Branes María C, Contreras Jorge E, Sáez Juan C
Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
Med Sci Monit. 2002 Aug;8(8):BR313-23.
During inflammatory responses activated polymorphonuclear cells (PMNs) adhere to each other and form clusters within the vasculature or injured tissues. We hypothesized that conditions that partially mimic the chemical environment of inflammatory foci induce the expression of functional gap junctions (GJs) between cultured PMNs.
MATERIAL/METHODS: Human PMNs were treated with bacterial lipopolysaccharide (LPS), TNF-a, LPS plus medium conditioned by LPS-treated endothelial cells (ECs) or TNF-a plus ECs conditioned medium. Gap junctional communication was evaluated with the dye coupling technique using a permeant and an impermeant GJ fluorescent dye and GJ blockers. The expression of connexins, GJ protein subunits, was evaluated by immunocytochemistry and immunoblotting. Cytochalasin-D and nocodazole were used to evaluate the involvement of cytoskeleton in the induction of dye coupling.
Treatment with LPS or TNF-a induced the formation of PMN aggregates, but cells were not dye coupled. If the latter protocols occurred in medium conditioned by LPS-treated ECs or resting ECs, respectively, intercellular transfer only of the GJ permeant molecule was observed in most clustered cells. Dye coupling was reversibly inhibited by GJ blockers and prevented by cytochalasin-D, a microfilament disrupter, but not by nocodazole, a microtubule disrupter. Treatments that induced dye coupling also induced connexin43 and connexin40, but not connexin32 immunoreactivity. None of these connexins was detected in circulating cells.
EC-derived factor(s) and microfilament integrity are required for dye coupling between LPS- and TNF-a-treated PMNs. GJ formation between PMNs is correlated with the presence of connexins 43 and 40, but not 32 and requires intact microfilaments.
在炎症反应过程中,活化的多形核白细胞(PMN)相互黏附并在血管系统或受损组织内形成细胞簇。我们推测,部分模拟炎症病灶化学环境的条件可诱导培养的PMN之间功能性间隙连接(GJ)的表达。
材料/方法:用人源PMN分别用细菌脂多糖(LPS)、肿瘤坏死因子-α(TNF-α)、LPS加经LPS处理的内皮细胞(EC)条件培养基或TNF-α加EC条件培养基处理。使用可渗透和不可渗透的GJ荧光染料及GJ阻滞剂,通过染料偶联技术评估间隙连接通讯。通过免疫细胞化学和免疫印迹评估连接蛋白(GJ蛋白亚基)的表达。用细胞松弛素-D和诺考达唑评估细胞骨架在染料偶联诱导中的作用。
用LPS或TNF-α处理可诱导PMN聚集体形成,但细胞未发生染料偶联。如果后两种方案分别在经LPS处理的EC或静息EC的条件培养基中进行,则在大多数聚集细胞中仅观察到GJ可渗透分子的细胞间转移。染料偶联可被GJ阻滞剂可逆性抑制,并被微丝破坏剂细胞松弛素-D阻止,但未被微管破坏剂诺考达唑阻止。诱导染料偶联的处理也诱导了连接蛋白43和连接蛋白40,但未诱导连接蛋白32的免疫反应性。在循环细胞中未检测到这些连接蛋白中的任何一种。
LPS和TNF-α处理的PMN之间的染料偶联需要EC衍生因子和微丝完整性。PMN之间GJ的形成与连接蛋白43和40的存在相关,但与连接蛋白32无关,并且需要完整的微丝。