Interleukin 1 (IL-1)
10.2 Interleukin 1 (IL-1)
IL-1 is known as endogenous pyrogen, catabolin and haematopoietin 1. Almost every nucleated cell type can be induced to produce IL-1 but it is produced in greatest amount by activated mono- cytes and macrophages. IL-1 has stimulatory and regulatory effects in terms of growth and differ- entiation of numerous cell types, has profound effects on the immune system, regulating both T and B cells, and is a mediator of inflammatory responses.
Two distinct genes have been identified (IL-1α and IL-1β) whose gene products produce IL-1 activity that is essentially identical and which both bind to the IL-1 receptor. The IL-1 precursors have a MW r of around 33 000, but when proteolytically cleaved produce mature proteins with a MW r of around 17 000. The Il-1α precursor protein is biologically active, but the IL-1β must be processed in order to exert any activity. Yet most of the biological activity in the circulation is exerted by IL-1β.
10.2.1 Thymocyte costimulator assay
Thymocytes from young (6–10-week-old) mice are cultured in the presence of a submitogenic concentration of phytohaemagglutinin (PHA) that is potentiated by IL-1.
MATERIALS AND EQUIPMENT Mouse thymocytes Tissue culture medium Fetal bovine serum (FBS) Phytohaemagglutinin (PHA)
3 H-thymidine 96-well microtitre plates (round-bottomed wells)
Automated cell harvester Beta spectrometer
METHOD
1 Remove the thymus from a freshly killed mouse and prepare a single-cell suspension, using aseptic technique.
2 Wash the cells twice by centrifugation and resuspend in medium at 1.5 × 10 7 /ml.
3 Dispense 100 µl aliquots into individual wells of a 96-well microtitre plate.
4 Add 50 µl of PHA (diluted 1 : 250 in tissue culture medium containing 10% FBS) to each culture. The final dilution of 1 : 1000 should be submitogenic, i.e. it should not give a significant proliferative response in the absence of IL-1. If it does induce a mitogenic response, check
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10.2INTERLEUKIN 1 (IL-1)
5 Add the IL-1 samples under test at a range of dilutions, using 50 µl aliquots of each, to triplicate wells.
6 Appropriate controls are: cells + medium alone cells + PHA alone cells + IL-1 alone in each case in a final volume of 200 µl.
7 Incubate the plates at 37°C for 48–72 h and pulse with 18.5 × 10 3 Bq of 3 H-thymidine (74 × 10 10 Bq/mol) for 4–6 h prior to harvesting.
8 Harvest the plates using an automated cell harvester and process the samples for b scintillation counting (see Section 9.5.1).
Assessment of results
A standard IL-1 preparation is available from the National Institute for Biological Standards and Controls, UK. Then a standard curve can be plotted of proliferation ( 3 H-thymidine uptake) against IL-1 concentration and units of activity shown by the unknown samples read off the curve. Alternatively, you may wish to assign an arbitrary number of units to the dilution giving 50% of maximal stimulation.
10.2.2 Calcium ionophore costimulation
Interleukin 1 plus calcium ionophore can stimulate certain IL-1-sensitive cell lines to produce IL-
2 (see Section 10.3). The IL-2 produced is then measured using an IL-2-dependent T-cell line. MATERIALS AND EQUIPMENT
EL4.6.1 cell line (IL-1 sensitive, IL-2 producer) IL-2-dependent T-cell line, e.g. CT-6, CTLL-D, DIOS Tissue culture medium Fetal bovine serum (FBS) Calcium ionophore, e.g. ionomycin or A6137 Dimethyl sulphoxide (DMSO) 96-well microtitre plates
3 H-thymidine (74 × 10 10 Bq/mol)
Recombinant IL-2 Beta scintillation counter
Preparation in advance
1 Make a stock solution of calcium ionophore at 1 × 10 –3 m in DMSO and store at –20°C.
2 Prepare subcultures of the two cell lines to be used 24–48 h in advance of the assay, to ensure that they are in their log phase of growth.
C H A P T E R 1 0: The cytokines
METHOD
1 Add 100 µl aliquots of IL-1 samples, at a range of dilutions and in triplicate, to 96-well microtitre plates.
2 Add 50 µl of calcium ionophore diluted to 1 × 10 –6 M in complete medium to each well.
3 Harvest EL4 cells from an actively growing culture, wash twice by centrifugation and resuspend at 4 × 10 6 /ml.
4 Add 50 µl of the cell suspension to each well and incubate overnight at 37°C in a humidified atmosphere containing 5% CO 2 in air.
Controls should include calcium ionophore plus cells alone, the highest concentration of IL-1 plus cells alone and cells alone.
5 Remove 100 µl of medium from each well and transfer to the corresponding well of a second microtitre plate.
6 Harvest the CTLL-D (or equivalent) cells from an actively growing culture, wash three times to remove IL-2 from the growth medium and resuspend the cells at 1 × 10 5 /ml.
7 Add a 100 µl aliquot of cell suspension to each microtitre well containing the transferred supernatants. Be sure to include cultures containing CTLL-D cells with doubling dilutions of recombinant IL-2, for the construction of a standard curve.
8 Incubate the plates overnight at 37°C in a humidified atmosphere of 5% CO 2 in air.
9 Add 18.5 × 10 3 Bq of 3 H-thymidine to each well 4–6 h before harvesting the plates with an automated cell harvester.
10 Process the samples for liquid scintillation counting.
TECHNICAL NOTES • The colorimetric MTT assay using the tetrazolium salt, 3-(4,5 dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (see Section 9.5.2) can also be used to assay for cell proliferation. • The DIOS murine helper T-cell line may also be used to measure IL-2 in a proliferative assay.