to treatment does not rule out the role of antineural www.thelancet.com/neurology Vol 9 April 2010
complement-fixing autoantibodies in the pathogenesis of autoimmune encephalitis. We have no conflicts of interest.
Anna Paola Batocchi, Giacomo Della Marca, Giovanni Frisullo [email protected] Department of Neurosciences, Catholic University, Rome, Italy 1
2
3
Lancaster E, Lai M, Peng X, et al. Antibodies to the GABAB receptor in limbic encephalitis with seizures: case series and characterisation of the antigen. Lancet Neurol 2010; 9: 67–76. Batocchi AP, Della Marca G, Mirabella M, et al. Relapsing-remitting autoimmune agrypnia. Ann Neurol 2001; 50: 668–71. Frisullo G, Della Marca G, Mirabella M, et al. A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia. Exp Neurol 2007; 204: 808–18.
Authors’ reply We thank Batocchi and colleagues for their comments; however, they seem to have misunderstood us:1 the antibodies of our patients did not recognise a 110 kDa band in denaturing immunoblot. Patients’ antibodies precipitated the B1/B2 subunits of the GABAB receptor, which was confirmed with immunoblots of the immunoprecipitate by use of commercially available specific antibodies, indicating that the target epitope is present in its functional conformation. Additional analyses with cultured neurons showed that the epitope was on the cell surface and that patients had no other antibodies to cell-surface antigens. Our patients’ phenotype resembled that of animals with disrupted GABAB receptors. Therefore, the disorder they had was not only clinically but also immunologically different from that of the patient described by Batocchi and colleagues.2,3 The authors indicate that their patient’s symptoms resulted from antibodies to an intracellular GABAB receptor epitope that competed with a commercial antibody, via complement activation. However, controls to identify the antibody specificity and studies to rule out other antibodies were missing.
For example, reactivity of patient’s IgG with non-transfected cells was not assessed; normal IgG showed some reactivity with transfected Chinese hamster ovary cells, and competition experiments between normal IgG and the commercial antibody were not done.3 Moreover, to model the disorder the authors injected patient’s or control IgG into the cisterna magna of mice at concentrations ranging from 800 to 4000 times the normal levels of IgG in human CSF. Data from these mice showed deposits of IgG mainly inside Purkinje cells—a pattern different from that of their patient’s antibody reactivity, which, according to the authors, spared Purkinje cells2,3—and deposits of complement that did not colocalise with the patient’s IgG (figure 5 in the paper by Frisullo and colleagues3). These findings, along with the well-known property of Purkinje cells to take up proteins from CSF, raises concern about the specificity of the IgG and complement deposits. Furthermore, the symptoms of the mice3 only partially resembled the patient’s syndrome.2 The rapid recovery of the mice is also puzzling in that Purkinje cells are unlikely to recover from supposed complementmediated cytotoxicity and complement activation is unlikely to occur after intracellular penetration of an antibody. More consistent with the reversibility of their patient’s symptoms is that other (undefined) antibodies to cell-surface antigens were involved; these would not activate complement because the data of Batocchi and colleagues2 did not show cell-surface linear deposition of complement. As we suggested,1 the occurrence of antibodies that can fix complement does not demonstrate that this mechanism is operational. JD has received royalties from a patent related to Ma2 autoantibody test and has filed patent applications for NMDA and GABAB receptor autoantibody tests. JD has received funding from Euroimmun for projects unrelated to the study referred to in these letters. All other authors have no conflicts of interest.
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