Abstract
BackgroundImmunoglobulin G (IgG) antibodies reactive with intracellular neuronal proteins have been described in paraneoplastic and other autoimmune disorders. Because neurons have been thought impermeable to immunoglobulins, however, such antibodies have been considered unable to enter neurons and bind to their specific antigens during life. Cerebellar Purkinje cells - an important target in paraneoplastic and other autoimmune diseases - have been shown in experimental animals to incorporate a number of molecules from cerebrospinal fluid. IgG has also been detected in Purkinje cells studied post mortem. Despite the possible significance of these findings for human disease, immunoglobulin uptake by Purkinje cells has not been demonstrated in living tissue or studied systematically.MethodsTo assess Purkinje cell uptake of immunoglobulins, organotypic cultures of rat cerebellum incubated with rat IgGs, human IgG, fluorescein-conjugated IgG, and rat IgM were studied by confocal microscopy in real time and following fixation. An IgG-daunorubicin immunotoxin was used to determine whether conjugation of pharmacological agents to IgG could be used to achieve Purkinje cell-specific drug delivery.ResultsIgG uptake was detected in Purkinje cell processes after 4 hours of incubation and in Purkinje cell cytoplasm and nuclei by 24-48 hours. Uptake could be followed in real time using IgG-fluorochrome conjugates. Purkinje cells also incorporated IgM. Intracellular immunoglobulin did not affect Purkinje cell viability, and Purkinje cells cleared intracellular IgG or IgM within 24-48 hours after transfer to media lacking immunoglobulins. The IgG-daunomycin immunotoxin was also rapidly incorporated into Purkinje cells and caused extensive, cell-specific death within 8 hours. Purkinje cell death was not produced by unconjugated daunorubicin or control IgG.ConclusionPurkinje cells in rat organotypic cultures incorporate and clear host (rat) and non-host (human or donkey) IgG or IgM, independent of the immunoglobulin's reactivity with Purkinje cell antigens. This property permits real-time study of immunoglobulin-Purkinje cell interaction using fluorochrome IgG conjugates, and can allow Purkinje cell-specific delivery of IgG-conjugated pharmacological agents. Antibodies to intracellular Purkinje cell proteins could potentially be incorporated intracellularly to produce cell injury. Antibodies used therapeutically, including immunotoxins, may also be taken up and cause Purkinje cell injury, even if they do not recognize Purkinje cell antigens.
Highlights
Immunoglobulin G (IgG) antibodies reactive with intracellular neuronal proteins have been described in paraneoplastic and other autoimmune disorders
We demonstrate that IgG uptake is not limited to immunoglobulins of the host species, that fluorochrome-conjugated IgGs can be used to follow IgG uptake in real time, and that targeted Purkinje cell destruction can be achieved following uptake of an immunotoxin whose IgG is not reactive with Purkinje cell antigens
Confirmation of IgG and IgM integrity and lack of reactivity with Purkinje cells To preclude the possibility that immunolabeling for IgG or IgM might be due to potential IgG breakdown fragments, the normal rat IgG, the IgG monoclonal GK 1.5, and the normal rat IgM used in these experiments were analyzed by SDS-PAGE
Summary
Immunoglobulin G (IgG) antibodies reactive with intracellular neuronal proteins have been described in paraneoplastic and other autoimmune disorders. Antibodies to cytoplasmic components of cerebellar Purkinje cells have been repeatedly described in sera and cerebrospinal fluid (CSF) of patients developing paraneoplastic cerebellar degeneration in the setting of systemic cancer, as well as in systemic lupus erythematosus and certain other disorders [1,2,3,4,5,6,7] Despite their frequent detection, the roles of such antibodies in the pathogenesis of neuronal injury have been uncertain. Uptake of IgG has been suggested by the detection of kappa and lambda light chains in Purkinje and certain other neurons of a patient dying of multiple myeloma [16] Despite these observations, the ability of viable Purkinje cells to incorporate antibody has never been studied systematically, and the use of post mortem material in all published studies does not exclude the possibility of entry of IgG into neurons after death. The ability of Purkinje and related neurons to take up antibody is important because of the possible role of autoantibodies in disease causation and because cerebellar injury and Purkinje cell destruction have been described in animals and human patients receiving IgG-conjugated immunotoxins [17,18,19,20]
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