C-X-C chemokines

7IPIO Monocytes

MIP-2 Neutrophils

CTAP-III Neutrophils, fibroblasts

Chemotaxis Chemotaxis Chemotaxis

Other monocyte chemoattractants Other PMN chemoattractants Other chemoattractants of the read-out assay, the extent to which other cytokine activities overlap and the ease with which their contribution can be eliminated. In general the multiple functions of individual cytokines are associated with the same epitope and therefore properties of individual molecules do not vary with respect to each other, e.g. the collagenase activity of IL-1 is parallel to its I cell activity. The properties which are usable in assays may be divided into two groups: (stimulation and inhibition of) proliferation and function. The former may be assayed by growth stimulation (usually [3H]thymidine incorporation) into sensitive cells or by assaying mitochondrial function as an indicator of cell viability, e.g. by cleaving 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) to form a dark blue formazan product. Such assays have the advantage of similarity of technique for a variety of cytokines and therefore many can be assayed with semiautomated equipment.

Functional assays are much more heterogeneous. They are necessary when the cytokine to be tested has no growth related properties (e.g. IL-8 must be assayed in neutrophil migration assays), or where the functional properties are more specific than the growth-related properties. Some cytokines can only be specifically bioassayed in organ culture (e.g. Mul-lerian inhibitory substance by assaying regression of 14-day fetal rat embryo urinogenital tracts) or in vivo (e.g. EGF-induced eyelid opening in neonatal mice or MIP1- and 2-induced neutrophil chemotaxis in mouse cerebrospinal fluid).


It has already been stated that many biological effects are mediated by more than one cytokine, e.g. bone resorption is induced both by II.-l and TNF« and many cclls respond to more than one cytokine as a growth factor, e.g. mouse thymocytes respond to IL-1, II.-4, IL-6 and II -7 to varying degrees, in addition the test preparations may contain both stimulatory and inhibitory cytokines, e.g. TGF|3 is inhibitory in many of the IL-1 assays and assays where early hematopoietic cells are the target, and interferon y inhibits many IL-4 assays.

Specificity may be increased by a variety of tech niques:

1. Selection of assay systems where two cytokines with overlapping properties are likely to coexist, nonoverlapping properties may be selected for assay, e.g. human GM-CSF and G-CSF may be distinguished as the former has growth stimulatory properties on the megakaryoblastic leukemia cell line Mo7E.

2. The cells may not have linear responses to the different cytokines at the same dilutions of the test material, therefore the assay conditions can be arranged to favor the cytokine under test.

3. Use of mouse target cells may reduce the range of human cytokines influencing the assay (see above).

4. When known cytokines are likely to influence the assay addition of excess neutralizing antibodies (plus appropriate control antibodies for monoclonal antibodies or serum for polyclonal antibodies) may be used to enhance specificity.

5. To further ensure that the activity assayed is attributed to the correct cytokine, neutralizing antibodies to the cytokine must completely abrogate all bioactivity.

Inhibitor proteins

Specific physiological inhibitors of, for example, IL-1 and TNF of the relevant cytokine influence bioassays.

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