Study On The Biomarkers Of NMO From The B Cell Perspective And The Effects Of LPS-priming On Pathogenicity Of AQP4-antibody

Objective：1. To detect the concentration of B cell activating factor (BAFF) in serum andcerebrospinal fluid (CSF) from patients with neuromyelitis optica(NMO), andexplore BAFF whether as a biomarker that plays a role in disease activity and thediagnosis of NMO versus multiple sclerosis(MS).2. To detect the propotion of CD1d (hi) CD5+CD19+regulatory B cell(Bregs) in NMO, and explore CD1d (hi) CD5+CD19+Bregs whether as abiomarker that plays a role in the diagnosis of NMO versus MS and providesadditional evidence about the distinct immunological nature of NMO and MS.3. To establish a NMO model, and confirm the pathogenicity ofAQP4-antibody and provide an opportunity to conduct further studies.4. To study the effects of LPS-priming on the pathogenicity ofAQP4-antibody, and explore the role of microgial activation in NMO and tounderstand the possible mechanisms.Methods：1. From April2012to October2013,44NMO patients and38MS patientswho treated at the Chinese People’s Liberation Army General Hospital wereenrolled. All of the patients were in the acute phase. Thirty healthy controls and15controls with noninflammatory neurological diseases were recruited. Theconcentration of BAFF in serum and CSF were measured by ELISA. Thepropotion of CD1d (hi) CD5+CD19+Bregs were evaluated by flow cytometry.2. The exposure of primary cultured new-borned rat cortical cells to the seraof AQP4-antibody positive with NMO induced changes of astrocytes, neurons andmicrogials, which were evaluated by the cellular immunofluorescence staining of GFAP, MAP-2and CD11b. A model of NMO was established.3. The effects of LPS-priming on cultured rat cortical cells exposed to thesera of AQP4-antibody positive with NMO were also evaluated by the cellularimmunofluorescence staining of GFAP, MAP-2and CD11b. The levels of TNF-αand IFN-γ in cultured supernatants were measured by ELISA respectively.Results：1. Mean BAFF in serum was250.2±126.9pg/ml for NMO,249.6±130.7pg/ml for MS, and222.9±126.1pg/ml for controls. There was no statisticaldifference among the three groups(P＞0.05). Mean BAFF in CSF was525.8±230.0pg/ml for NMO,298.4±141.9pg/ml for MS, and141.4±76.2pg/mlfor controls. The levels of BAFF in CSF were higher in NMO group and MSgroup compared with the controls (P <0.05). The levels of BAFF in CSF weremuch higher in NMO group compared with MS group (P <0.05). Both NMO andMS group showed a trend to an increased EDSS scores with increased CSF BAFF.However, for NMO, CSF BAFF was not associated with AQP4-antibody titer.2. The propotion of CD1d (hi) CD5+CD19+Bregs out of the CD19+Bcells was（7.94±4.16）%for NMO（,20.61±11.13）%for MS, an（d19.35±8.04）%for controls. Out of the lymphocytes was（1.61±0.89）%,（2.70±1.21）%, and（2.52±1.27）%, respectively. The proportions were significantly lower in NMOgroup compared with MS and control group（P <0.05）, but were not significantlydifferent between MS and controls(P＞0.05). The percentage of CD1d (hi) CD5+CD19+Bregs out of the CD19+B cells was （6.8%，1.33-14.2%） forAQP4-Ab-positive NMO patients, and（8.8%，3.5-20.0%）for AQP4-Ab-negativeNMO patients. Out of the lymphocytes was（1.43%，0.2-1.9%）and（2.60%，1.07-4.32%）, respectively. The percentages were lower in AQP4-Ab-positiveNMO patients than those of AQP4-Ab-negative NMO patients（P <0.05）.3. Induced by AQP4-Ab positive sera of patients with NMO, primarycultured rat cortical cells changed. The astrocytes exhibited cellular swelling andthe neurons exhibited pyknosis. The microglias showed cellular swelling and pseudopodia apophysis. Compared with blank control group and normal serumgroup, the center focus areas of GFAP and CD11b immunoreactive cells increasedsignificantly, and the center focus areas of MAP2immunoreactive cells reduced inAQP4-Ab positive serum group（P <0.05）. The neurite lengths of GFAP andCD11b immunoreactive cells increased, and The neurite lengths of MAP2immunoreactive cells reduced（P <0.05）.4. LPS-priming increased the pathogenicity of AQP4-antibody. Weobserved the apoptosis of astrocytes and neurons. The center focus areas of GFAPand MAP2immunoreactive cells reduced significantly. The neurite lengths ofGFAP and MAP2immunoreactive cells shortened or disappeared（P <0.05）.However, for CD11b immunoreactive cells, the center focus areas did notchange(P＞0.05), and the neurite lengths increased（P <0.05）. In culturedsupernatants the levels of TNF-α were higher than that of control group（P <0.05）,while the IFN-γ levels did not show any increase (P＞0.05).Conclusion：1. BAFF in CSF may be a useful biomarker that plays a role in diseaseactivity and the diagnosis of NMO versus MS.2. CD1d (hi) CD5+CD19+Bregs may be a biomarker that plays a role inthe diagnosis of NMO versus MS. At the same time the results define the distinctimmunological nature of NMO and MS, AQP4-Ab-positive NMO andAQP4-Ab-negative NMO.3. AQP4-antibody positive serum stimulation of primary cultured rat corticalcells can establish a modle of NMO.4. Treated with LPS-priming，the microglia can be activated and can makethe pathogenicity of AQP4-antibody enhanced. The immunological mechanismsmay be involved and proinflammatory cytokine TNF-α plays an important role inthe process.