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Mitochondrial superoxide generation enhances P2X7R-mediated loss of cell surface CD62L on naive human CD4+ T lymphocytes.

ABSTRACT: Migration of naive CD4(+) T lymphocytes into lymphoid tissue is essential for their activation and subsequent roles in adaptive immunity. The adhesion molecule L-selectin (CD62L), critical for this process, is highly expressed on naive CD4(+) T lymphocytes and is downregulated upon T lymphocyte activation. We demonstrate protein expression of P2X7R on naive CD4(+) T lymphocytes and show functional channel activity in whole-cell patch clamp recordings. CD62L downregulation occurs rapidly in response to extracellular ATP, a process that is blocked by selective antagonists of P2X7R. This loss of surface CD62L expression was not associated with externalization of phosphatidylserine. While investigating the mechanisms for this process, we revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R-dependent CD62L downregulation by increasing ATP potency. Enhanced superoxide generation in the mitochondria of rotenone- and antimycin A-treated cells was observed and may contribute to the enhanced sensitivity of P2X7R to ATP. P2X7R-dependent exposure of phosphatidylserine was also revealed by preincubation with mitochondrial uncouplers prior to ATP treatment. This may present a novel mechanism whereby P2X7R-dependent phosphatidylserine exposure occurs only when cells have enhanced mitochondrial reactive oxygen species generation. The clearance of apoptotic cells may therefore be enhanced by this mechanism which requires functional P2X7R expression.

SUBMITTER: Foster JG

PROVIDER: S-EPMC3672848 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Mitochondrial superoxide generation enhances P2X7R-mediated loss of cell surface CD62L on naive human CD4+ T lymphocytes.

Foster John G JG Carter Edward E Kilty Iain I MacKenzie Amanda B AB Ward Stephen G SG

Journal of immunology (Baltimore, Md. : 1950) 20130114 4

Migration of naive CD4(+) T lymphocytes into lymphoid tissue is essential for their activation and subsequent roles in adaptive immunity. The adhesion molecule L-selectin (CD62L), critical for this process, is highly expressed on naive CD4(+) T lymphocytes and is downregulated upon T lymphocyte activation. We demonstrate protein expression of P2X7R on naive CD4(+) T lymphocytes and show functional channel activity in whole-cell patch clamp recordings. CD62L downregulation occurs rapidly in respo ...[more]

PMID: 23319734

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Project description:BACKGROUND:Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. AIM:The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes. METHODS:Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFN?, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. RESULTS:Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFN?, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFN?, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4+ lymphocytes. CONCLUSION:For the first time, the immunomodulatory capacity of CHF5633 on CD4+ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4+ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.

Dataset Information

Mitochondrial superoxide generation enhances P2X7R-mediated loss of cell surface CD62L on naive human CD4+ T lymphocytes.

Publications

Mitochondrial superoxide generation enhances P2X7R-mediated loss of cell surface CD62L on naive human CD4+ T lymphocytes.

Similar Datasets

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