Neutrophil function tests

7.2 Neutrophil function tests

In vitro assays are available for many of the key neutrophil activities, e.g. chemotaxis, phago- cytosis and microbicidal activity. However the precise relationship between the parameter being measured and its in vivo expression is not always clear. The nitroblue tetrazolium reduction assay described below can be used to measure both phagocytosis (this is the only way in which the dye enters the cell) and one of the metabolic pathways responsible for microbial killing (hexose monophosphate shunt activation).

C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity

7.2.1 Nitroblue tetrazolium (NBT) test

Addition of the yellow NBT dye to plasma results in the formation of a NBT–heparin or NBT–fibrinogen complex, which may be phagocytosed by neutrophils. Normal neutrophils show little incorporation of the complex unless they are ‘stimulated’ to phagocytic activity, e.g. by the addition of endotoxin.

This technique may be used to measure the degree of ‘stimulation’ of untreated cells or their capacity for phagocytosis after stimulation.

Stimulated neutrophils incorporate the dye complex into phagosomes and, after lysosomal fusion, intracellular reduction results in the formation of blue insoluble crystals of formazan. The percentage of phagocytic cells may be determined using a light microscope or, as described below, the total dye reduction may be quantified spectrophotometrically after dioxan extraction.

MATERIALS AND EQUIPMENT Sample of fresh venous blood in heparin (20 IU/ml) Distilled water Phosphate-buffered saline (PBS) Escherichia coli endotoxin (1 mg/ml in PBS) 4m M nitroblue tetrazolium (NBT) dye in PBS containing 340 m M sucrose Dioxan

0.1 M HCl Nylon wool, 100 mg in siliconized Pasteur pipette Water bath at 70°C Spectrophotometer

METHOD

1 Obtain blood sample in heparin (20 IU/ml) by venepuncture. Use a sample for total and differential leucocyte counts (see Figs 7.1 & 7.2). The NBT reduction activity of the sample must be determined within 60 min of venesection.

2 Add 15 µl of endotoxin solution (1 mg/ml in PBS, initial concentration) to 1.5 ml of blood and incubate at 37°C for 10 min.

3 Add 0.1 ml of freshly prepared NBT dye solution and mix gently.

4 After 20 min at 37°C, add blood dropwise to a nylon wool column.

5 Once the sample has entered the column wash twice with 2 ml of PBS and then 2 ml of distilled water. The distilled water will lyse any residual erythrocytes.

6 Add 2 drops of HCl to the column, to stop further reduction of the intracellular dye, and wash with 2 ml of distilled water.

7 Remove the nylon wool with forceps and place in 5 ml dioxan (in a glass container).

8 Incubate at 70°C with occasional vigorous shaking until the nylon wool returns to its original white colour (about 20 min).

9 Centrifuge the dioxan extract to remove any precipitate or nylon fibres (1000 g for 10 min at room temperature).

10 Measure the extinction at 520 nm using a spectrophotometer (use a dioxan standard). The unstimulated control value is obtained by a parallel incubation of untreated blood, i.e. add

15 µl PBS alone at step 2, then assay as steps 3–10.

7.2NEUTROPHIL FUNCTION TESTS

207

Fig. 7.1 Light microscopy of non-lymphoid blood leucocytes. Monocytes are the largest of the blood leucocytes and, in viable cell preparations, are not easily distinguished from cells of the lymphoid lineage. Granulocytes are classified according to the staining reaction of their granules in response to histological dyes, e.g. May–Grünwald/Giemsa staining. (a) Monocytes have a C-shaped nucleus and grey cytoplasm with

a few azurophilic granules on May–Grünwald/Giemsa staining. They are the largest of the blood leucocytes and, on entering the tissues, differentiate into macrophages. (b) Neutrophils are polymorphonuclear cells with neutrophilic cytoplasm. Older cells have a more segmented nucleus and, in general, it is difficult to discern their cytoplasmic granules (except during infection aso-called toxic granulation). These cells account for about 90% of circulating granulocytes; they are about 15 µm in diameter and highly phagocytic. (c) There are very small numbers of basophils in circulation (< 1.0% of blood granulocytes). The characteristic deep blue granules obscure the nucleus and contain histamine avery important in type I anaphylactic-type hypersensitivity reactions. (d) The bilobed nucleus shown in the photograph is typical of a human eosinophil; the bright red granules make identification easy. These cells make up about 2–5% of blood leucocytes, but their frequency increases greatly in parasitic infections and allergic reactions. Typically these cells kill invading organisms by secretion of toxic cationic granules (exocytosis) rather than phagocytosis. Lymphocytes circulate for months, or even years; granulocytes circulate in the blood for about 7 h and thereafter are around in the tissues for only a few days.

TECHNICAL NOTES • All glassware must be siliconized to prevent adherence of phagocytes.

• Both neutrophils and monocytes ingest NBT by phagocytosis. • The conversion factor for the calculation of moles of formazan from extinction coefficient must

be calculated for a sample of each batch of dye, after chemical reduction, as below.

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C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity

Fig. 7.2 Morphology of lymphoid cells under the light microscope. (a) and (b) show the morphology of typical small lymphocytes. The cells have a diameter of about 10 µm and are characterized by a large nucleus : cytoplasm ratio atypical of G0 or ‘resting’ cells. In May–Grünwald/Giemsa staining, the cells have a deeply staining nucleus, condensed chromatin and a thin rim of blue cytoplasm. (c) Large granular lymphocyte. Cells with this morphology have been associated with the majority of natural killer, lymphokine-activated killer and antibody-dependent cell-mediated cytotoxicity activity due to peripheral blood mononuclear cells. Their azurophilic granules contain perforins, which become integrated into the membrane of target cells during cytotoxic killing. These cells have a characteristic density which facilitates their purification from other lymphoid cells by density gradient centrifugation. (d) Reactive lymphocyte from a patient with infectious mononucleosis or glandular fever. Viable cell phenotyping of this cell would have shown it to be a T lymphocyte, in this case reacting to Epstein–Barr virus infection of B lymphocytes. The cell has extensive blue cytoplasm and an ‘open’ chromatin structure, as evidenced by the apparent holes in the nucleus shown in the photograph. T lymphocytes stimulated in vitro with an antigen or phytomitogen have a similar morphology and the same close ‘wrapping’ around the exterior of adjacent erythrocytes. (e) and (f ) Plasma cells showing the typical characteristics of: large cytoplasm containing an eccentric nucleus with a ‘cartwheel’ chromatin structure; deep blue cytoplasm rich in RNA; and a lucid zone near the nucleus, corresponding to the Golgi apparatus. The cytoplasm is frequently vacuolated, presumably due to intracellular antibodies about to be secreted.

7.2NEUTROPHIL FUNCTION TESTS

7.2.2 Determination of conversion factor

MATERIALS AND EQUIPMENT Ascorbic acid 4m M nitroblue tetrazolium (NBT) in distilled water containing 340 m M sucrose

0.1 M sodium hydroxide containing 24 m M sodium bicarbonate Distilled water Dioxan Waterbath at 70°C Spectrophotometer

METHOD

1 Add 150 µmol ascorbic acid to 0.2 ml of NBT solution and mix.

2 Add 2 ml of 0.1 M sodium hydroxide containing 24 m M sodium bicarbonate.

3 Incubate for 10 min at room temperature and add 5 ml distilled water.

4 Centrifuge at 1000 g for 15 min at room temperature.

5 Wash once in water by centrifugation (1000 g for 15 min at room temperature). Remove the supernatant and resuspend the blue insoluble formazan precipitate in 10 ml dioxan.

6 Dilute 1 ml of the suspension with 9 ml dioxan and incubate at 70°C for 20 min.

7 Cool to room temperature, and measure the extinction at 520 nm using a spectrophotometer (use a dioxan blank).

8 Calculate the conversion factor from the extinction value. As a rough guide, the conversion factor should be approximately 1 extinction unit (E 520 nm ) = 40 nmol of formazan.

7.2.3 Calculation of NBT uptake by phagocytes

1 Using the conversion factor determined above, determine the number of moles of formazan extracted from the untreated and endotoxin-stimulated blood.

2 Calculate the number of potential phagocytes used per assay (the percentage of the absolute count due to neutrophils and monocytes).

3 Express results as moles of formazan per phagocyte. Normal range: untreated blood, 0.92–

3.62 fmol/phagocyte; endotoxin-stimulated blood, 2.52–4.90 fmol/phagocyte.

7.2.4 Neutrophil chemotaxis

The assay for neutrophil chemotaxis is a good guide to neutrophil function and is discussed in Section 10.9.

7.2.5 Continuous-flow cytometry for phagocytosis

A fluorescent dye is used which allows the use of the flow cytometer to carry out the measure- ments. 2′7′-dichlorofluorescein diacetate was the first dye to be used for this purpose (Bass et al. 1983; Vuorte et al. 1996) and gives a green fluorescent product on oxidation following the oxidative burst within the neutrophils.

C H A P T E R 7: Phagocytosis, complement and antibody-dependent cytotoxicity

MATERIALS 2′7′-dichlorofluorescein diacetate (DCF-DA) 20 m M in ethanol; store below 0°C in the dark Heparinized whole blood Dulbecco’s phosphate-buffered saline (PBS) (calcium- and magnesium-free), containing 5 m M

glucose, 1% gelatin, 5 m M sodium azide and DCF-DA at 250 m M final concentration EDTA 350 ng/ml phorbol myristic acetate (PMA) in ethanol

METHOD

1 Dilute 100 µl heparinized whole blood or cell suspension with Dulbecco’s PBS containing DCF-DA.

2 Mix for 20 min at 37°C in a shaking water bath.

3 Add 0.5 ml EDTA and 350 ng/ml phorbol myristate acetate (PMA) in ethanol.

4 Treat with ice-cold distilled water for 20 s.

5 Centrifuge and resuspend in PBS–gelatin–glucose without DCF-DA.

6 Examine by flow cytometry.

TECHNICAL NOTES • Azide inhibits enzymatic decomposition of H 2 O 2 by cellular catalase and myeloperoxidase and does not impair H 2 O 2 production. • Ethanol depresses DCF fluorescence and so should be kept to a minimum concentration. • Neutrophils produce between 50 and 70 nmol of superoxide/min 10 7 neutrophils in response to PMA. • Other fluorescent indicators such as dihydrorhodamine 123 and hydroethidine have also proved useful.