BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated.MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex assay.RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-alpha- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation.DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation / Marraccini, Chiara; Merolle, Lucia; Casali, Emanuela; Baricchi, Roberto; Pertinhez, Thelma A. - In: BLOOD TRANSFUSION. - ISSN 2385-2070. - 20:2(2022), pp. 120-126. [10.2450/2020.0208-20]

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation

Casali, Emanuela;Pertinhez, Thelma A
2022-01-01

Abstract

BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated.MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex assay.RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-alpha- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation.DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.
Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation / Marraccini, Chiara; Merolle, Lucia; Casali, Emanuela; Baricchi, Roberto; Pertinhez, Thelma A. - In: BLOOD TRANSFUSION. - ISSN 2385-2070. - 20:2(2022), pp. 120-126. [10.2450/2020.0208-20]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2893102
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