Interleukin 8 (IL-8)

10.9 Interleukin 8 (IL-8)

Interleukin 8 is an inflammatory cytokine that exerts chemoattractant activity for neutrophils. It has activating properties and can be assayed by measuring either neutrophil chemotaxis or activation.

Chemotaxis assay is classically performed in Boyden chambers in which isolated cells to be tested are placed on one side of a chamber (Fig. 10.2) and separated by a membrane from medium containing the chemotactic stimulus. After incubation the membrane is stained and examined microscopically to determine how far the cells have migrated towards the stimulus. Multiwell forms of the chambers are now available for examining several samples. Cellulose ester filters, 150 µm thick, with 3–5-µm pores are suitable for neutrophils.

It is an assay requiring no special apparatus: cells are placed in small wells cut in agarose gels and allowed to migrate under the agarose towards a chemotactic signal placed in an adjacent well.

10.9.1 Agarose migration assay for human neutrophil chemotaxis

MATERIALS AND EQUIPMENT Neutrophils (see Chapter 7) IL-8 or other chemotactic test sample

Medium containing

Chamber

cells

containing chemotactic agent

150-µm polycarbonate membrane

Fig. 10.2 Chamber for testing neutrophil or monocyte chemotaxis.

C H A P T E R 1 0: The cytokines

Plastic tissue culture dishes, 6-cm diameter Agarose Tissue culture medium, 10 × concentrated Eagle’s MEM with HEPES buffer Stainless steel punch 3 mm internal diameter with inside bevel Glutaraldehyde 2.5% w/w in phosphate-buffered saline (PBS) May–Grünwald and Giemsa stains

METHOD Preparation of agarose plate

1 Dissolve 0.2 g agarose in 10 ml distilled water by careful boiling, e.g. in a water bath or microwave oven and cool to 56°C.

2 Warm double-strength medium, 10 ml containing 2 ml 10 × concentrated Eagle’s MEM,

2 ml heat-inactivated fetal bovine serum (FBS) and 6 ml distilled water, to 56°C.

3 Mix the two solutions together.

4 Pour 6 ml portions of agarose/medium solution into plastic culture dishes on a level surface and allow to cool.

5 When set transfer to a refrigerator for 15–30 min to allow the gel to harden.

6 Cut the pattern (Fig. 10.3) with the steel punch.

7 Remove agarose plugs with a Pasteur pipette attached to a vacuum pump.

TECHNICAL NOTES • Heat-inactivated FBS is included to cut down on absorption of cytokine to the agarose and to

improve the gel handling properties. It must be tested to check that is does not have chemo- tactic activity as this would increase the random migration of the neutrophils. It is necessary to inactivate the serum as agarose will activate the alternative complement pathway in fresh serum.

• Care must be taken not to scratch the plate when punching the wells.

Fig. 10.3 Pattern for cell migration under agar.

10.9INTERLEUKIN 8 (IL-8)

• Do not worry at slight lifting of gel when removing plugs as this will aid the neutrophils in mov- ing under the gel. • It is advisable to prepare a template corresponding to the pattern in Fig. 10.3. If this is con- structed in Perspex, about 3 mm thick, mounted on side supports, this will guide the punch to produce vertical wells.

• Any liquid that collects in the wells after punching should be carefully removed with a Pasteur pipette.

Chemotaxis assay

1 Fill the middle well of each set of three with 10 µl neutrophils, 5 × 10 7 /ml.

2 Add 10 µl of control solution, e.g. medium, to inner well of three.

3 Add 10 µl of test solution or standard IL-8 solution to outer well of three.

4 Incubate plate in humidified chamber at 37°C for 2 h.

5 Terminate migration by flooding plates with 2.5% glutaraldehyde for 1 h at room temperature or overnight at 4°C.

6 Carefully remove the agarose with a small spatula taking care not to rotate the gel.

7 Stain cells with May–Grünwald and Giemsa solutions.

8 Determine migration by either: (i) counting the number of cells which have migrated out of the well; or (ii) measuring the distance migrated by the cells.

TECHNICAL NOTES • A positive control for migration may be obtained by using agarose-activated fresh serum.

Simply place fresh human serum in a well 1 h before rest of samples. • Longer incubation times may be used but all materials will need to be prepared and used under sterile conditions.

Calculation of results The migration distance for the positive samples must be corrected for random migration. This is

accomplished by subtracting the distance moved towards the control well. The result may be expressed as corrected distance moved or as a stimulation index (SI):

SI = Corrected distance moved Random distance moved