Interleukin 6 (IL-6)

10.7 Interleukin 6 (IL-6)

Interleukin 6 is also known as B-cell differentiation factor 2 (BCDF-2) or interferon β2 (IFN-β2) and can be detected in the circulation following Gram-negative bacterial infections. It has several demonstrable activities in vitro; for example, when assayed for its ability to induce B-cell differen- tiation, it increases surface IgM expression and secretion of µ chains.

Il-6 enhances cytotoxic T lymphocytes and is a potent growth factor for myeloid cells. It also stimulates hepatocytes to produce acute-phase reactant proteins and therefore has a role in inflammation; it is also said to have nerve growth factor-like activity.

IL-6 can be produced in vitro by stimulating human fibroblasts with polyribonucleotides or cyclohexamide plus actinomycin D and is constitutively produced by the leukaemia virus- transformed human T-cell line TCL-Na1. IL-6 can also stimulate hybridoma cells used in mono- clonal antibody production (see also Chapter 2).

10.7.1 ELISA assay for immunoglobulin secretion by an IL-6-responsive cell line

MATERIALS AND EQUIPMENT IL-6-responsive cell line, e.g. SKW6-CL4 or CESS Tissue culture medium Fetal bovine serum (FBS) 96-well microtitre plates Reagents for anti-IgM (using SKW6-CL4) or anti-IgG (CESS) ELISA (see Section 5.4.3) ELISA reader

Preparation in advance Subculture the indicator cell of choice 24–48 h before the assay to ensure that it is in log-phase

growth.

METHOD

1 Harvest the indicator cells, wash twice by centrifugation and resuspend in tissue culture medium containing 10% FBS. Adjust the cell concentration to 4 × 10 4 /ml (SKW6-CL4) or

6 × 10 4 /ml (CESS).

2 Dispense 100 µl aliquots of either cell line into each well of a microtitre plate.

3 Prepare a range of dilutions of the supernatant under test and add 100 µl aliquots to wells in triplicate.

Continued

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

4 Incubate the plates in a humidified atmosphere of 5% CO 2 in air at 37°C for 72 h.

5 Transfer 100 µl aliquots of culture supernatant from each well to the corresponding well in

a second microtitre plate.

6 Assay these supernatants using an anti-IgM or anti-IgG ELISA as appropriate for the cell line used (see Section 5.4.3).

Assessment of results The amount of immunoglobulin secreted is proportional to the IL-6 concentration in the sample

under test. A plot of concentration of the standard IL-6 versus optical density (OD) (immunoglobu- lin secretion) can be used to determine the activity of the sample. If a standard preparation is not available, assign arbitrary units to the concentration that gives half maximal stimulation.

10.7.2 Reverse plaque-forming cell techniques

Instead of determining the amount of immunoglobulin (IgM or IgG) secreted by CESS or SKW6- CL4 cells by an ELISA assay, one can assay activation by determining the number of IgM or IgG plaque-forming cells in a reverse plaque assay.

Reverse plaque-forming cell assay

MATERIALS AND EQUIPMENT As for Section 10.7.1 but with reagents for reverse plaque assay.

METHOD

As for Section 10.7.1, but with the following modifications:

1 Incubate the cells for 48 h in a humidified atmosphere of 5% CO 2 in air.

2 Enumerate the antibody-forming cells in a reverse plaque assay, remembering to assay for IgM plaques for SKW6-CL4 cells and IgG plaques for CESS cells.

Assessment of results The number of plaques is proportional to the IL-6 concentration in the sample under test. A plot

of concentration of the standard IL-6 versus the number of plaque-forming cells can be used to determine the activity of the sample. If a standard preparation is not available, assign arbitrary units to the concentration which gives half maximal plaque numbers.

Assay for IL-6 by hybridoma proliferation

A highly sensitive assay for IL-6 utilizes hybridoma B9 cells (Aarden et al. 1987). In each microtitre

plate well 5 × 10 3 cells are cultured in 200 µl Iscove’s modified Dulbecco’s medium supplemented with 50 µM 2-mercaptoethanol, 5% v/v fetal bovine serum, plus streptomycin and penicillin. Following addition of IL-6, and culture for 44 h, 7.4 kBq (0.2 µCi) 3 H-thymidine is added and 4 h later the thymidine incorporation is determined following cell harvesting and β-scintillation counting (see Section 9.1).

10.7INTERLEUKIN 6 (IL-6)