Name: fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples
Uploaded: 2016-07-28T15:00:00
Description: Watch this Scientific Journal Video about Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples at JoVE.com

This work was made possible by funding from the German Federal Ministry of Education and Research (BMBF, 1315883) as well as by the S&#228;chsische Aufbaubank (SAB) project 100116526 from a funding of the European Regional Development Fund (ERDF).

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 o...

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...

Polymorphonuclear leukocytes (PMNs, also called granulocytes) and monocytes represent important cellular components of the innate immune system in the blood1,2. 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 response2-4. 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 arthritis6,7. 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 inflammation9-11.
In the named immune cells two closely related enzymes, namely myeloperoxidase (MPO, neutrophils and monocytes) and eosinophil peroxidase (EPO, eosinophils) can be found12,13. 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 properties14-16. Under physiological conditions MPO mainly forms hypochlorous acid (HOCl) and hypothiocyanite (-OSCN) while the latter and hypobromous acid (HOBr) are formed by EPO17-19. New results suggest that this (pseudo-)halogenating enzyme activity may also contribute to the regulation of inflammatory responses and to the termination of immune reactions20,21. In fact, the HOCl production by MPO and derived products were shown to suppress T cell-based adaptive immune responses22-24.
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 used25-27. In contrast to the application of unspecific peroxidase staining methods28,29, this approach allows the selective detection of the halogenating peroxidase activity, which is often impaired at severe inflammation30,31.

<table border="0" cellpadding="0" cellspacing="0" width="738">
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		<tr height="21">
			<td height="21" style="height:21px;width:213px;">materials/equipment</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">15 ml centrifugation tubes</td>
			<td style="width:203px;">VWR/Corning</td>
			<td style="width:120px;">734-0451</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">1.5 ml sample tubes</td>
			<td style="width:203px;">VWR/Eppendorf</td>
			<td style="width:120px;">211-2130DE</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:213px;">Pipettes for volumes up to 5 ml</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">e.g. 3120000070</td>
			<td style="width:203px;">We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 &#181;l, 10-100&#181;l, 100-1000 &#181;l an 500-5000&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">laminar flow bench</td>
			<td style="width:203px;">Thermo Electron Corperation</td>
			<td style="width:120px;">HeraSafe</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Vortex mixer</td>
			<td style="width:203px;">Bender &#38; Hobein AG</td>
			<td style="width:120px;">Vortex Genie 2</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tabletop centrifuge</td>
			<td style="width:203px;">Kendro Laboratory Products</td>
			<td style="width:120px;">Laborfuge 400R</td>
			<td style="width:203px;">The centrifuge should be able to be used at 450x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Small centrifuge</td>
			<td style="width:203px;">eppendorf</td>
			<td style="width:120px;">5415D</td>
			<td style="width:203px;">The centrifuge should be able to be used at 400x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Incubator</td>
			<td style="width:203px;">Heraeus</td>
			<td style="width:120px;">cytoperm 2</td>
			<td style="width:203px;">Settings: 37 &#176;C, 95% humidity, 5 % CO2 content</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">UV-Vis spectrophotometer</td>
			<td style="width:203px;">Varian</td>
			<td style="width:120px;">Cary 50 bio</td>
			<td style="width:203px;">A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Flow cytometer</td>
			<td style="width:203px;">Becton, Dickinson</td>
			<td style="width:120px;">BD Facs Calibur</td>
			<td style="width:203px;">Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (e.g 488 nm argon laser)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:203px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Chemicals</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate buffered saline (PBS)</td>
			<td style="width:203px;">amresco</td>
			<td style="width:120px;">K812</td>
			<td style="width:203px;">sterile solution, ready to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;"></td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">P4417</td>
			<td style="width:203px;">tablets for solving in 200 ml millipore water</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Hanks balanced salt solution (HBSS) with Ca(2+)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1387</td>
			<td style="width:203px;">970 mg/100 ml, carefully check and adjust the pH value to 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:203px;">Merck Millipore</td>
			<td style="width:120px;">1.09057.1000</td>
			<td style="width:203px;">1M solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Sodium hydroxide</td>
			<td style="width:203px;">Riedel-deHa&#235;n</td>
			<td style="width:120px;">30620</td>
			<td style="width:203px;">Solid pellets. For a 1M solution solve 4 g/100 ml Millipore water</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Aminophenyl fluorescein</td>
			<td style="width:203px;">Cayman</td>
			<td style="width:120px;">10157</td>
			<td style="width:203px;">5 mg/ml solution (11.81 mM) in methyl acetate, aliquotes of e.g. 100 &#181;l should be prepared and stored at -20 &#176;C</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:213px;">Hydrogen peroxide</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1009</td>
			<td style="width:203px;">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</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">4-aminobenzoic acid hydrazide (4-ABAH)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">A41909</td>
			<td style="width:203px;">A first stock solution of 1 M should be prepared in DMSO a second one of 100 mM by 1:10 dilution in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DMSO</td>
			<td style="width:203px;">VWR chemicals</td>
			<td style="width:120px;">23500.26</td>
			<td style="width:203px;">-</td>
		</tr>
	</tbody>
</table>

fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples

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 &#181;l 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.

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...

Watch this Scientific Journal Video about Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples at JoVE.com

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

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.

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 overall goal of this erythrocyte depletion method is to achieve a quick and simple leukoctye-enrichment from a small blood sample. Many advantages of this technique are that it is cheap, reliable, and fast. Moreover, it can be applied to human and non-human blood samples.Before beginning the lysis procedure, adjust the pipette for water administration to two milliliters, and the pipette for PBS to five milliliters. Then place open flasks of water and PBS under a laminar flow hood at room temperature. As the hypotonic lysis is a time-critical process, everything should be prepared previously.Moreover, the lysis proceudre should always be done in the same way, to have compatible results. Next, transfer 100 microliters from each blood sample into a 15 milliliter conical tube. And add two milliliters of distilled water to each sample with vortexing at the medium setting.Rest the cells for 60 seconds, then add five milliliters of PBS. And mix the samples by vortexing. Now, collect the cells by centrifugation, and decant the supernatants.Invert the tubes and tap the openings on a paper towel to remove as much liquid as possible. Then repeat the hypotonic lysis procedure as just demonstrated. After obtaining a dry pellet for the second time, re-suspend each sample in 500 microliters of HBSS, until a homogeneous solution is obtained.And transfer the cells into 1.5 milliliter, micro-centrifuge tubes on ice. To check the peroxidase specificity of the samples after amino phenol fluorescein, or APF staining, first add five microliters of the heme peroxidase inhibitor, 4-ABAH, to the appropriate samples for a 15 minute incubation at 37 degrees Celsius. Next, add five microliters of APF working solution to each tube, and gently vortex the cells on the medium setting.Incubate all of the samples at 37 degrees Celsius for 30 minutes. Then add five microliters of hydrogen peroxide working solution to each sample, and gently vortex the tubes. After 60 minutes at 37 degrees Celsius, centrifuge the cells, and carefully remove the supernatant without disturbing the pellets.Finally, re-suspend the cells in 250 microliters of HBSS, and store the samples in the dark until their analysis, to avoid photo bleaching. Here, a representative example of the leukocyte populations after hypotonic lysis and APF staining as just demonstrated is shown. By plotting cell size versus granularity, the robust depletion of the erythrocytes can be observed, with the red blood cell and debris population accounting for only 22%of the events.Further, the intensity distribution of the APF-derived fluorescence facilitates a clear discrimination of the peroxidase-negative erythrocites and lymphocytes, with a moderate APF oxidation observed, for the peroxidase-positive monocytes and eosinophils, and a robust peroxidase oxidation exhibited by the neutrophils. Once mastered, this technique can be applied to up to 50 blood samples in parallel if it is performed properly. Following this procedure, other methods like antibody staining can be performed to identify the cell subsets of interest.After its development, this technique paved the way for researchers in the field of small animal studies to explore the role of halogenating activity of MPO and EPO in animal models for chronic inflammatory diseases.

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