A flow cytometric method for the detection of intracellular basic proteins in unseparated peripheral blood and bone marrow eosinophils

Eosinophils and their basic proteins play a major role in allergic disease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for the detection of intracellular eosinophil
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  JOURN L OF IMMUNOOGIC L METHODS zyxwvutsrqponmlkji ELSEVIER Journal of Immunological Methods 190 ( 1996) 245-254 A flow cytometric method for the detection of intracellular basic proteins in unseparated peripheral blood and bone marrow eosinophils zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM N rug a**, .M. Thurau a, P. Lackie d, J. Baier b, G. Schultze-Werninghaus ‘, C.H.L. Rieger a, U. Schauer a a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA epurtment zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB f aediatrics. Ruhr University, Bochum, Germany b Department of nternal Medicine (Division of ncology). Ruhr University, Bochum. Germany ’ Department of Internal Medicine (Division of Pneumology and Allergology). Ruhr Uniuersity, Bochum, Germany d mmunopharmacology Group. Southampton General Hospital. Southampton, UK Received 28 December 1994; revised 29 September 1995; accepted 21 November 1995 zyxwvutsrqponmlkjihgfedcbaZ Abstract Eosinophils and their basic proteins play a major role in allergic disease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for the detection of intracellular eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in unseparated clinical samples. After fixation with parabenzoquinone and permeabilization with n-octyl-P-c-glucopyranoside, the detection of intracellularly stored proteins was achieved using of monoclonal antibodies against ECP (EGl, EG2) and EPO in combination with an FITC-labeled second step antibody. Confocal microscopy was used to demonstrate the intracellular srcin of the fluorescent signal. Fixation with parabenzoquinone was superior to a previously described protocol using paraformaldehyde, since it reduces non-specific binding of FITC to the basic proteins in eosinophils. Fixation and permeabilization do not alter the light scatter characteristics of eosinophils in contrast to other leukocytes and thus permit gating on eosinophils without prior purification. Furthermore, the procedure does not alter the detection of cell surface antigens on eosinophils and simultaneous measurements of surface antigens and intracellular proteins is possible. We have used different clinical samples (peripheral blood, bone marrow cells) to demonstrate differences in the expression of ECP and EPO. We conclude that the detection of intracellular eosinophil proteins by flow cytometry is a rapid, easy and semiquantitative procedure which may be used to study their expression in diseases where eosinophils are involved. Keyord.5: Flow cytometry; Eosinophil granulocyte; Intracellular immunofluorescence; Parabenzoquinone Abbreviations: ECP, eosinophi cationic protein; EDTA, ethylenediaminetetraacetic acid; EPO, eosinophil peroxidase; zyxwvutsrqponmlkjihgfe PX eosinophi] protein X; FACS. fluorescence activated cell sorter. FITC, fluorescein isothiocyanate; Fl, fluorescence; FSC, forward scatter; LTB~, leuliotriene B4; MBP, major basic protein; MFI. mean fluorescence intensity; OG, n-octyl-pr>glucopyranoside; PBQ, parabenzquinone; PBS, phosphate buffered saline; PE. phycoerythrin; PFA, paraformaldehyde; SSC, side scatter. * Corresponding author. At: Department of Respiratory Medicine, Medical School of Hannover, 30623 Hannover, Germany. Tel.: + 49-5 I I-532-3530; Fax: + 49-51 l-532-3353. 0022.1759/96/.$I5.00 0 1996 Elsevier Science B.V. All rights reserved SSDl 0023 1759(95)00272-3  246 N. Krug et ul./Jourd of Immunological Metho 190 119961245-254 zyxwvutsrqponmlkjihgfedcbaZYXWV 1. Introduction 2. Material and methods Eosinophils play a major pathophysiological role in allergic disease. The release of their basic proteins (ECP, MBP, EPO, EPX) causes tissue damage and this contributes to chronic allergic inflammation (Weller, 1991). Consequently, methods to measure their expression in eosinophils are of particular inter- est in order to monitor the ongoing disease process. 2.1. Processing of cells and intracellular staining While the expression of basic proteins in eosinophils is usually assessed by immunohisto- chemistry and the concentrations of the released proteins in various tissue fluids determined by RIA or ELISA, a new flow cytometric method of intracel- lular ECP detection has recently been introduced (Hed and Hallden, 1993): After cell fixation with paraformaldehyde (PFA) and permeabilization with n-octyl-/?-D-glucopyranoside (OG) monoclonal anti- bodies against ECP and an FITC-labeled second step antibody were used to determine the intracellular ECP expression of eosinophils in whole blood sam- ples. However, autofluorescence of eosinophils (Weil and Chused, 1981; Mayeno et al., 1992) and espe- cially nonspecific binding of negatively charged flu- orescein molecules (e.g. FITC) to eosinophilic cationic proteins (Floyd et al., 1983; Detlefs et al., 1987) are known to give false positive results when analysing eosinophils. Several attempts have been made to prevent non-specific binding using immuno- histochemical procedures and in in situ hybridization (Johnston and Bienenstock, 1974; Kingston and Pearson, 1981; Fredens et al., 1986; Patterson et al., 1989). However, this problem has not been ad- dressed in the flow cytometric analysis of eosinophils. In this report we show that the use of the fixative parabenzoquinone (PBQ) instead of PFA is able to reduce the nonspecific binding of FITC and FITC- labeled antibodies to permeabilized eosinophils. The fixation procedure does not alter the detection of typical eosinophil surface markers and light scatter characteristics of eosinophils are well preserved. We demonstrate that this method is suitable for determin- ing the expression of ECP and EPO in different unseparated clinical samples. Clinical samples (peripheral blood or bone mar- row cells) were obtained from normal subjects (II = 24), mild asthmatic patients (n = 2 1) and patients with idiopathic hypereosinophilia (n = 4) and col- lected into EDTA-containing tubes. Informed con- sent was obtained from all participating subjects. Erythrocytes were lysed by 10 min incubation (room temperature) of 150 ~1 blood or bone marrow with 2 ml of lysing solution (Becton Dickinson, Heidelberg, Germany). After centrifugation at 300 X g for 5 min leukocytes were washed in PBS. Cells were fixed either in 0.4% parabenzoquinone (PBQ) (Sigma, St. Louis, USA) in 0.01 M PBS or in 4% paraformalde- hyde (PFA) (Riedel-deHaen, Seelze, Germany) in 0.15 M PBS for 10 min. After a further wash in PBS cells were permeabilized by incubation in 0.74% n-octyl-P-D-glucopyranoside (OG) (Sigma) in PBS for 6 min. Permeabilized cells were washed in PBS and monoclonal antibodies against ECP (EGl and EG2, mouse IgGl, Pharmacia Diagnostics AB, Upp- sala, Sweden), EPO (mouse IgG2a, Oncogene Sci- ence. Uniondale, USA), vimentin (mouse IgGl, Sigma) and isotype-matched nonspecific control an tibodies (Coulter, Krefeld, Germany) were added in optimal concentrations. After 30 min of incubation at 4”C, cells were washed in PBS and incubated with FITC-labeled goat anti-mouse IgGl or IgG2a (Southern Biotechnology Associates, Birmingham, USA) as a secondary antibody for 30 min. In some experiments FITC (Molecular Probes, Eugene, USA) was added to the permeabilized cells. After a final wash in PBS, the cells were resuspended in PBS and kept at 4°C in the dark. 2.2. Staining of cell surface antigens and double staining of surface and intracellular antigens To detect the influence of fixation and permeabi- lization on eosinophil surface marker expression EDTA blood was incubated with labeled antibodies (anti-CD1 la-FITC, anti-CD1 lb-PE, anti-CDl8-FITC (all Becton Dickinson) anti-CD9-FITC (Dako. Den-  N. Krug et al./Journal of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF mmunological Methods 190 (1996) 245-254 247 mark), anti-CD 16-Tricolor (Medac, Hamburg, Ger- many) and anti-CD49d-FITC (Endogen, Boston, USA)) for 30 min at 4°C. Erythrocytes were lysed (Lysing solution, Becton Dickinson), leukocytes washed in PBS and cells were either directly anal- ysed by flow cytometry or treated with PBQ and OG as described above and then analysed. In a few experiments surface antigens were la- beled with anti-CD1 lb-PE (mouse IgG2a, Becton Dickinson) or anti-CD49d (mouse IgGl, Dianova, Hamburg, Germany) + goat anti-mouse IgGl-Tri- color (Medac). Erythrocytes were lysed, cells were treated with PBQ and OG and intracellular staining was performed with EG2 + goat anti-mouse IgGl- FITC or anti-EPO + goat anti-mouse IgG2aFITC respectively. 2.3. Flow cytometric analysis A FACScan flow cytometer (Becton Dickinson, Mountain View, USA) equipped with filter settings for FITC (530 nm) @l-l), PE (585 nm) (Fl-2) and Tricolor (Medac, Hamburg, Germany) emitting in the deep red (> 650 nm) (Fl-3) was used. 15 000 cells were computed in list mode and analysed using Lysis II software (Becton Dickinson). To identify eosinophils in non-permeabilized leukocyte samples a gate was set on granulocytes with high granularity on the forward (FCS) and side (SSC) scatter plot, and nonstaining cells for CD16- Tricolor (Medac, Hamburg) falling within the gated area were identified by the detection of Fl-3. The surface markers on these eosinophils were then anal- ysed by detection of Fl-1 and Fl-2 using double gates on Fl-3-negative cells and granulocytes with high granularity. Cells staining positively for the surface markers were expressed as a percentage of eosinophils. This method for identifying eosinophils in unseparated blood samples has been recently eval- uated and a very good correlation (r = 0.92, p < 0.0001) between the percentage of eosinophils de- tected by conventional eosinophil staining methods and the percentage of CDl6- granulocytes in the granulocyte population has been demonstrated (Thurau et al., 1996). In permeabilized leukocyte samples eosinophils were easily identified by their preserved scatter properties in the FCS-SSC plot (see Section 3). For surface marker detection a gate was set on this cell population and surface markers were then analysed by detection of Fl-1 and Fl-2 positive cells falling in this gate. Positive detection was recorded as percentage expression by eosinophils. For intracellular detection of basic proteins in permeabilized eosinophils a gate was set on cells with preserved scatter properties in the FCS-SSC plot (see Section 3). The expression of antigen in eosinophils was then quantified as mean fluores- cence intensity (MFI) of Fl-1. The MFI with control mAb was substracted from the MFI for each specific antibody. This mode of quantification was chosen instead of giving percentages of positive cells, since almost all eosinophils in peripheral blood or bone marrow stained positively for ECP and EPO. In order to ensure standardized conditions, FITC-fluo- rescent microspheres with a definite fluorescence intensity (Fluoresbrite, Polyscience, Eppelheim, Ger- many) were used before each experiment to calibrate the flow cytometer. 2.4. Eosinophil puri’cation To demonstrate that the cell population with pre- served scatter properties were in fact eosinophils, eosinophils were purified from the peripheral blood of patients with hypereosinophilia using discontinu- ous Percoll density gradients as described by Klo- progge et al. (1989) (purity 2 90%). For the demon- stration of intracellular staining by confocal mi- croscopy eosinophils were further purified (> 99%) using the magnetic cell separation system with nega- tive selection of CD16- granulocytes described by Hansel et al. (1991b). 2.5. Confocal microscopy To confirm and visualize the intracellular stain- ing of eosinophils confocal microscopy was em- ployed. Purified eosinophils were treated with PBQ and OG as described above and cells were stained with EG2 followed by the FITC-labeled anti-mouse antibody. Propidium iodide (25 pg/ml in PBS, Sigma) was used as a nuclear counter stain immedi- ately prior to microscopy. Cells suspended in PBS were mounted in a chamber on a microscope slide for examination with a Leica TCS 4D confocal microscope equipped with a krypton/argon laser  248 N. Krug et al./Journul oj’Immunologica1 Methods 190 1996) 245-254 zyxwvutsrqponmlkjihgfedcbaZY before fixation permeabilization after fixation permeabilization FSC zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA SC w, , d’. “.’ ” dO 4lhl sdo sbo FSC FSC Fig. I Panels on the left side show forward (FSC) and side scatter (SSC) plots of peripheral blood samples from A) a normal SubJect (3% eosinophils) and (B) a patient with hypereosinophilia (27% eosinophils) and (C) purified eosinophils (90% with IO% contamining neutrophils). The right side shows the corresponding dot plots after fixation (parabenzoquinone) and permeabilization (n-octyl-Pogluco- pyranoside), demonstrating unchanged scatter characteristics of the eosinophils after treatment. lhis procedure provides clear separation of eosinophils from other leukocytes.  operating at 488 nm and 568 nm. Dual channel (numeral aperture 1.4) was used to scan adjacent imaging with two photomultipliers used an FITC horizontal sections through selected cells. Images band pass filter and a long pass (590 nm) filter for were generated using the integral computer and dis- propidium iodide. A 63 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA   oil immersion objective play software. IgGl -Control + goat-anti- mouse-FITC EG2 f goat-anti- mouse-FITC FITC Paraformaldehyde e FSC FL1 Fig. 7. Pcr~pheral blood Isulocytes were fixed with parafommaldehydc left) or parabcnqu~none right) and pctrmcahthzed by rt-octyl-P-t> glucopyranosidr. .A scatter characteristics of rosmophtls were better preserved by fixation with parabenLvquinune_ R pnrahcnt.oquinonc but not parafotmaldehyde) prevented nonspecific bmding of FITC-labeled control antibodies to eosinophils. C. the slgnal/bachground r3tio of specific staining with FITC-labeled anti-ECP was increased with parabenzoqumone. D: nonspecIfic bmdinp of unconjugated FITC to Ieuhocytes was prevented by parabenzoquinonc. Parabenzoquinone 17 FL1 w * zyxwvutsrqponmlkji FL1
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