正常大白鼠松果腺內巨噬細胞/小神經膠細胞的特性及經細菌毒素刺激或頸上神經節摘除後其免疫反應之研究

Studies on features and immune responses of pineal macrophages/microglia in the intact rats and those challenged with bacterial toxins or superior cervical ganglionectomy

江謝雅芬(Ya-Fen Jiang-Shieh)

指導教授 ： 謝正勇 ； 溫振源

國立臺灣大學/醫學院/解剖學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00620

摘要

1. 大白鼠松果腺內的巨噬細胞/小神經膠細胞免疫分子特性的區域分布
本研究用巨嗜細胞專一性的抗體(OX-42，OX-6，ED-1 及ED-2)去辨識松果腺巨噬細胞/小神經膠細胞，試著去檢視成鼠松果腺內巨噬細胞/小神經膠細胞是否顯示不同免疫型態分佈於特別區域。每一種抗體(OX-42，OX-6，及ED-1)識別松果腺內巨噬細胞/小神經膠細胞其中一群細胞，然而非常少見ED-2反應的巨噬細胞/小神經膠細胞。本實驗計量研究顯示松果腺內巨噬細胞/小神經膠細胞表現補體第三型接受體(被OX-42標誌)比主要組織相容第二型抗原(被OX- 6標誌)或未知細胞質/溶小體抗原(被ED-1標誌)更多。以此種免疫組織化學染色法，我們發現巨噬細胞/小神經膠細胞廣泛的分佈在整個松果腺，尤其是被OX-42標誌的巨噬細胞/小神經膠細胞，該細胞均勻地分散在松果腺的背-腹側方向。在此腺體同樣的方向，被OX-42，OX-6標誌的巨噬細胞/小神經膠細胞主要位於中央區域。並且無論是何種免疫型態的巨噬細胞/小神經膠細胞很少分佈於松果腺遠端。很有趣地，被OX-6標誌的松果腺巨噬細胞/小神經膠細胞，其細胞密度明顯的呈現近-遠端梯度。除了不同區域的松果腺巨噬細胞/小神經膠細胞不同免疫型態，在大型血管周圍常發現明顯細胞聚集。經相鄰切片染色分析，這些聚集細胞大部分是圓形帶有不同免疫分子所組成。以參重免疫標誌及共軛焦掃瞄顯微鏡觀察，進一步證實松果腺巨噬細胞/小神經膠細胞可能擁有兩種不同抗原(ED-1+/OX-6+、OX-42+/OX-6+或OX-42+/ED-1+)。很明顯地，大多數巨噬細胞/小神經膠細胞均表現所有檢視的抗原(ED-1+/OX-6 +/OX-42+)。本研究結果指出成鼠松果腺巨噬細胞/小神經膠細胞免疫分子是異質性的，其分佈的不同也是依據不同的免疫表型。因此，我們認為松果腺巨噬細胞/小神經膠細胞免疫表型的不同是根據其所在的不同區域分佈，而這特殊性可能與松果腺免疫調節功能有關。
2. 葛蘭氏陰性及陽性菌產物對大白鼠松果腺內神經膠與松果腺細胞的影響
酯多醣是葛蘭氏陰性菌的主要發炎組成份，眾所周知會引起敗血症及活化中樞神經系統內的小神經膠細胞。相反的，葛蘭氏陽性菌產物對小神經膠細胞的作用，尤其是在沒有血腦障壁環境內的小神經膠細胞是值得探討。本實驗以一系列抗體OX-6、OX-42及ED-1研究，靜脈注射酯多醣或壁酯酸後，大白鼠松果腺巨噬細胞/小神經膠細胞的反應。這些抗體分別認識巨噬細胞的主要組織相容第二型抗原、補體第三型接受體及未知溶小體蛋白質。在大白鼠注射酯多醣(50 μg/kg) 48 小時之後犧牲，大白鼠松果腺巨噬細胞/小神經膠細胞密度及其OX-6、OX-42與ED-1免疫分子表現顯著增加。在大白鼠接受高劑量壁酯酸(20 mg/kg)後，松果腺巨噬細胞/小神經膠細胞OX-6及OX-42免疫活性也增加，但是ED-1並沒改變。在此同時，這兩種毒素皆會刺激標誌星狀神經膠細胞的神經膠絲酸蛋白增加。給予酯多醣或壁酯酸處理後，有一個有趣現象就是血清內的褪黑激素減少，而血清素免疫標誌卻是增加。電子鏡觀察下，松果腺細胞內有液泡化的現象，這些酯多醣或壁酯酸引發的液泡很顯然是來自於顆粒性內質網及高爾基氏體小囊。本實驗認為酯多醣及壁酯酸可以引發松果腺免疫反應，使松果腺巨噬細胞/小神經膠細胞及星狀神經膠細胞活化。另外，松果腺細胞的代謝及分泌活性可被葛蘭氏陽性及陰性菌產物所改變。
3. 摘除單側頸上神經節對於大白鼠松果腺巨噬細胞/小神經膠細胞的影響
普遍認為切斷周邊神經在神經受傷處及其遠端會發生一系列生化變化。由單核球分化而來的巨噬細胞侵入受傷區域以因應神經切斷的傷害。正常情況就存在該區域的其他巨噬細胞也會出現此處，在上述的變化過程中扮演重要的角色。本實驗乃探討大白鼠松果腺的巨噬細胞/小神經膠細胞因應單側摘除上頸神經節時的可能反應。在單側摘除上頸神經節後，松果腺巨噬細胞/小神經膠細胞的表面抗原，例如補體第三型接受體、組織相容第二型抗原及溶小體未知功能的蛋白質會全面的增加，特別是表現在松果腺去除神經支配的一側，而且每一種免疫分子呈現獨特的表現型式。有趣的是，顯著的免疫分子改變也發生在松果腺仍有神經支配的一側。然而，免疫分子的改變在同一松果腺切片上有神經支配與去除神經支配處之間沒有顯著差異。摘除上頸神經節造成松果腺內色氨酸水解酵素免疫反應快速減少，而在摘除後兩周恢復正常。這同時也造成松果腺細胞內調節褪黑激素合成的主要酵素--轉乙醯酵素的下降。單側摘除上頸神經節後，卻提高松果腺內類澱粉前驅蛋白表現，後者是神經纖維受傷的一個指標。在單側摘除上頸神經節所引發的種種改變中，松果腺巨噬細胞/小神經膠細胞活化的時程演變似乎與類澱粉前驅蛋白表現變化較一致。因此，結論松果腺巨噬細胞/小神經膠細胞對單側摘除上頸神經節的反應可能與交感神經纖維的退化有關。

關鍵字

松果腺 ； 巨噬細胞/小神經膠細胞

並列摘要

Regional heterogeneity in immunoreactive macrophages/microglia in the rat pineal gland
Using specific macrophage antibodies (OX-42, OX-6, ED-1 and ED-2), this study attempted to examine the distribution of macrophages/microglia in the pineal gland of adult rats. Except for ED-2, all antibodies labeled distinct subpopulations of macrophages/ microglia in the gland; ED-2 labeling was hardly detectable. The quantitative study showed that the pineal macrophages/microglia expressing complement type 3 receptors (OX-42) were more numerous than those expressing the major histocompatibility complex class II antigen (OX-6) or unknown cytoplasmic/lysosomal antigens (ED-1). The pineal macrophages/ microglia were ubiquitous, especially the OX-42 labeled cells which were distributed from the dorsal to the ventral aspect of the gland. The macrophages/microglia labeled with OX-6 or ED-1 were localized mainly in the intermediate portion. Immunolabeled cells were sparsely distributed in the distal portion of the pineal gland. A notable feature was that the OX-6 labeled macrophages/microglia showed a proximal-distal gradient in cell density. Another interesting feature was the occurrence of prominent cell aggregations around the larger blood vessels. These cells were mostly round and exhibited different immunomolecules. Confocal microscopic study with triple immunolabeling further revealed that individual pineal macrophage/microglial cell possessed two or more different antigens (ED-1+/OX-6+, OX-42+/OX-6+ or OX-42+/ED-1+). Remarkably, a large population of them co-expressed ED-1+/OX-6+/ OX-42+. The present results have shown that the expression of immune molecules of pineal macrophages/microglia varies with the topographical distribution of the cells. It is suggested that this may be linked to their immunoregulatory functions in the gland.

Reactive changes of interstitial glia and pinealocytes in the rat pineal gland challenged with cell wall components from from gram-positive and –negative bacteria
Lipopolysaccharide (LPS), the major proinflammatory component of gram-negative bacteria, is well known to induce sepsis and microglial activation in the CNS. On the contrary, the effect of products from gram-positive bacteria especially in areas devoid of blood-brain barrier remains to be explored. In the present study, a panel of antibodies, namely, OX-6, OX-42 and ED-1 was used to study the response of microglia/macrophages in the pineal gland of rats given an intravenous LPS or lipoteichoic acid (LTA). These antibodies recognize MHC class II antigens, complement type 3 receptors and unknown lysosomal proteins in macrophages, respectively. In rats given LPS (50 mg/kg) injection and killed 48h later, the cell density and immunoexpression of OX-6, OX-42 and ED-1 in pineal microglia/macrophages were markedly increased. In rats receiving a high dose (20 mg/kg) of LTA, OX-42 and OX-6, immunoreactivities in pineal microglia/macrophages were also enhanced, but that of ED-1 was not. In addition, both bacterial toxins induced an increase in astrocytic profiles labelled by glial fibrillary acid protein. An interesting feature following LPS or LTA treatment was the lowering effect on serum melatonin, enhanced serotonin immunolabelling and cellular vacuolation as studied by electron microscopy in pinealocytes. The LPS- or LTA-induced vacuoles appeared to originate from the granular endoplasmic reticulum as well as the Golgi saccules. The present results suggest that LPS and LTA could induce immune responses of microglia/macrophages and astroglial activation in the pineal gland. Furthermore, the metabolic and secretory activity of pinealocytes was modified by products from both gram-positive and -negative bacteria.

Responses of the pineal macrophages/microglia following unilateral superior cervical ganglionectomy
It is well known that a series of biochemical changes occurs at the site of the lesion of transected peripheral nerve and distal to it. Macrophages differentiated from monocytes that invade the area in response to transection. Other macrophages normally present in situ also play important roles in these changes. This study was to examine whether pineal macrophages/microglia in rats respond to unilateral superior cervical ganglionectomy (USCGx). After USCGx, pineal macrophages/ microglia showed an overall increase in all antigens examined such as major histocompatibility complex class II antigen (MHC II), complement receptor type 3 (CR3), and lysosomal protein (ED1) of unknown function. Each immunomolecule displayed specific expression pattern during USCGx processes, in particular at the denervated site of pineals. Interestingly, a significant change of immunomolecules also occurred at innervated site of the pineal. However, there was no specific difference between the innervated and denervated site in the same pineal section examined. USCGx caused a rapid decrease of tyrosine hydroxylase immunoreactivity that backed to the normal amount at two weeks after USCGx. The latter also resulted in a decline of N-acetyltransferease, a key enzyme regulating the rate of melatonin synthesis but enhanced β-APP protein expression, which was a general marker for axonal injury. Among USCGx-induced alterations, the chorological changes of pineal macrophages/microglia activation seem to be parallel to those of β-APP protein expression. It was therefore concluded that responses of pineal macrophages/microglia to unilateral superior cervical ganglionectomy may be related to the axonal degeneration of sympathetic fibers.

並列關鍵字

pineal gland ； macrophages/microglia

參考文獻

Adachi A, Natesan AK, Whitfield-Rucker MG et al.( 2002) Functional melatonin receptors and metabolic coupling in cultured chick astrocytes. Glia 39:268-278

Ahlgren S, Li GL, Olsson Y. (1996) Accumulation of beta-amyloid precursor protein and ubiquitin in axons after spinal cord trauma in humans: immunohistochemical observations on autopsy material. Acta Neuropathol (Berl). 92(1):49-55.

Aldskogius H and Kozlova EN. (1998) Central neuron-glial and glial-glial interactions following axon injury. Prog Neurobiol. 55(1):1-26.

Azzi G, Bernaudin JF, Bouchaud C et al. (1990) Permeability of the normal rat brain, spinal cord and dorsal root ganglia microcirculations to immunoglobulins G. Biol Cell 68:31-36.

Bernatowicz A, Kodel U, Frei K et al.(1995) Production of nitrite by primary rat astrocytes in response to pneumococci. J Neuroimmunol 60:53-61.