FcγRIIB在急性缺血性中風及再灌流損傷的角色

Abstract

越來越多的證據顯示急性缺血性中風及再灌流的發炎反應，是神經元損傷和功能修復的關鍵決定因素。然而免疫系統在大腦中風後，如何影響神經發炎及功能預後仍未完全被了解。重要的是，針對腦部缺血性中風後再灌流的病人尚未出現有效的治療。 FcγRIIB為一個廣為人知的抑制型接受器，可與IgG (免疫球蛋白G)的Fc位置結合。此外，FcγRIIB也廣泛的表達在各種免疫細胞上，並且容易在神經細胞上被偵測到。因為急性腦部缺血和再灌流會導致神經元細胞死亡和無菌性神經發炎，接下來的組織修復對於功能恢復是必要且關鍵的。近期，循環的白血球已被提出可在大腦血腦障蔽層(BBB)被破壞時進入大腦，並在發炎及其緩解上扮演重要的角色。然而，免疫細胞上的FcγRIIB是否對於腦部的發炎及修復的過程中有所影響，幾乎沒有被探討過。 為了回答這個問題，我們在野生型(WT) 和FcγRIIB缺陷小鼠身上執行暫時性的中大腦動脈堵塞手術(MCAO)，以研究循環的白血球在急性缺血及再灌注損傷中的作用。我們的實驗結果顯示，在24小時的再灌流後，全身缺乏FcγRIIB的小鼠比野生型(WT)的小鼠增加兩至三倍的腦部梗死量。有趣的是，在骨髓免疫細胞(LysM-cre)或B細胞(CD19-cre)上缺乏FcγRIIB的小鼠，也比野生型(WT)的小鼠顯著呈現較大的腦部梗死量，暗示了急性缺血性中風再灌流損傷中，在循環免疫細胞上的FcγRIIB扮演了一個保護的角色。然而，在樹突細胞(CD11c-cre)上FcγRIIB缺陷的小鼠顯示與野生型(WT)小鼠在腦部梗死量上沒有太大差異，因此樹突狀細胞上的FcγRIIB可能不會影響灌流後的急性缺血性中風。 為了瞭解B細胞和巨噬細胞之間的交互作用，我們給予小鼠clodronate liposome用以消除體內的巨噬細胞。在野生型(WT)小鼠中，是否消除體內巨噬細胞的老鼠在腦部梗死量沒有顯著差異。然而在B細胞(CD19-cre)中具有FcγRIIB缺陷的小鼠中，與具有正常巨噬細胞功能的小鼠相比，消除巨噬細胞小鼠的腦部梗死量略微減少。暗示B細胞上FcγRIIB的保護作用，可能與抑制巨噬細胞的發炎功能有關。 基於上述研究結果，我們接著實驗是否靜脈注射免疫球蛋白(IVIG)這個臨床上用於增加FcγRIIB表現的免疫治療，能夠改善急性缺血性中風後的再灌流損傷。在野生型(WT)小鼠中，中風再灌流後使用安慰劑小鼠的腦部梗死量大於給予注射免疫球蛋白(IVIG)的小鼠。而在I232T小鼠中，使用安慰劑或是注射免疫球蛋白(IVIG)的小鼠在腦部梗死量沒有顯著差異。此結果顯示IVIG的神經保護作用是通過FcγRIIB。FcγRIIB在免疫細胞上的抑制功能如何影響腦部梗死量及腦部缺血和再灌流損傷的背後機轉，將會再進行進一步的研究。

As a vascular disease, mounting evidence show that inflammation after acute ischemic stroke and reperfusion is a pivotal determinant in neuronal damage and functional repair. However, how immune system affects neuroinflammation and functional outcome following cerebral stroke remains incompletely understood. Importantly, there is no effective treatment for patients with reperfusion injury after ischemic stroke of brain. FcγRIIB is a well-known inhibitory receptor that binds to the Fc region of IgG. In addition to wide expression in various immune cells, FcγRIIB is readily detected in neural cells in the brain. Because acute cerebral ischemic and reperfusion results in neuronal cell death and sterile neuroinflammation, subsequent tissue repair is necessary and critical for functional recovery. Recently, circulating leukocytes have been shown to influx into brain when the integrity of blood-brain barrier (BBB) is disrupted and they play a crucial role in inflammation and resolution. However, whether FcγRIIB on immune cells plays a role in the inflammatory and reparative processes in the brain is virtually unexplored. To answer this question, we performed transient middle cerebral artery occlusion (MCAO) in wild-type (WT) and FcγRIIB-deficient mice to investigate the contribution of circulating leukocytes in acute ischemia and reperfusion injury. Our results showed that mice with systemic FcγRIIB deficiency displayed a two- to three-fold increase of infarct volume than WT mice after 24 hours of reperfusion. Interestingly, mice with FcγRIIB deficiency in either myeloid cells (LysM-cre) or B cells (CD19-cre) also exhibited significantly larger infarct volume than that of WT mice, suggesting a protective role of FcγRIIB of circulating immune cells in reperfusion injury after acute ischemic stroke. However, mice with FcγRIIB deficiency in dendritic cells (CD11c-cre) showed no difference infarct volume when compared with WT mice. Indicating that FcγRIIB on dendritic cells may not affect acute ischemic stroke after reperfusion. To determine the interaction between B cells and macrophages, clodronate liposomes was treated to mice with FcγRIIB deficiency in B cells (CD19-cre) to deplete macrophages. The infarct volume was no significant different between normal and depleted macrophages of WT mice. In FcγRIIB-CD19-cre knockout mice, the infarct volume of macrophages depleted mice is slight decrease in comparison to the mice, which has normal function of macrophages. The result indicates that FcγRIIB on B cells may plays a protective role to suppress the pro-inflammation function of macrophages. Based on these findings, we next examined whether the intravenous immunoglobulin (IVIG) therapy, a clinical method to enhance FcγRIIB expression for immunotherapy, could ameliorate reperfusion injury following acute ischemic stroke. In wild-type mice, the infarct volume of mice treated with vehicle is larger than mice treated with IVIG after stroke reperfusion. While the infarct volume was no significant different between I232T mice treated with vehicle and that treated with IVIG. The result indicates that the neuroprotective effects of IVIG are through FcγRIIB. The underlying mechanisms on how the inhibitory function of FcγRIIB on immune cells could influence the formation of infarct zone and the outcome of ischemia and reperfusion injury of brain will be investigated in detail.