Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

Summary

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Abstract

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 µl makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.

Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.

Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

Introduction

Polymorphonuclear leukocytes (PMNs, also called granulocytes) and monocytes represent important cellular components of the innate immune system in the blood^1,2. They contribute to the primary defense against pathogens as well as to the activation of the acquired immune system and the initiation of a systemic inflammatory response^2-4. Yet especially neutrophils, the most abundant type of granulocytes, and monocytes also significantly contribute to the regulation and termination of acute inflammatory events⁵. Therefore these cells may also play an important role in chronic inflammatory diseases like rheumatoid arthritis^6,7. In....

Protocol

All human blood samples were obtained from healthy volunteers, and the applied leukocyte enrichment protocol follows the guidelines of ethics commission of the Medical Faculty of the University of Leipzig. The experiments with rat blood were approved by the responsible local ethical committee (Landesdirektion Sachsen, Referat 24), according to the German guidelines on animal care and use.

1. Experimental Setup

NOTE: As the hypotonic lysis procedure for the depletion of erythrocytes from the blood samples is a time-critical procedure, prepare all necessary equipment (e.g., buffers) for this part of the prot....

Results

As reported previously the method described above turned out to be applicable both to human and to non-human material³². Moreover as shown for mice with asthmatic symptoms the APF staining may be a suitable tool to detect differences in the systemic pro-inflammatory status. Therefore in a subsequent study we used this protocol to repeatedly evaluate the halogenating activity of MPO (and EPO) in female Dark Agouti rats with pristane-induced arthritis (PIA). A representa.......

Discussion

As neutrophils are the most abundant leukocytes in human blood the isolation of peroxidase-positive cells often only focuses on these cells and includes a separation of neutrophils from other leukocytes by density gradient centrifugation³⁸. Yet as neutrophils are much less abundant in murine blood samples³⁹ for the latter more complicated methods have to be used⁴⁰. Moreover both methods also lead to the removal of peroxidase-positive monocytes from the samples and, due to the need of larg.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials/Equipment
15 ml centrifugation tubes	VWR/Corning	734-0451	-
1.5 ml sample tubes	VWR/Eppendorf	211-2130DE	-
Pipettes for volumes up to 5 ml	Eppendorf	e.g., 3120000070	We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 µl, 10-100 µl, 100-1,000 µl an 500-5,000 µl
laminar flow bench	Thermo Electron Corperation	HeraSafe	-
Vortex mixer	Bender & Hobein AG	Vortex Genie 2	-
Tabletop centrifuge	Kendro Laboratory Products	Laborfuge 400R	The centrifuge should be able to be used at 450 x g
Small centrifuge	Eppendorf	5415D	The centrifuge should be able to be used at 400 x g
Incubator	Heraeus	cytoperm 2	Settings: 37 °C, 95% humidity, 5% CO2 content
UV-Vis spectrophotometer	Varian	Cary 50 bio	A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied
Flow cytometer	Becton, Dickinson	BD Facs Calibur	Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (e.g., 488 nm argon laser)
Name	Company	Catalog Number	Comments
Chemicals
Phosphate buffered saline (PBS)	amresco	K812	sterile solution, ready to use
Phosphate buffered saline (PBS)	Sigma-Aldrich	P4417	tablets for solving in 200 ml millipore water
Hanks balanced salt solution (HBSS) with Ca2+	Sigma-Aldrich	H1387	970 mg/100 ml, carefully check and adjust the pH value to 7.4
Hydrochloric acid	Merck Millipore	1.09057.1000	1 M solution
Sodium hydroxide	Riedel-deHaën	30620	Solid pellets. For a 1 M solution solve 4 g/100 ml Millipore water
Aminophenyl fluorescein	Cayman	10157	5 mg/ml solution (11.81 mM) in methyl acetate, aliquots (e.g. 100 µl) should be prepared and stored at -20 °C
Hydrogen peroxide	Sigma-Aldrich	H1009	This 30% stock solution corresponds to a concentration of about 8.8 M. Further dilutions have to be freshly prepared in distilled water immediately prior to use and quantified by absorbance measurements
4-aminobenzoic acid hydrazide (4-ABAH)	Sigma-Aldrich	A41909	A first stock solution of 1 M should be prepared in DMSO a second one of 100 mM by 1:10 dilution in HBSS
DMSO	VWR chemicals	23500.26	-