Although many cells contribute to inflammation, eosinophils are noted for their contribution to late phase allergic-type disorders. Eosinophils make up less than 10% of the circulation leukocyte population, yet they are known to be extremely important in the inflammatory response to allergic and parasitic agents (1). When activated within tissues, eosinophils release highly basic preformed mediators such as eosinophil peroxidase and major basic protein. These substances are toxic to parasites and damaging to the surronding tissue, including smooth muscle constriction, increased vascular permeability and superoxide-mediated tissue destruction. While eosinophils have been associated with these inflammatory reactions, the soluble mediators that influence eosinophil availability and function have only recently been identified. Interleukin 5 (IL-5) along with the chemokine eotaxin are key players in the coordination of eosinophil-based inflammatory responses (1-3). See references 3-7 for reviews on IL-5.
Human Il-5 is a 135 amino acid (aa) polypeptide with a predicted mass of 12.5kD. It is secreted by a restricted number of mesenchymal cell types (8,9). In its native state, mature IL-5 is synthesized as a 115 aa, highly glycosylated 22 kD monomer that forms a 40-50kD disulfide-linked homodimer (8-10). Although the content of carbohydrate is high, carbohydrate is not needed for bioactivity (10). Monomeric IL-5 has no activity; a homodimer is required for function (6). This is in contrast to the receptor-related cytokines IL-3 and GM-CSF, which exist only as monomers (13, 14). Just as one IL-3 and GM-CSF monomer binds to one receptor, one IL-5 homodimer is able to engage only one IL-5 receptor (7). It has been suggested that IL-5 (as a dimer) undergoes a general conformational change after binding to one receptor molecule, and this change precludes binding to a second receptor (15). Human and mouse mature IL-5 are 71% identical at the aa level (8, 16). While mouse IL-5 is highly active on human cells, (6) human IL-5 is only marginally active on mouse cells (10, 17).
The receptor for IL-5 consists of a ligand binding alpha-subunit and a non-ligand binding (common) signal transducing beta-subunit that is shared by the receptors for IL-3 and GM-CSF (18).
CD125/IL-5 Ra: The human IL-5 Ra subunit is a 60kD, 400 aa type I transmembrane glycoprotein that binds IL-5 with low affinity (19,20). In the human, the Kd for IL-5 Ra is reported to be anywhere from 600-2000 pM (19, 20, 21) while the Kd for mouse Il-5 binding to mouse IL-5Ra is 2-10nM (23). Consistent with other cytokine receptors, the IL-5 Ra has two tandem barrel structures (N-and C- domain) that are made of seven anti-parallel b-strands. Within the N-domain are four conserved cysteine residues, and within the C-domain is the cytokine-associated WSXWS motif; N-terminal to both barrel structures is a fibronectin type III domain (19, 24). Although the a chain has been described as a non-signal transducing subunit, evidence suggests that the membrane-proximal portion of the 55 aa cytoplasmic region either signals, or significantly contributes to signaling (25-28). Solubel, 50kD forms of IL-5 Ra have been identified. (20-23, 29) and when present are inhibitory to IL-5 activity (21). Mouse IL-5 Ra is 70% identical to human IL-5a at the aa level (19,23). Cells known to express Il-5 Ra include CD5+ B cells, eosinophils, mast cells, CD34+ stem cells and basophils.
CD131/bc: The human IL-5Rb-subunit (bc) is a 120-130kD, type I transmembrane glycoprotein that serves as the major signal transducing subunit for the IL-3, IL-5 and GM-CSF receptors (18, 30, 31). In this role, it is analogous to gp 130 and IL-2 Rg (32). The human molecule is 871 aa long, and is divided into a 422 aa extracellular region, a 27 aa transmembrane segment, and a 432 aa cytoplasmic domain (30). Like IL-5 Ra, bc contains N- and C- domains that are duplicated; unlike IL-5Ra, there is no N- terminal fibronectin domain (18, 24, 32). Mouse bc (also known as AIC2B) is 55% identical to human bc (30,33). Cells known to express bc include basophils, eosinophils, neutrophils, monocytes and dendritic cells, endothelial cells, mast cells and macrophages, CD5+ B cells and CD 34+ stem cells.
The kinetics of IL-5 binding are still under investigation. Assuming equality with the IL-3 system, (homodimeric) IL-5 binds noncovalently to one IL-5 Ra subunit, which then noncovalently recruits one bc subunit, forming a temporary noncovalently linked trimer (32, 35, 36). At this point, a second, newly generated IL-5 R complex can be engaged with the IL-5 Ra subunit from complex #1 forming a disulfide bond with bc if complex #2. This is followed by disulfide bonding of the two remaining unlinked receptor components. It is likely that the two bc subunits also become disulfid-linked, creating a functional signal transducing complex, with a stoichiometry of 2/2/2 (32, 36). A newly formed IL-5 R trimer also may link with naturally preformed GM-CSF Ra/bc complexes to form hybrid IL-5 R/GM-CSF R complexes (37). It is unknown what function these complexes may play in IL-5 activity.
As with many cytokines and growth factors, IL-5 has an approximately 15 aa NLS in the body of the molecule (38). IL-5 with its receptor can be transported into the nucleus following its binding on the cell surface (39, 40). It is suggested that STATs, which are associated with the receptor, actually enter the receptor via the IL-5 NLS (41).
Cells known to express IL-5 include eosinophils, NK cells, TC2 CD8+ T cells, mast cells, CD45+ CD4+ T cells, gd T cells and IL-1b activated endothelial cells. IL-5 is best known for its activity on B cells and eosinophils. Relative to cells, IL-5 appears to indue the differentiation of activated conventional B-2 cells (42). In mice, IL-5 promotes production of IgA, IgE and IgG1 (42).
IL-5 appears to perform a number of functions on eosinophils. These include the down modulation of Mac-1 (43), the upregulation of receptors for IgA and IgG (44), the stimulation of lipid mediator (leukotriene C4 and PAF) secretion (45) and the induction of granule release (47). IL-5 aslo promotes the growth and differentiation of eosinophils. The exact role that IL-5 plays, however, is unclear; it may act in an adjunct fashion (48). Finally, there is a great deal of interest in the interaction between IL-5 and the CC chemokine eotaxin. Studies suggest that inflammatory-induced and locally produced IL-5 and eoxtaxin may act on the bone marrow in a cooperative manner.
This IL-5 ELISA is a 4.5 hour solid phase immunoassay readily applicable to measure IL-5 levels in serum, plasma, cell culture medium, and urine in the range of 31.25pg/ml to 1000pg/ml. It showed no cross reactivity with other cytokines tested.
Principle of the Assay
This IL-5 enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to IL-5. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated monoclonal antibody preparation specific for IL-5 and incubated. IL-5 if present, will bind and become immobilized by the antibody pre-coated on the wells and then be "sandwiched" by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound IL-5 and other components of the sample. In order to quantitate the amount of IL-5 present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits that each have a high affinity binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain IL-5, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm � 2nm.
Kit Components:
1. IL-5 Microtiter Plate, 1x 96 wells
2. Biotin Conjugate, 1x7ml
3. Avidin Conjugate, 1x14ml
4. IL-5 Standard 2x1 vials
5. Calibrator Diluent I, 1x22ml
6. Calibrator Diluent II, 1x 22ml
7. Wash Buffer (20X), 1x 60ml
8. Substrate A, 1x10ml
9. Substrate B, 1x10ml
10. Stop Solution, 1x14ml, 2N Sulphuric Acid (H2SO4).
Sensitivity: <7.5pg/ml
Range: 7.5-1000pg/ml
Storage and Stability:
Store components at 4�C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.�
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