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 fact, asthma, a chronic inflammatory airway disease, is characterized by an impaired apoptosis of eosinophils, the second most granulocyte type in the blood⁸. Yet the apoptosis of granulocytes and their quick removal by macrophages are two essential steps during the cellular termination of inflammation^9-11.

In the named immune cells two closely related enzymes, namely myeloperoxidase (MPO, neutrophils and monocytes) and eosinophil peroxidase (EPO, eosinophils) can be found^12,13. These heme peroxidases are classically related to the humoral immune response as they two-electronically oxidize (pseudo-)halides to the corresponding hypo(pseudo)halous acids which are known for their bactericidal properties^14-16. Under physiological conditions MPO mainly forms hypochlorous acid (HOCl) and hypothiocyanite (^-OSCN) while the latter and hypobromous acid (HOBr) are formed by EPO^17-19. New results suggest that this (pseudo-)halogenating enzyme activity may also contribute to the regulation of inflammatory responses and to the termination of immune reactions^20,21. In fact, the HOCl production by MPO and derived products were shown to suppress T cell-based adaptive immune responses^22-24.

In order to gain more insights into the immunological role of leukocytes from the innate immune system at chronic inflammatory diseases and to determine the contribution of MPO and EPO to this physiological function we developed a method to quickly enrich leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity in these cells. For erythrocyte depletion we have chosen a standardized method including two-subsequent hypotonic lysis steps with distilled water, which leads to a quick leukocyte enrichment at low material costs. For the subsequent determination of the halogenating MPO and EPO activity the HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) was used^25-27. In contrast to the application of unspecific peroxidase staining methods^28,29, this approach allows the selective detection of the halogenating peroxidase activity, which is often impaired at severe inflammation^30,31.

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 protocol in advance.

1. Label one 15 ml centrifuge tube for hypotonic lysis and one 1.5 ml sample tube for subsequent aminophenyl fluorescein (APF) staining per blood sample.
   1. If the leukocyte enrichment will be performed under sterile conditions, perform this labeling under a flow box.
2. Prepare about 12 ml phosphate-buffered saline (PBS, 10 mM) per sample. Depending on whether the leukocytes shall be isolated under sterile conditions or not, prepare PBS either by using sterile ready solution from a supplier or by dissolving PBS tablets in distilled water. Check the pH value and, if necessary, adjust to pH 7.4 by using small amounts of 0.1 M HCl and NaOH solutions.
   1. Dissolve PBS tablets (see Table of Materials) in 200 ml distilled water to obtain a non-sterile buffer solution sufficient for about 16 samples. If desired, sterilize this solution by sterile filtration.
3. Prepare about 1 ml Hanks balanced salt solution (HBSS) supplemented with Ca²⁺ per sample.
   1. Dissolve 970 mg of HBSS salt in 100 ml (final volume) double-distilled water. Before filling up to the final volume, check the pH value and adjust it to pH 7.4. Due to its low buffer capacity, prepare this buffer freshly each day and perform the pH adjustment with special care by using small amounts of 0.1 M HCl and NaOH solutions.
      NOTE: Sterile solution may be used, depending on the experiment. The solution may be sterilized by sterile filtration.
4. Besides the two buffers, label and prepare a tube (e.g., 50 ml centrifugation tube) or flask (e.g., 250 ml measuring cup) with double-distilled water for the hypotonic lysis procedure in advance. Prepare about 10 ml water per sample.
5. Prepare a container with crushed ice for the APF staining (section 3), which is performed on ice.
6. Prepare aliquots of APF in advance to allow the quick preparation of working solutions used during the staining (step 3.2) and to avoid repeatedly freezing and thawing of the APF solution.
   NOTE: APF is commonly obtained as a 5 mg/ml (11.81 mM) stock solution in methyl acetate.
   1. Freeze aliquots of 10-100 µl in 0.5 ml tubes. For the APF staining a final dye concentration of 10 µM is used (step 3.8).

2. Hypotonic Lysis of the Erythrocytes

NOTE: Perform the hypotonic lysis steps (except the centrifugation steps) under a laminar flow bench to avoid contamination. Perform the whole procedure at room temperature. As the hypotonic lysis is a time critical process, adjust the pipettes for water (2 ml) and PBS (5 ml) addition in advance and open the water and PBS flasks before starting the procedure. Store the PBS solution and the distilled water at room temperature before usage.

1. From each blood sample transfer 100 µl to the appropriate 15 ml centrifugation tube.
   NOTE: In our experiments blood samples usually contain 10 U/ml of heparin in order to avoid coagulation. Yet due to different sources of the sample material the nature and concentration of the anti-coagulant may differ. Its influence on the APF staining should be tested by comparing the results to results obtained from blood samples supplemented with other types or concentrations of anti-coagulant.
2. Add 2 ml distilled water and mix the samples by using a vortex mixer set to a medium level. Incubate the samples for 60 sec, then add 5 ml PBS per sample and mix using the vortex mixer.
   NOTE: Up to about 5 samples can be prepared in parallel. Keep the sample order the same for the addition of water and PBS to achieve comparable incubation times.
3. After regeneration of isotonic conditions by PBS addition (step 2.2) pellet the remaining intact cells in all samples by centrifugation for 6 min at room temperature and 450 x g.
4. Remove the supernatant by pouring it into a table waste bin. Remove as much liquid as possible by inverting the centrifugation tube and tapping the opening onto a paper towel. Ensure that the cell pellet is as dry as possible.
5. Repeat the hypotonic lysis procedure as described before (step 2.2).
   NOTE: In principle this hypotonic lysis procedure (steps 2.3 to 2.5) could be repeated more than once, depending on the purity of the mixed leukocyte fraction to be obtained. After two lysis steps the share of remaining erythrocytes in the obtained mixed cell fraction is about 25%.
6. Pellet the remaining intact cells by centrifugation for 6 min at room temperature and 450 x g. Remove the supernatant (see step 2.4). Add 500 µl HBSS to the pellet and gently dissolve it until a homogenous solution is obtained. Transfer each sample to the accordingly labeled 1.5 ml sample tube.
7. Depending on the experiment directly analyze obtained samples (e.g., via flow cytometry)²⁵, stain with fluorescence-labeled antibodies (for identification of single cell types)³², incubate with cell stimuli (for cell activation experiments)³³ or stored on ice and use later. Depending on the aim of the study, immediately perform subsequent experiments on the mixed leukocyte fraction since granulocytes have a fairly short half-life of about 20 hr.
   NOTE: This also holds for the peroxidase activity staining described below.

3. Halogenating Peroxidase Activity Staining

NOTE: HOCl- and HOBr-production by the blood-derived heme peroxidases MPO and EPO is quantified by using APF, which is oxidized to fluorescein by the named hypohalous acids. Therefore if cell labeling with fluorescence-labeled antibodies is performed in combination with APF staining, avoid fluorophores that interfere with the emission signal of fluorescein.

1. Perform APF staining at 4 °C on ice.
2. For preparing a working solution of APF thaw an appropriate aliquot of the dye (e.g., 10 µl, 11.81 mM) and dilute it with HBSS to exactly 1 mM (e.g., add 108.1 µl buffer to the 10 µl).
   1. For each sample (500 µl cell solution) prepare 5 µl of the described APF working solution. Accordingly, use one 10 µl aliquot of APF (11.81 mM), which yields 118.1 µl APF working solution (1 mM) to prepare about 20 samples. Due to loss during pipetting prepare about 10% more APF working solution than calculated.
3. In order to check the peroxidase specificity of the APF staining, prepare control samples with the heme peroxidase inhibitor 4-aminobenzoic acid hydrazide (4-ABAH)³⁵.
   1. Prepare a 1 M stock solution of 4-ABAH (151.17 g/mol) in dimethyl sulfoxide (DMSO) by diluting 75.6 mg 4-ABAH in 500 µl solvent. Further dilute 4-ABAH 1/10 in HBSS.
4. For the preparation of a H₂O₂ working solution label two 1.5 ml centrifugation tubes with "70 mM H₂O₂" and "7 mM H₂O₂", respectively.
   1. In the tube labelled "70 mM H₂O₂", freshly dilute 10 µl of a 30% stock solution (8.8 mM) of H₂O₂ using 990 µl distilled water to obtain a concentration of about 88 mM. Store on ice and use within 4 hr.
      NOTE: H₂O₂ is typically delivered as a 30% stock solution by the suppliers. When stored at 4 °C, it is stable for about 3 years. Perform dilutions of H₂O₂ in distilled water to avoid decomposition. H₂O₂ dilutions could also be prepared in 0.1 M NaOH solution.
   2. For the determination of the exact H₂O₂ concentration prepare a further 1 to 10 dilution from the first H₂O₂ stock solution (about 88 mM) by adding 100 µl from this solution to 900 µl distilled water in the 1.5 ml tube labeled "7 mM H₂O₂".
   3. Record a spectrum in the near UV range (e.g., 200-300 nm) by using an UV-Vis photospectrometer and use the absorbance at 240 nm (ε₂₄₀ = 34 M^-1 cm^-1) to determine the actual H₂O₂ concentration in the second H₂O₂ stock solution³⁴. Ensure that the reference cuvette contains only distilled water. For the measurement use quartz cuvettes as a spectrum in the UV range is recorded.
   4. From this result, calculate the exact concentrations of both H₂O₂ stock solutions. Adjust the concentration of the first stock solution in the "70 mM H₂O₂^" sample tube to exactly 70 mM by adding an appropriate amount of distilled water. Store the obtained working solution on ice and use within one hour.
5. Add 5 µl of the 4-ABAH working solution (100 mM) to the appropriate samples for a final concentration of 1 mM. Incubate the samples for 15 min at 37 °C in the incubator before APF addition.
6. Add 5 µl APF working solution (1 mM) to each 500 µl sample to obtain a final dye concentration of 10 µM. Mix the samples gently (e.g., by using the vortex mixer on a medium setting) and incubate for 30 min at 37 °C.
7. Add 5 µl H₂O₂ working solution (70 mM) to each 500 µl sample for a final concentration of 700 µM. Gently mix the samples and incubate them for 60 min at 37 °C. Perform appropriate control measurements in parallel.
   NOTE: Control measurements showed that 700 µM H₂O₂ are not toxic for the cells. Nor were any significant cellular responses observed.
   NOTE: The APF staining may also be performed by omitting the addition of H₂O₂, depending on the scientific question. In the absence of hydrogen peroxide only the basal chlorinating MPO and EPO activity is determined while the addition of H₂O₂ allows the detection of the maximal HOCl and HOBr production by the cells. The latter means a significantly higher fluorescent signal.
8. For pelleting the cells, centrifuge the samples for 10 min at room temperature and 400 x g. Thoroughly remove the supernatant without destroying the pellet and add 250 µl HBSS to resuspend the cells.
   NOTE: The samples are now ready for analysis via flow cytometry²⁵.
9. Store the samples in the dark until analysis to avoid bleaching. After excitation at 480-490 nm detect the emission of fluorescein at about 525 nm³⁷.

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	-