Heparan sulfate depletion amplifies TNF-α-induced protein leakage in an in vitro model of protein-losing enteropathy
Heparan sulfate depletion amplifies TNF-α-induced protein leakage in an in vitro model of protein-losing enteropathy
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DOI：
10.1152/ajpgi.00461.2004
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发表时间：
2005-05-01
影响因子：
4.5
通讯作者：
Freeze, HH
中科院分区：
文献类型：
--
作者：
Bode, L;Eklund, EA;Freeze, HH
蛋白丢失性肠病（PLE）是血浆蛋白通过肠道的过度丢失，通常与肠上皮细胞基底外侧表面硫酸乙酰肝素（HS）蛋白聚糖（HSPG）的偶发性丢失相关。PLE发作通常与促炎状态相关。我们研究了HS的丧失或促炎细胞因子TNF-α的治疗是否直接导致蛋白质渗漏，以及两者的结合是否加剧了这一过程。我们建立了第一个PLE的体外模型，并测量了白蛋白/FITC通过在transwell上生长的单层肠HT 29或Caco-2细胞的通量，并通过跨上皮电阻（TER）确定了完整性。通过乙酰肝素酶消化或通过抑制HS合成从基底外侧表面损失HS，使白蛋白通量增加1.58 +/- 0.09倍，并使TER降低23.4 +/-6.5%。TNF-α治疗使白蛋白通量增加4.04 +/- 0.03倍，使TER降低75.7 +/- 4.7%，但仅轻微降低HS含量。HS损失和TNF-α治疗的综合作用不仅是相加的，而且是协同的，白蛋白通量增加7.00 +/- 0.11倍，TER减少83.9 +/- 8.1%。TNF-α与可溶性HS或肝素的共孵育消除了这些协同作用。基底外侧HS的丧失直接导致蛋白质渗漏并放大促炎细胞因子TNF-α的作用。我们的研究结果表明，HSPGs的损失使患者更容易受到PLE，并提供了一个潜在的解释，一些PLE患者对肝素治疗的有利反应。
Protein-losing enteropathy (PLE), the excessive loss of plasma proteins through the intestine, often correlates with the episodic loss of heparan sulfate (HS) proteoglycans (HSPG) from the basolateral surface of intestinal epithelial cells. PLE onset is often associated with a proinflammatory state. We investigated whether loss of HS or treatment with the proinflammatory cytokine TNF-alpha directly causes protein leakage and whether a combination of both exacerbates this process. We established the first in vitro model of PLE and measured the flux of albumin/FITC through a monolayer of intestinal HT29 or Caco-2 cells grown on transwells and determined the integrity by transepithelial electrical resistance (TER). Loss of HS from the basolateral surface, either by heparanase digestion or by inhibition of HS synthesis, increased albumin flux 1.58 +/- 0.09-fold and reduced TER by 23.4 +/- 6.5%. TNF-alpha treatment increased albumin flux 4.04 +/- 0.03-fold and reduced TER by 75.7 +/- 4.7% but only slightly decreased HS content. The combined effects of HS loss and TNF-alpha treatment were not only additive, but synergistic, with a 7.00 +/- 0.11-fold increase in albumin flux and a 83.9 +/- 8.1% reduction of TER. Coincubation of TNF-alpha with soluble HS or heparin abolished these synergistic effects. Loss of basolateral HS directly causes protein leakage and amplifies the effects of the proinflammatory cytokine TNF-alpha. Our findings imply that loss of HSPGs renders patients more susceptible to PLE and offer a potential explanation for the favorable response some PLE patients have to heparin therapy.