Dataset Information

Toward the Proteome of the Human Peripheral Blood Eosinophil.

ABSTRACT: Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric focusing (Zoom(®) Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3,141 proteins which had Mascot expectation scores of 10(-3) or less. Of these 426 were unique and non-redundant of which 231 were novel proteins not previously reported to occur in eosinophils. Ingenuity Pathway Analysis showed that some 70% of the non-redundant proteins could be subdivided into categories that are clearly related to currently known eosinophil biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the eosinophil. This dataset of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

SUBMITTER: Straub C

PROVIDER: S-EPMC2967046 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Toward the Proteome of the Human Peripheral Blood Eosinophil.

Straub Christof C Pazdrak Konrad K Young Travis W TW Stafford Susan J SJ Wu Zheng Z Wiktorowicz John E JE Haag Anthony M AM English Robert D RD Soman Kizhake V KV Kurosky Alexander A

Proteomics. Clinical applications 20090101 10

Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric ...[more]

PMID: 21048890

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Project description:Objectives: Eosinophils cultured with all-trans RA (ATRA) and 9-cis RA showed dramatically induced cell survival, and the efficacy of RAs (10-6 M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Using a gene microarray, we studied mRNA profile regulated by RAs. Methods: Peripheral venous blood was obtained from subjects with mild eosinophilia. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll (Pharmacia, Uppsala, Sweden) density gradients. The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified eosinophils were resuspended in RPMI 1640 medium (Gibco, Grand Island, NY) with 10% FCS, and incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 × 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) as per the manufacturer’s instructions. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis. Results: There was little variation between the samples treated by 9-cisRA and ATRA. The comparison between vehicle control and 9-cisRA, or vehicle control and ATRA, identified an increase (greater than twofold change over control) in 19 and 11 transcripts, respectively. The number of decreased transcripts (less than 0.5-fold) resulting fromby 9-cisRA or ATRA exposure wasere 19 and 8 transcripts, respectively. In these transcripts, the expression changes relative to vehicle control were smaller in withthe ATRA compared with 9-cis RA, suggesting that 9-cis RA induced gene transcription more effectively rather than ATRA. Of note, among the ten genes with the biggestmost decrease relative tofrom vehicle control, we found an apoptosis- related gene, caspase-3. Conclusions: We screened several genes involved in RA-induced prolonged eosinophil survival.

Project description:Objectives: Eosinophils cultured with all-trans RA (ATRA) and 9-cis RA showed dramatically induced cell survival, and the efficacy of RAs (10-6 M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Using a gene microarray, we studied mRNA profile regulated by RAs. Methods: Peripheral venous blood was obtained from subjects with mild eosinophilia. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll (Pharmacia, Uppsala, Sweden) density gradients. The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified eosinophils were resuspended in RPMI 1640 medium (Gibco, Grand Island, NY) with 10% FCS, and incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 Ã 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) as per the manufacturerâs instructions. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis. Results: There was little variation between the samples treated by 9-cisRA and ATRA. The comparison between vehicle control and 9-cisRA, or vehicle control and ATRA, identified an increase (greater than twofold change over control) in 19 and 11 transcripts, respectively. The number of decreased transcripts (less than 0.5-fold) resulting fromby 9-cisRA or ATRA exposure wasere 19 and 8 transcripts, respectively. In these transcripts, the expression changes relative to vehicle control were smaller in withthe ATRA compared with 9-cis RA, suggesting that 9-cis RA induced gene transcription more effectively rather than ATRA. Of note, among the ten genes with the biggestmost decrease relative tofrom vehicle control, we found an apoptosis- related gene, caspase-3. Conclusions: We screened several genes involved in RA-induced prolonged eosinophil survival. Purified peripheral blood eosinophils were incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 Ã 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis.

Dataset Information

Toward the Proteome of the Human Peripheral Blood Eosinophil.

Publications

Toward the Proteome of the Human Peripheral Blood Eosinophil.

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