Expression of the Human Resistin in Neutrophil

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ * resistin / Anti-hresistin IgG-Biotin-PE RTqPCR RNA Anti-hresistin IgG-FITC anti-hnl IgG-Biotin-PE Q78 Expression of the Human Resistin in Neutrophil GAO Zhen-Qiu 1 ZHANG Qiao 1 XU Tong 2 TIAN Hui 1 REN Gui-Ping 1 LI Lu 1 LIU Xiang-Yu 1 LI De-Shan 1 * 1 Bio-pharmaceutical Laboratory College of Life Sciences Northeast Agricultural University Harbin China 2 Clinical Laboratory Heilongjiang Province Hospital Harbin China Abstract Resistin is a secreted protein with high level of Cys expressed from mouse white adipose tissue Recently many studies show that expression patterns of human and mouse resistin are different Mouse resistin is primarily expressed in white adipose tissue whereas human resistin is highly expressed in PBMCs / macrophages and bone marrow But the cellular localization of the protein in bone marrow is not clear In the present study in order to identify the resistin-expressing cells in bone marrow we detected the expression of resistin in bone marrow by immunofluorescence The result showed that human resistin was dominantly expressed in mature neutrophils with rod and segment-shaped nuclei and the No No 2008RFQXS017 * Tel deshanli@ 163 com Received July Accepted October Supported by Research Foundation of Heilongjiang Educational Committee No Scientific and Technological Innovative Talents Foundation of Harbin No 2008RFQXS017 * Corresponding author Tel deshanli@ 163 com GAO Zhen-Qiu and ZHANG Qiao contributed equally to this work

2 1 49 expression level was higher in the cells with rod-shaped nuclei than the segment-shaped ones Antihresistin IgG-Biotin-PE stained cells were sorted and analyzed by Wright dye staining The results showed that human resistin was primarily stored in neutrophils and less in eosinophils of peripheral WBCs Moreover human resistin was present in cytoplasm RT-qPCR analysis revealed that monocytes and neutrophils were the primary cells of the human peripheral WBCs In order to confirm the cell type that expressed resistin we detected bone marrow cells and peripheral WBCs expressing both HNL and resistin by two-color fluorescence analysis and the results indicated that the dominant resistin-expressing cells were neutrophils Hence we conclude that the majority of the resistin-expressing cells in human are neutrophils which sheds light on the relationship between inflammation and the expression of human resisitin Key words human resistin bone marrow peripheral blood neutrophil resistin Steppan anti-hresistin IgG-Biotin S-PE R&D FITC IgG anti-hresistin IgG- FITC IgG mouse anti-rabbit 1 IgG-Biotin IgG IgG-Biotin IgG anti-hnl IgG-Biotin CD3-FITC CD19-PE 2 CD14-FITC 3 Patel FITC IgG IgG-FITC 3 hoechest Triton X-100 Tween 20 BSA Sigma PCR 2 Invitrogen 5 -GTCGCCGGCTCCCTAATATTT GTGACGGCGAAGCCTCG-3 5 GAPDH 5 -GCCAAAAGGGTCATCATCTC-3 6 ~ 9 5 -GGCCATCCACAGTCTTCT Leica BD FACS Aria TM Cell Sorter PCR ABI WBCs 1 ml 5 ml ACK 150 mmol / L NH 4 Cl 10 mmol / L KHCO 3 1 mmol / L EDTA 5 min r / min 10 min WBCs a 1 2 PBS ph 7 4 WBCs 2 1 ml PBS 1 5 4% PBS 30 min b 0 1% Tween 20 PBS

3 % Triton X-100 PBS 30 min 3 5 min c 1% BSA PBS 37 1 h d antihresistin IgG-Biotin 1 μg / ml mouse anti-rabbit IgG-Biotin CD19-PE CD14-FITC anti-hnl IgG-Biotin 1 μg / ml min 1 μg S-PE 4 40 min 3 e 3 RNA Oligo dt 15 M- 10 mg / L hoechst MLV cdna 1 15 min Quant SYBR Green PCR Thermal 3 5 min Cycler DiceTM Real Time PCR PCR ABI Anti-hresistin IgG-Biotin-PE PCR 95 2 min s s s 40 WBCs PCR 3 3 a 4% Ct Ct PBS 40 min PBS ΔCt ΔCt r / min 5 min 3 b min PBS 3 c 1% BSA PBS min PBS 3 d 1 μg / ml mouse antihresistin IgG- Biotin mouse anti-rabbit IgG-Biotin 4 40 min PBS μg S-PE 40 min PBS 3 e 500 μl 2% PBS hoechst anti-hresistin IgG- mouse anti-rabbit IgG- Bio-S-PE 3 PBS r / min 5 min Fig 1A3-a b 1 7 Anti-hresistin IgG-FITC anti-hnl IgG- Fig 1A3-c d Biotin-PE Fig 1A3-e f WBCs -A Fig 1A3-g HNL -B resistin -C -D Fig 1A3-h IgG-Biotin A anti-hnl IgG-Biotin B D 1 μg / ml 4 40 min PBS 3 1 μg S-PE 1 μg / ml IgG-FITC A 1 μg S-PE B 1 μg / ml antihresistin IgG-FITC C 1 μg S-PE 1 μg / ml antihresistin IgG-FITC D 40 min IgG-FITC IgG- Bio-S-PE 1 8 RT-qPCR mrna CD3-FITC 3 Trizol ΔCt ΔΔCt 2 - ΔΔCt 1 t 2 Biotin-PE Fig 1B1 anti-rabbit IgG-Biotin-PE Fig 1B2 2 2 WBCs anti-hresistin IgG- PBS μl 2% PBS Biotin S-PE

4 1 51 Fig 1 Immunofluorescence of bone marrow 400 White blood cells were isolated from human bone marrow adjusted to / ml in PBS and divided into two groups control group B and sample A All cells were treated as the following procedure 1 4% paraformaldehyde PFA was added to fix the cells 2 0 5% Triton X-100 in PBS was added to permeabilize the cell membrane 3 The cells were incubated for 5 min with 5% BSA in PBS to block the nonspecific binding 4 Antibody and dye were added to each group respectively The cell nuclei were stained with hoechest33258 in blue and resistin-positive cells were stained with PE in red A1 B1 Chromatin stained with hoechst blue A2 Human resistin-expressing cells stained with anti-hresistin IgG-Biotin-PE red B2 Cells stained with IgG-Biotin-PE A3 Merged figure of A1 and A2 Letter notes a Typical human resistin-expressing cell with big single and circular nuclei b Typical human resistin-expressing cell with big single and long-circular nuclei c Typical human resistin-expressing cell with kidney-shaped nuclei d Typical human resistinexpressing cell with horseshoe-shaped nuclei e Typical human resistin-expressing cell with rod-shaped nuclei f Typical human resistin-expressing cell with segment-shaped nuclei g h Human reisitin negative cells Fig 2 Anti-hresistin IgG-Biotin-PE one-color fluorescence analysis and Wright dye of the human resistin-expressing cells of peripheral WBCs from healthy volunteer The WBCs of the human peripheral blood were isolated from human bone marrow adjusted to / ml in PBS and divided into two groups The control group was stained with the mouse anti-rabbit IgG 1 μg / ml and the sample was stained with the mouse anti-human resistin antibody 1μg / ml After analyzed by flow cytometry the resistin positive cells were sorted and centrifuged 250 μl of the sorted cell was placed on two microscope slides One was analyzed with microscopy The other was open-air dried and stained with Wright Geimsa at RT for 10 min washed with deionized water and analyzed by optical microscope A Histograms show human resistin-expressing cells before sorted grey histogram control sample white histogram human resistin positive stained sample B Histograms show human resistinexpressing cells after sorted by flow cytometry C Observation of the sorted cells with optical microscope 400 D Observation of the sorted cells with fluorescence microscope 400 E The sorted cells were stained with Wright dye 1000 a Rod-shaped neutrophil b Segment-shaped neutrophil c Eosinophil

5 52 27 HNL 74% Fig 2A 69 8% 97 1% Fig 2B 30 HNL HNL Fig 2C Fig 4A 0 1% Fig 4A-Q1 Fig 2D HNL 71 2% Fig 4B- Q1 0 2% Fig 4A- 2 ~ 4 Q4 70 6% Fig 4C-Q4 HNL Fig 2E a b 0 3% Fig 2E c Fig 4A-Q2 HNL 2 3 RT-qPCR 68 1% Fig 4D-Q2 mrna 70% ΔΔCt 1 3 mrna WBCs HNL Fig 3 Fig 3 Analysis of resistin mrna expression in the human peripheral WBCs from healthy volunteer by RTqPCR The lymphocyte monocyte and neutrophil cells of the human peripheral blood were sorted by flow cytometry RNA of these cells were extracted and reverse transcribed to cdna Resistin mrna of monocytes and neutrophils were assessed by RT-qPCR related to GAPDH mrna expression and shown as fold compared to lymphocyte indicated statistically significant difference compared to lymphocyte RT-qPCR HNL IgG-FITC IgG-Biotin-PE Fig 5A HNL 63 7% Fig 5B-Q1 65 4% Fig 5B-Q4 WBCs HNL 0 2% Fig 5A-Q2 HNL 64 8% Fig 5D-Q2 63 5% 30 P < mrna 64 4 % % 10

6 1 53 Fig 4 Anti-hresistin IgG-FITC and anti-hnl IgG-Biotin-PE two-color flow cytometry analysis of the human resistinexpressing cells of human bone marrow The WBCs of the human bone marrow were isolated from human bone marrow adjusted to / ml in PBS and divided into four groups control group HNL-one-color group hresistin one-color group and twocolor group Antibody and dye were added to each group respectively All groups were resuspended in 500 μl 2% V / V formaldehyde fixation buffer for flow cytometry analysis A Cells incubated with IgG-FITC and IgG-Biotin-PE control B Cells stained with anti-hnl IgG-Biotin-PE sample C Cells stained with anti- hresistin IgG-FITC sample D Cells double-stained with anti-hresistin IgG-FITC and anti-hnl IgG-Biotin-PE sample Fig 5 Anti-hresistin IgG-FITC and anti-hnl IgG-Biotin-PE two-color flow cytometry analysis of the human resistinexpressing cells of peripheral WBCs from healthy volunteer The WBCs of the human peripheral blood were isolated from human peripheral blood and adjusted to / ml in PBS A Cells incubated with IgG-FITC and IgG-Biotin-PE control B Cells stained with anti-hnl IgG-Biotin-PE sample C Cells stained with anti-hresistin IgG-FITC sample D Cells double-stained with anti-hresistin IgG-FITC and anti-hnl IgG-Biotin-PE sample mrna 1 / WBCs 3 U937 HL60 11 / Kunnari RT-qPCR Kunnari 12 mrna DELFIA mrna 3

7 54 27 References 3 1 Steppan C M Bailey S T Bhat S et al The hormone resistin links obesity to diabetes J Nature McTernan C L McTernan P G Harte A L et al Resistin central obesity and type 2 diabetes J Lancet Patel L Buckels A C Kinghorn I J et al Resistin is expressed 3 activators J et al cross-sectional and interventional studies in normal insulin- resistant and diabetic subjects J 4 ~ 5 d individuals with acute inflammatory disease J Sch ffler A Ehling A Neumann E et al synovial fluid J JAMA Fagerberg B Fagerlund C Hulthe J HNL levels of leptin in human macrophages and directly regulated by PPAR gamma Biochem Biophys Res Commun Vozarova de Courten B Degawa-Yamauchi M Considine R V High serum resistin is associated with an increase in adiposity but not a worsening of insulin resistance in Pima indians J Diabetes Lee J H Chan J L Yiannakouris N et al Circulating resistin levels are not associated with obesity or insulin resistance in humans and are not regulated by fasting or leptin administration J Clin Endocrinol Metab 6 Stejskal D Adamovskáa S Bartek J et al Resistinconcentrations in persons with type 2 diabetes mellitus and in Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub Kidney Int Karmiris K Koutroubakis I E Xidakis C et al antihresistin IgG-FITC anti-hnl IgG-Biotin-PE resistin functional implications J Yang R Z Huang Q Xu A et al 68 1% 64 8% Biochem Biophys Res Commun % 63 5% PMNs modulated by LPS and TNFα J 50% ~ 70% PMNs 13 Xu SY Carlson M Engstr m et al secondary granules of human neutrophils J Invest Sevéus L Amin K Peterson C G et al 15 IL-6 TNF-α Cell Biol PMNs PMNs neutrophils in atherosclerosis J Adipocytokines in Resistin and GFR J Circulating adiponectin resistin and ghrelin in inflammatory bowel disease J Inflamm Bowel Dis Ghosh S Singh A K Aruna B et al The genomic organization of mouse resistin reveals major differences from the human Gene Comparative studies of resistin expression and phylogenomics in human and mouse J 12 Kunnari A M Savolainen E R Ukkola O H et al The expression of human resistin in different leucocyte lineages is Regul Pept Purification and characterization of a human neutrophil lipocalin HNL from Scand J Clin Lab Human neutrophil lipocalin HNL is a specific granule constituent of the neutrophil granulocytes Studies in bronchial and lung parenchymal tissue and peripheral blood cells J Histochem 15 Zernecke A Bot I Djalali-Talab Y et al Protective role of CXC receptor 4 / CXC ligand 12 unveils the importance of Circ Res