Lesional Antibody Synthesis and Complement Deposition Associate With De Novo Antineuronal Antibody Synthesis After Spinal Cord Injury

Abstract

Background and objectives: Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane.

Methods: This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored.

Results: Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier.

Discussion: This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes.

Figure 1. Antibody Binding of Sera of Patients With Spinal Cord Injury (SCI) to Native Spinal Cord Epitopes as Detected by Tissue-Based Assays (TBA) and Binding to Living Dorsal Root Ganglia Cells (DRG)

Sera of patients with SCI are applied to native rat spinal cord sections and incubated for 3 hours at 37°C (1:200 in blocking solution). A representative SCI patient serum from the acute phase (1 week post-SCI) (A) and 2 representative vertebral fracture control sera (B, C) illustrate a negative staining pattern. By contrast, 10 weeks after SCI, de novo neuropil labeling of the Rexed Laminae II and III in the dorsal horn becomes apparent (arrowheads; D: follow-up serum of A; E, F: 2 representative sera of patients with SCI). It contains the substantia gelatinosa, a less myelinated region characterized by high synaptic density, which receives input from A-delta (mechanoreceptor) or C fibers (nociceptor). The dorsal horn is considered crucial for sensory-motor integration and serves as an entry gate for propriospinal information mediating considerable effects of neurorehabilitation.^, The role of the dorsal horn is further emphasized by evidence demonstrating even descending corticospinal tract (CST) fibers to synapse on dorsal horn neurons. Last, corresponding dorsal horn laminae contain Calcitonin Gene-Related Peptide positive fibers as candidates involved in neuropathic pain formation and autonomic control. This pattern is recapitulated by several SCI patient sera and demarcates a characteristic recognition pattern emerging after SCI. The targeted gray matter neuropil is composed of a dense unmyelinated fiber network of neuronal process, containing synapses, axons, and dendrites. The neuropil represents an established target for antibody-mediated autoimmune disease. Tissue-based assays are an established and validated diagnostic mean to determine autoantibodies in neurologic disease.^, Extending from autoantibodies recognizing spinal cord epitopes, the binding to living primary DRG cultured cells is considered relevant for functional relevance. The presence of autoantibodies binding to the membrane of DRG neurons and glia cells in patient sera is visualized by antihuman IgG antibodies (AF488, green) and counterstained with 4′,6-Diamidin-2-phenylindol (DAPI, blue). Sera from patients with SCI demonstrate binding of human IgG antibodies to the DRG cells (G–I). The surface staining pattern is absent in the serum of a control patient (J–L). Scale bar A–F = 500 μm, scale bar G–L = 10 μm.

Figure 2. Dynamic Emergence of Autoantibodies Detecting Epitopes of the Spinal Cord and Living Dorsal Root Ganglia (DRG) Cells in Patients After Spinal Cord Injury (SCI) Compared With That in Controls

Tissue-based assay (TBA) and DRG screening at baseline (median [interquartile range] 8 [7–9] days after injury) and follow-up (median [interquartile range] 70 [66–74] days after injury). Applying 2 diagnostic assessments testing for different aspects of autoantibody binding, patients with SCI develop in 16% of the total SCI sample autoantibodies as being double positive in TBA and DRG assays, as visualized in a Venn-like diagram depicting the overlap of TBA and DRG immune reactivity relative to the total SCI or vertebral fracture (VF) group (A) Patient sera that contain autoantibodies detecting spinal cord epitopes in situ (TBA) and having the ability to bind to living DRG cells constitute higher odds for functional relevance than those being immune positive in a singular assay alone (A, left).^, By contrast, in the vertebral fracture control cohort, no patient has been detected with double-positive serum (A, right). Neurogenic SCI-lesion-associated patterns of autoantibody binding were analyzed further by segregating SCI severity graded using the American Spinal Injury Association impairment scale (AIS) (“motor complete SCI” [AIS A/B]; “motor incomplete SCI” [AIS C/D]; “vertebral fracture without SCI” [VF]) (B) or for neurologic level of injury (“cervical SCI” [c SCI]; “thoracic, lumbar or sacral SCI” [th/l/s SCI]; “vertebral fracture without SCI” [VF]) (C). Applying 2 diagnostic assay patients testing for different aspects of autoantibody binding, patients with motor complete SCI develop in 22% autoantibodies as being double positive in TBA and DRG. By contrast, in the motor incomplete group, 1 patient (5%) and in the control cohort, no patient has been detected with double-positive serum (B). In the thoracic, lumbar, or sacral SCI group, 7 patients (30%) revealed double reactivity, whereas only 2 sera of patients with cervical SCI (5%) were double positive (C). Relative frequencies of TBA⁺, DRG⁺ double immune reactivity and single reactivity are expressed as percentages with absolute frequencies indicated in the bars. Comparison of TBA and DRG double-assay reactivity (TBA⁺, DRG⁺) vs single assay or no reactivity (TBA⁺, DRG− or TBA−, DRG⁺ or TBA−, DRG−) using the χ² test: At baseline by injury severity, p = 0.070; at follow-up by injury severity, p = 0.031 (B); at baseline by neurologic level, p = 0.26; at follow-up by neurologic level, p = 0.003 (C).

Figure 3. Medication Administered for the Treatment of Strong Pain in the Cohort Study

Patients with spinal cord injury and antibody binding detected in tissue-based assays (TBA) and dorsal root ganglia (DRG) cells (TBA⁺, DRG⁺ double positive) received earlier and more frequently gabapentinoid (pregabalin or gabapentin) medication for the specific treatment of neuropathic pain compared with TBA⁻, DRG⁻ double-negative patients with SCI and VF. Regarding opioid medication used mainly for strong non-neuropathic but also for neuropathic pain, no clear differences between the groups were observed. Patients with SCI were matched for American Spinal Injury Association impairment scale, neurologic level, age, and sex. Patients with VF were matched for age and sex.

Figure 4. B Cells Extravasate, Convert Into Plasma Cells, and Synthesize Antibodies in Subacute Spinal Cord Injury (SCI) Lesions After Human SCI

B cells leave the vessel lumen (extravasate) and populate the perivascular space (2 weeks after SCI). These findings are corroborated by the detection of 2 different B cell antigens, CD20 (A) and CD79a (B) (black arrows). B cells can convert into plasma cells as the appearance of CD138⁺ plasma cells coincided with the presence of B cells. CD138⁺ plasma cells are not restricted to the perivascular space and are interspersed in the reorganizing lesion milieu characterized by spongiotic tissue changes (C) (asterisk). Plasma cells synthesize IgG (D) and IgM antibodies (E). IgG synthesis is localized in the deposition areas of activated complement (C9neo antigen) (F).

Figure 5. Intrathecal De Novo IgM Synthesis Emerging After Spinal Cord Injury (SCI) Coinciding a Late Reboosted Inflammatory Milieu

Consecutive CSF diagnostics over time in a 55-year-old female patient with motor-sensory complete SCI of the neurologic level C4 illustrated in absolute measures (A) and plotted as Reiber diagram (B).^, Three weeks after SCI, despite a remaining mild disturbance of the blood-spinal cord barrier (BSB), no intrathecal antibody synthesis can be detected (IgG, IgA, and IgG CSF/serum ratios are within the normal range relative to the albumin CSF/serum ratio, as the asterisks are below the solid line in each graph on the left). Within the subsequent 4 weeks, a dynamic autoimmune process develops at the lesion site until week 7. Evolving post-traumatic CNS autoimmunity is characterized by a (1) reopening of the BSB (3-fold albumin CSF/serum ratio) indicative for a mild BBB disturbance and (2) remarkable de novo antibody synthesis detected in the CSF compartment only (IgG, IgA, and IgM CSF/serum ratios are substantially increased relative to the only slightly increased albumin CSF/serum ratio (asterisks are above the solid line in each graph on the right). A predominant IgM class response confirms its novel onset (de novo synthesis).