Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research

Veterinary Immunology and Immunopathology 144 (2011) 417–422

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Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research Jella Wauters a,∗ , Thierry Franck b , Frederik Pille c , Ann Martens c , Kristel Demeyere a , Stanislas Sys d , Didier Serteyn b , Frank Gasthuys c , Evelyne Meyer a a

Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylane 133, 9820 Merelbeke, Belgium Department of Large Animal Surgery and Center for Oxygen Research and Development, University of Liège, Bât. B42, Sart Tilman, 4000 Liège, Belgium Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylane 133, 9820 Merelbeke, Belgium d Department of Internal Medicine and Clinical Biology of Large Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylane 133, 9820 Merelbeke, Belgium b c

a r t i c l e

i n f o

Article history: Received 15 July 2011 Received in revised form 9 September 2011 Accepted 26 September 2011 Keywords: Flow cytometry Horse Neutrophils Cellular Surface Myeloperoxidase

a b s t r a c t Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two secondary antibodies and two linking-fluorochromes, respectively. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Myeloperoxidase (MPO) is a specific heme enzyme of the azurophilic granules of polymorphonuclear leukocytes or neutrophils (Borregaard et al., 2007). During neutrophil activation, azurophilic granules release their contents at the site of oxidant generation so that MPO can react with its substrate H2 O2 , generating HOCl and related compounds (Edwards et al., 1988). Myeloperoxidase is considered to be selectively expressed in cells committed to the granulomonocytic differentiation (Borregaard and Cowland, 1997) and its expression is associated with the promyelocytic stage of neutrophil differentiation (Leong et al., 2004). Therefore, MPO has been initially selected as a marker to

∗ Corresponding author. Tel.: +32 92 64 73 56; fax: +32 92 64 74 97. E-mail address: [email protected] (J. Wauters). 0165-2427/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2011.09.009

measure and quantify the granulocyte content in a variety of tissues (Bradley et al., 1982; Mullane et al., 1984, 1985; Xia and Zweier, 1997). Nowadays, MPO is studied intensively due to its involvement in different human pathologies such as atherosclerosis, Alzheimer’s disease and multiple sclerosis (Brennan et al., 2001; Hoy et al., 2002; Gray et al., 2008; Schindhelm et al., 2009). Recently, MPO has also been validated as a diagnostic biomarker in equine gastrointestinal (Grulke et al., 1999, 2008), pulmonary (Art et al., 2006) and orthopedic (Fietz et al., 2008) diseases. In these studies, several options for the extracellular detection of equine MPO were described. These include an MPO-specific enzymatic method based on the conversion of o-dianisidine, a horse-specific enzyme-linked immunosorbent assay (ELISA) technique and a specific immunological extraction followed by enzymatic detection (SIEFED) method (Franck et al., 2005, 2006; Fietz et al., 2008).

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Although these extracellular detection methods can also be applied for intracellular detection after cell lysis, no options are available for the detection of MPO on the single cell level. Nevertheless, such detection might offer an alternative and innovative opportunity of monitoring equine pathologies. Disease is frequently accompanied by inflammation which is characterized by migration and subsequent degranulation of neutrophils. The latter is usually restricted to the phagolysosome. Hence, cellular degranulation is often responsible for an increase in extracellular MPO levels and a concomitant decrease of intracellular MPO is to be expected. Recently, the electrostatic binding of human MPO to the surface of senescing and degranulating neutrophils was reported (Flemmig et al., 2008). Therefore, cellular MPO is defined as the sum of both the intracellular and surface-bound MPO. The aim of the current study was to develop a single cell flow cytometric detection technique based on a polyclonal anti-equine MPO antibody. The cell pre-treatment for surface MPO detection, requiring only fixation, and for cellular MPO detection, requiring additional permeabilization, is first discussed. Subsequently, an indirect labeling protocol comparing two fluorochrome-labeled secondary antibodies is presented. Finally, a direct staining method was developed by labeling the primary antibody with two candidate fluorochromes. Validation parameters such as the variability are additionally included. 2. Materials and methods 2.1. Isolation of equine white blood cells Peripheral blood was collected into EDTA-containing vacutest tubes (Kima srl., Arzergrande, Italy) via jugular venipuncture from clinically healthy horses. The protocol was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (number EC2008/035). Two ml of blood was incubated with 14 ml red blood cell (RBC) lysis buffer (1×) diluted from a 10× solution in 1 l aqua dest of 1.68 M NH4 Cl, 0.1 M KHCO3 , 1 mM tetrasodium EDTA (all from Sigma–Aldrich, St. Louis, MO, USA) during 5 min (min) on ice. A washing step, i.e. centrifugation during 5 min at 250 × g (Beckman GS-6KR centrifuge, GMI Inc., Ramsey, MN, USA) at 4 ◦ C, with 30 ml Dulbecco’s phosphate balanced salt solution (dPBS, Gibco Brl., Scotland, UK) was included to evaluate the effectiveness of lysis. The remaining RBCs were eliminated by another short (3 min) lysing step with 5 ml lysing buffer. Finally, two washing steps with 5 ml dPBS were carried out and the white blood cells were counted (Coulter Counter, Analis, Suarlée, Belgium) and prepared for flow cytometric analysis. 2.2. Cellular pre-treatment Each sample contained 2 × 105 cells and all manipulations were performed in adequate flow cytometer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Surface MPO was detected after fixation of cells with 100 ␮l 4% Formaldehyde (Sigma–Aldrich, Steinheim, Germany) during 10 min at 4 ◦ C. Detection of the cellular MPO after fixation followed by permeabilization was achieved by incubation during 15 min at room temperature with 100 ␮l Fix and 100 ␮l Perm (Fix and Perm, Invitrogen, Molecular Probes, Carlsbad, CA, USA) respectively deprived from light. Each fixation and permeabilization step was followed by three washing steps with 250 ␮l Dulbecco’s modified Eagle’s medium (DMEM, Gibco Brl., Scotland, UK) supplemented with 1% bovine serum albumin (BSA, Merck KGaA, Darmstadt, Germany).

2.3. MPO staining 2.3.1. Indirect labeling protocol In the indirect labeling or two-step staining protocol, detection of the MPO protein is performed by use of an antibody-tandem, consisting of a primary antibody against MPO and a fluorochrome labeled secondary antibody. The rabbit anti-equine MPO antibody of which the manufacturing can be consulted in Franck et al. (2005) and which was formerly successfully implemented in the ELISA and SIEFED techniques, was provided at a concentration of 1.26 mg/ml in dPBS at pH 7. Two goat anti-rabbit secondary antibodies were selected based on their intracellular labeling capacity, namely the Alexa Fluor 488 antibody (F(ab )2 fragment, IgG, Invitrogen, Molecular Probes, Eugene, OR, USA) and the allophycocyanin (APC) antibody (F(ab )2 fragment, IgG, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Optimization experiments, including antibody titrations and specificity controls, i.e. the use of a primary antibody isotype and of the secondary antibody alone, resulted in following final protocol for indirect MPOlabeling. After pre-treatment, the cells were incubated with 100 ␮l blocking medium containing supplemented DMEM enriched with 20% goat serum during 30 min at room temperature, followed by one washing step. The primary antibody was added at 30 ␮g/ml in 100 ␮l supplemented DMEM for 30 min at room temperature. Before addition of the secondary antibody, three washing steps were performed. The secondary antibody, labeled with either Alexa Fluor 488 or APC, was suspended in 100 ␮l supplemented DMEM at 25 ␮g/ml and incubated during 30 min at 4 ◦ C deprived from light. Again three washing steps were carried out prior to resuspending the cells in 400 ␮l of 1× BD CellFIX (Becton, Dickinson and Company, Erembodegem, Belgium) and analysis by a FACSCanto flow cytometer (Becton, Dickinson and Company, Erembodegem, Belgium). To assess the efficacy of the surface and cellular pretreatment reagents, staining patterns of indirectly labeled neutrophils were analyzed by fluorescence microscopy (Leica 291 DM RBE, Leica Microsystems GmbH, Wetzlar, Germany). Nuclear staining was performed by incubating the cells during 20 min at 4 ◦ C with 300 ␮l dPBS containing propidiumiodide (PI, Sigma–Aldrich, St. Louis, MO, USA) at 8 ␮g/ml followed by two washing steps. Stained

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neutrophils were studied after cytocentrifugation (Shandon, Southern Products Ltd., Runcorn, UK). 2.3.2. Direct labeling protocol The direct labeling or one-step staining was achieved by initial linking of the primary antibody to a chosen fluorochrome. For this purpose, the DyLight 649 Microscale Antibody Labeling kit (Pierce Protein Research Products, Thermo Fisher Scientific, Wilmington, DE, USA) and the lightning-link Atto633 conjugation kit (Innova Biosciences Ltd., Cambridge, UK) guidelines were followed. The antibody concentrations and fluorochrome/antibody ratios were assessed using the NanoDrop 1000 V37 (Thermo Fisher Scientific, Wilmington, DE, USA). After pre-treatment of the white blood cells, blocking was performed using 100 ␮l purified polyclonal rabbit IgG (PRABP01, AbD serotec, Oxford, UK) at 2 mg/ml during 30 min at room temperature. The residual blocking antibody was removed by three washing steps. Staining was carried out using 100 ␮l direct labeled primary antibody at 50 ␮g/ml during 30 min at 4 ◦ C in the dark. The optimal working concentration of the two direct labeled primary antibodies was selected after their titration. After washing of the pellet, cells were resupended in BD CellFIX and analyzed as described for the indirect staining protocol. 2.3.3. Variability parameters The variability of the signal emitted by the fluorochromes, involved in either indirect (Alexa Fluor 488/APC) or direct (DyLight 649/Atto633) MPO labeling, was expressed by the mean coefficient of variation (CV) obtained from 4 horses. The CVs were calculated on the level of the horses (between-horse variability) as well as on the level of the neutrophils from one horse (within-horse variability). The variability of the flow cytometric method was monitored by duplicating the indirect labeling with the Alexa Fluor 488 labeled secondary antibody on the level of white blood cell isolation (within-day variability). The instrument’s variability for both indirectly (Alexa Fluor 488) and directly (DyLight 649) labeled neutrophils was evaluated by analyzing the neutrophils’ MPO signal with a time-between sampling of a few days (between-day variability) to a few months (between-months variability; 5 months and 2 months for indirectly and directly labeled neutrophils, respectively). Both the method’s and the instrument’s variability were reported as mean of the CVs obtained for 4 horses. 3. Results and discussion A technique for the detection of MPO on the cellular level is currently lacking in equine diagnostic research. Therefore, a flow cytometric protocol for the analysis of equine surface-bound and cellular MPO was defined in this study. Cellular pre-treatment, including fixation and permeabilization steps, is required for the detection of surface and cellular MPO. Such pre-treatments are reported to adversely affect cell morphology and result in altered flow cytometric scatter patterns (Lanza et al., 1997; Kappelmayer et al., 2000). Additionally, these reagents may influence fluorescence emission by pH-shifting and can

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Table 1 Summary of the variation coefficients reflecting the variability caused by the fluorochromes (within- and between-horses), the flow cytometric method (between-duplicates) and the instrument/reagents (betweendays and between-months) (4 horses). Fluorochrome

Indirect labeling Alexa Fluor 488 APC Direct labeling DyLight 649 Atto633

Variation coefficients Within-horses

Between-horses

23.1 52.7

7.3 11.7

17.0 31.8

3.0 14.3

Method

Variation coefficients Between-duplicates

Indirect labeling Alexa Fluor 488 Instrument/reagents

Indirect labeling Alexa Fluor 488 Direct labeling DyLight 649

7.4 Variation coefficients Between-days

Between-months

2.4

31.7

1.9

9.4

compromise antibody binding by affecting the MPO epitopes (Kalayci et al., 2000; Kingston et al., 2002; Krutzik and Nolan, 2003; Faldyna et al., 2007; Frampton et al., 2007). Particularly for surface detection, membrane-integrity should be guaranteed. Unfortunately, fixation products are well known to cause some degree of permeabilization (Schmid et al., 1991). For these reasons, light scatter and fluorescence characteristics were compared with nontreated control samples (Fig. 1). No major changes in forward nor side scattering were noticed for both pretreatment methods. Moreover, fluorescence microscopy demonstrated clearly the presence of membrane-integrity in formaldehyde fixed neutrophils, while membrane permeabilization was illustrated to be consistently performed in neutrophils treated with Fix and Perm (Fig. 2). After pre-treatment, the cells underwent either indirect or direct MPO staining. Both labeling protocols were initiated by a blocking step to minimize aspecific binding of the primary and secondary antibodies. Nevertheless, the presence of residual aspecificity was further evaluated by including an isotypic control and by monitoring the signal emitted by the inherently MPO-negative lymphocytes (Figs. S1 and S2). These background stainings were subsequently taken into consideration when determining the optimal working concentration of the primary antibody by titration in both the indirect and direct labeling strategies (Fig. S1). Evaluating the two selected secondary antibodies, the lower variability at the within- as well as the between-horse level of the Alexa Fluor 488 fluorochrome compared to the APC fluorochrome was remarkable (Table 1). Using the same criteria, the two selected direct labeling fluorochromes were compared (Table 1). The DyLight 649 fluorochrome proved to be superior compared to the Atto633 fluorochrome. Fig. 3 further illustrates two disjunct population peaks in Alexa

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Fig. 1. Forward and side scatter cytograms (FSC/SSC) (A) and fluorescence channel histograms, corresponding to the Alexa Fluor 488 (B) and DyLight 649 (C) fluorochrome respectively, of representative non-pre-treated isolated white blood cells (control) and isolated indirectly (Alexa Fluor 488) and directly (DyLight 649) stained white blood cells pre-treated with formaldehyde for surface MPO detection and with Fix and Perm for cellular MPO detection.

Fluor 488 and DyLight 649 labeled white blood cells that correspond with the MPO-negative lymphocyte population and the MPO-positive neutrophil population. In contrast, both populations show overlapping histograms after APC and Atto633 labeling. Because of the illustrated restrictions of both the APC and Atto633 fluorochromes, the method and instrument variability was only monitored in Alexa Fluor 488 and Alexa Fluor 488 and DyLight 649 labeled neutrophils, respectively

(Table 1). These data revealed a good repeatability of the flow cytometric technique (Table 1 and Fig. S3). However, measurements showed a rather high variation over time (between-months variability), despite the consistently low between-day variability. The non-aliquoted storage of secondary antibodies at fridge temperature might explain the more pronounced decrease in the fluorescence signal of Alexa Fluor 488 compared to DyLight 649. Indeed, the primary antibodies, whether labeled or not, were stored in

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Fig. 2. Fluorescence microscopic image of indirectly labeled isolated neutrophils treated for surface MPO detection with formaldehyde (A) and for cellular MPO detection with Fix and Perm (B).

Fig. 3. Fluorescence channel histograms of representative (Fix and Perm) pre-treated white blood cell samples stained with the indirect staining methods using either the Alexa Fluor 488 (A) or APC (B) labeled secondary antibodies, and the direct staining methods using either the DyLight 649 (C) or the Atto633 (D) stained primary anti-equine MPO antibodies.

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small aliquots to prevent repeated freeze–thaw cycles at −20 ◦ C and should therefore be less sensitive to degradation. The between-months variation may also be partly attributed to the ageing of the machinery and more specifically the fading of the lasers. Since fair repeatability was demonstrated within one week, a weekly validation of the system with standardized beads and adapted software should allow the comparison of results obtained over longer time periods. The comparison between fluorochromes, both for the indirect and the direct labeling strategy of equine cellular MPO, demonstrates the importance of a validation-based choice before switching to alternatives. Nevertheless, the availability of a plethora of anti-rabbit secondary antibodies and commercial labeling kits for the primary antibody labeling, should encourage other researchers to adapt flow cytometric protocols to their specific experimental needs. In summary, a new validated standard protocol for the flow cytometric detection of both surface and cellular equine MPO in neutrophils by indirect and direct labeling was defined. Those protocols are currently further explored in clinically relevant applications. A first application describes the elegant use of equine MPO in the flow cytometric identification of equine white blood cells, in particular for the difficult discrimination between blood monocytes and neutrophils (Wauters et al., submitted to Vet. Immunol. Immunopathol.). In an ongoing study, the dynamics of equine MPO in infectious and non-infectious arthritis are investigated by comparing extracellular (ELISA and SIEFED), surface-bound and cellular (flow cytometry) MPO in joint fluids of healthy and diseased horses. Acknowledgement Research funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.vetimm.2011.09.009. References Art, T., Franck, T., Lekeux, P., de Moffarts, B., Couetil, L., Becker, M., Kohnen, S., Deby-Dupont, G., Serteyn, D., 2006. Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction. Can. J. Vet. Res. 70, 291–296. Borregaard, N., Cowland, J.B., 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503–3521. Borregaard, N., Sorensen, O.E., Theilgaard-Monch, K., 2007. Neutrophil granules: a library of innate immunity proteins. Trends Immunol. 28, 340–345. Bradley, P.P., Priebat, D.A., Christensen, R.D., Rothstein, G., 1982. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 78, 206–209. Brennan, M.L., Anderson, M.M., Shih, D.M., Qu, X.D., Wang, X., Mehta, A.C., Lim, L.L., Shi, W., Hazen, S.L., Jacob, J.S., Crowley, J.R., Heinecke, J.W., Lusis, A.J., 2001. Increased atherosclerosis in myeloperoxidasedeficient mice. J. Clin. Invest. 107, 419–430.

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