Intravenous immunoglobulin (IVIg) dampens neuronal toll-like receptor-mediated responses in ischemia

10.1186/s12974-015-0294-8Journal of Neuroinflammation12

Neuronal low-density lipoprotein receptor-related protein 1 (LRP1) enhances the anti-apoptotic effect of intravenous immunoglobulin (IVIg) in ischemic stroke

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional and multi-ligand endocytic receptor abundantly expressed in neurons. Intravenous immunoglobulin (IVIg) is a purified preparation of plasma-derived human immunoglobulin used for the treatment of several neurological inflammatory disorders, and proposed for the treatment of stroke for its potent neuroprotective effects. LRP1 has been shown to be involved in the transcytosis of IVIg, and IVIg-LRP1 interaction leads to LRP1 tyrosine phosphorylation, which may contribute to the anti-inflammatory effects of IVIg. However, the question remains whether IVIg could induce its neuroprotective effects via LRP1 in neurons under ischemic stroke conditions. In cultured neurons and in a transient ischemic mouse model, ischemia decrease LRP1 levels and phosphorylation, and IVIg blocks these effects. In ischemic neurons, LRP1 antagonism by receptor associated protein (RAP) enhances the activation of pro-death signaling pathways such as nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and caspase-3, and IVIg reduces these effects. When applied to ischemic neuronal cultures, RAP induces a dramatic drop in Akt activation, and IVIg reverses this effect, as it does with the decrease in Bcl-2 levels caused by ischemic injury in the presence of RAP. Altogether, these results show evidence of LRP1 expression and activity modulation by IVIg, and support the role of LRP1 as a partner of IVIg in the execution of its neuroprotective effects

Intravenous immunoglobulin (IVIg) dampens neuronal toll-like receptor-mediated responses in ischemia

Background: Ischemic stroke causes a high rate of deaths and permanent neurological damage in survivors. Ischemic stroke triggers the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1), which activate toll-like receptors (TLRs) and receptor for advanced glycation endproducts (RAGE) in the affected area, leading to an exaggerated inflammatory response and cell death. Both TLRs and RAGE are transmembrane pattern recognition receptors (PRRs) that have been shown to contribute to ischemic stroke-induced brain injury. Intravenous immunoglobulin (IVIg) preparations obtained by fractionating human blood plasma are increasingly being used as an effective therapeutic agent in the treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke has been proposed, but little is known about the direct neuroprotective mechanisms of IVIg. We therefore investigate whether IVIg exerts its beneficial effects on the outcome of neuronal injury by modulating HMGB1-induced TLR and RAGE expressions and activations. Methods: Primary cortical neurons were subjected to glucose deprivation or oxygen and glucose deprivation conditions and treated with IVIg and recombinant HMGB1. C57/BL6J mice were subjected to middle cerebral artery occlusion, followed by reperfusion, and IVIg was administered intravenously 3 h after the start of reperfusion. Expression of TLRs, RAGE and downstream signalling proteins in neurons and brain tissues were evaluated by immunoblot. Results: Treatment of cultured neurons with IVIg reduced simulated ischemia-induced TLR2, TLR4, TLR8 and RAGE expressions, pro-apoptotic caspase-3 cleavage and phosphorylation of the cell death-associated kinases such as c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) as well as the p65 subunit of nuclear factor kappa B (NF-κB). These results were recapitulated in an in vivo model of stroke. IVIg treatment also upregulated the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) in cortical neurons under ischemic conditions. Finally, IVIg protected neurons against HMGB1-induced neuronal cell death by modulating TLR and RAGE expressions and signalling pathways. Conclusions: Taken together, these results provide a rationale for the potential use of IVIg to target inappropriately activated components of the innate immune system following ischemic stroke

Intravenous Immunoglobulin Protects Neurons Against Amyloid Beta-Peptide Toxicity And Ischemic Stroke By Attenuating Multiple Cell Death Pathways

Intravenous immunoglobulin (IVIg) preparations obtained by fractionating blood plasma, are increasingly being used increasingly as an effective therapeutic agent in treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke and Alzheimer\u27s disease has been proposed, but little is known about the neuroprotective mechanisms of IVIg. In this study, we investigated the effect of IVIg on downstream signaling pathways that are involved in neuronal cell death in experimental models of stroke and Alzheimer\u27s disease. Treatment of cultured neurons with IVIg reduced simulated ischemia- and amyloid βpeptide (Aβ)-induced caspase 3 cleavage, and phosphorylation of the cell death-associated kinases p38MAPK, c-Jun NH2-terminal kinase and p65, in vitro. Additionally, Aβ-induced accumulation of the lipid peroxidation product 4-hydroxynonenal was attenuated in neurons treated with IVIg. IVIg treatment also up-regulated the anti-apoptotic protein, Bcl2 in cortical neurons under ischemia-like conditions and exposure to Aβ. Treatment of mice with IVIg reduced neuronal cell loss, apoptosis and infarct size, and improved functional outcome in a model of focal ischemic stroke. Together, these results indicate that IVIg acts directly on neurons to protect them against ischemic stroke and Aβ-induced neuronal apoptosis by inhibiting cell death pathways and by elevating levels of the anti-apoptotic protein Bcl2. Intravenous immunoglobulin for Stroke Intravenous immunoglobulin(IVIg) is a therapeutic modality approved for the treatment of various condition. This study was performed in order to understand the mechanism ofhow IVIg elicits its neuroprotective effect in stroke and amyloid beta induced neuronal apoptosis. The findings from this study showed that IVIg elicits its neuroprotective effects by not only inhibiting the cell death pathways but also elevating the anti-apoptotic protein Bcl2. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry

EphA2^−/− mice have reduced post-stroke blood-brain barrier (BBB) leakage, edema and infiltrating immune cells.

<p>(A) Quantitative measurement of brain edema in ipsilateral brain hemisphere by the wet-dry method. EphA2^−/− mice have lower levels of post-stroke brain edema in comparison with wild type mice. (B) Quantitative measurement of Evans Blue dye extravasation in ipsilateral brain hemisphere. Evans Blue dye content was significantly lower in brains from EphA2^−/− mice compared with wild type. (C) Flow cytometry analysis of the ipsilateral hemisphere following 72 h of reperfusion showed significantly fewer infiltrating (CD45^high) immune cells in EphA2^−/− mice compared to vehicle treated controls. Data are means ± SEM from 12 to 13 animals. **<i>p</i>&lt;0.01 relative to wild type.</p

EphA2 expression is modulated following ischemic conditions <i>in vitro</i> and <i>in vivo.</i>

<p>Wild type primary cortical neurons were used to show expression profiles of EphA2 and its ligands - Ephrins A1, 2 and 3 - using immunocytochemistry. (A–D) Cortical neurons showed expression of EphA2 and Ephrins A1–3 under normal and GD conditions. The neuronal protein lysates obtained from normal and GD conditions were probed for changes in the above-mentioned proteins using immunoblotting. (E–I) Expression of EphA2 and its ligands ephrin-A1, ephrin-A2, and ephrin-A3, was increased following GD for 12–24 h when compared to normal conditions. (J–N) A similar increase in expression was observed <i>in vivo</i> following cerebral I/R when compared to sham conditions. Cell-specific expression profile of EphA2 in the ischemic (24 I/R) brain sections was analysed in peri-infarct regions (200X) (O–P). EphA2 was primarily localized to neurons and blood vessels in the peri-infarct regions. Protein lysates of cortical neuronal cultures subjected to GD for 24 h were analyzed for the apoptotic cell death marker cleaved caspase-3 (Q–R). EphA2^−/− neuronal cultures showed significantly lower levels of cleaved caspase-3 when compared to wild type cultures. Data are mean ± SEM, n = 3–5. *p&lt;0.05, **p&lt;0.001, ***p&lt;0.0001 relative to sham (<i>in vivo)</i> and normal controls (<i>in vitro)</i> for EphA2, Ephrin A1 or Ephrin A3 blots. Data are mean ± SEM, n = 3. *p&lt;0.05 relative to the wild type controls in Cl.Cas3 blots.</p

EphA2^−/− mice demonstrate less tight junction protein disruption following focal cerebral I/R.

<p>Cerebral I/R-induced tight junction disruption and BBB damage were analyzed with zona occludens-1 (ZO-1) and matrix metalloproteinase-9 (MMP-9) antibodies respectively, by immunoblotting. (A and B) EphA2^−/− mice demonstrate significantly lower levels of MMP-9 in I/R samples compared to the wild type group, suggestive of lower I/R-induced BBB damage. (A and C) EphA2^−/− mice demonstrate significantly higher levels of ZO-1 in sham and I/R samples when compared to the wild type group, indicative of less I/R-induced tight junction disruption. Data are mean ± SEM, n  = 4–6. *p&lt;0.05 or **p&lt;0.01 relative to the wild type controls.</p

Apoptotic neuronal death is reduced in EphA2^−/− mice following focal cerebral I/R.

<p>Ipsilateral cortical protein lysates from sham and I/R brains from both wild type and EphA2^−/− mice were analyzed for selected pro- and anti-apoptotic proteins using immunoblotting. (A–D) EphA2^−/− mice demonstrate significantly lower levels of apoptotic cell death (cleaved caspase-3; Cl. Cas3) and pro-apoptotic proteins (P-SAP/JNK, BAX) as compared to the wild type group following focal cerebral I/R. (A and E) EphA2^−/− mice demonstrate significantly higher levels of the anti-apoptotic protein, Bcl–2, compared to wild type following cerebral I/R. Data are mean ± SEM, n = 4–5. *p&lt;0.05, ***p&lt;0.0001 relative to wild type.</p

EphA2^−/− mice have reduced brain damage and better functional outcome after focal cerebral ischemia.

<p>Wild type and EphA2^−/− mice were subjected to 60 min MCAO, followed by 72 h reperfusion. (A) Quantified infarct volume was significantly smaller in EphA2^−/− compared with WT mice. (B) Representative ipsilateral stroke infarct volumes detected using TTC staining are shown. White (unstained) areas indicate infarction; red (stained) areas indicate normal tissue. (C) A five point neurological score was applied to the wild type and EphA2^−/− mice following ischemia and reperfusion. Data are means ± SEM from 10 to 12 animals. **<i>p</i>&lt;0.01 relative to wild type. (D) Regional cerebral blood flow in WT animals was recorded during and after ischemia and expressed as a percentage of the pre-ischemic value.</p