Multiplex Human Cytokine ELISA Kit (Th1/Th2/Th17)

RACTIVITY	Human
SENSITIVITY	Refer to inset
ASSAY RANGE	Refer to inset
REAGENTS PROVIDED	Refer to inset

INTENDED USE

This multiplex ELISA kit for Th1/Th2/Th17 cytokines is designed for semi-quantitative and simultaneous determination of cytokines relevant to T helper cell differentiation.  The kit simultaneously determines interferon-γ(IFN-γ), interleukin-2(IL-2), interleukin-4(IL-4), interleukin-10(IL-10), interleukin-13(IL-13), interleukin-17A(IL-17A), interleukin-22(IL-22), and tumor necrosis factor-α (TNF-α) in cell culture supernatant and other biological samples.  In combination with other Anogen quantitative cytokine ELISA kits, the Th1/Th2/Th17 cytokine multiplex ELISA kit is expected to be useful for the investigation of the relationship of cytokine expression, T helper cell differentiation in various disease models.  

The kit is intended FOR LABORATORY RESEARCH USE ONLY and should not be used in any diagnostic or therapeutic procedures. 

INTRODUCTION

T helper cells (Th cells) are a diversified group of CD4+ T cells that play an essential role in immune-response through cytokine secretion and cell to cell contact (surface molecule expression).  The importance of Th cells is manifested by the acquired immunodeficiency in HIV patients whose CD4+ T cells are selectively infected and destroyed by HIV virus.  The function of Th cells is multifaceted, including stimulation of CD8+ cytotoxic cells, promoting B cell proliferation, regulating antibody subclass switching, and activation of macrophages and other cells in innate immune system. 

T helper cells are originated from Naïve CD4+ T cells, which are generated in bone marrow and differentiate into effector Th cells in peripheral lymphoid organs and blood upon contact with cognate antigen that matches with the complementarity-determining region on T cell receptor. Th cells were classified into T helper 1 cells (Th1) and T helper 2 cells (Th2) in the mid-1980s based on their unique cytokine secretion patterns and their roles in cellular and humoral immunity.  Th1 cells secrete IFN-γ as their signature cytokine, and are also the primary producer of IL-2.  Th2 cells fail to make IFN-γ, but produce IL-4, IL-5, and IL-13 as their signature cytokines.  Th1 cells are needed in host defense against intracellular bacteria and virus, while Th2 cells are essential for humoral responses against extracellular parasites.  Cytokines play important roles in the development and maintenance of T helper cell lineage.  In the presence of IL-12, Naïve CD4+ cells tend to differentiate into Th1, whereas IL-4 stimulates Th2 development.  Interestingly, cytokines produced by one Th subset tends to inhibit the development of other Th subset(s).  For example, IL-10 secreted by Th2 can counteract the effect of IL-12 through suppressing Th1 and stimulating Th2 differentiation. 

In 2005, Harrington et al described a new subset of IL-17 secreting CD4+ T cells as T helper 17 (Th17) cells, which is involved with autoimmunity and clearance of extracellular bacteria and fungi.  It was observed that Th17 cells can be induced from Naïve CD4+ T cells by IL-23 in the absence of IFN-r and IL-4.  More recently, T follicular helper cells (Tfh), regulatory T cells (Treg), T helper 9 cells (Th9) and T helper 22 cells (Th12) were proposed. 

IL-2 is one of the key cytokines that drive adaptive immunity.  It is secreted by Naïve CD4+ cells immediately after T cell receptor is activated through binding with antigens presented on MHC by antigen-presenting cells.  IL-2 stimulates Th1 differentiation, clone expansion and acts as autocrine to stimulate its own production.  It also promotes the survival of antigen specific memory T cell, and stimulates the maturation of regulatory T-cells.  The cytokine also influences the activity of B cells, CD8+ T cells, gamma delta T cells, NK cells and LAK cells.

IL-10 is expressed by a variety of cell types and plays an important role in inflammatory and immune responses.   It down-regulates the expression of Th1 cytokines.  IL-10 also suppresses the production of pro-inflammatory cytokines by monocytes and neutrophils, down-regulates the expression of activating and co-stimulatory molecules on monocytes and dendritic cells, and stimulates the growth of B cells and mast cells.

Produced by Th1, CD8⁺ T cells and natural killer (NK) cells, IFN-g is known to stimulate Naïve CD4+ T cells to differentiate into Th1 cells.  It is both an inhibitor of viral replication and a regulator of numerous immunological functions.  IFN-g has been reported to induce its own expression. IFN-g has documented antiviral, antiprotozoal and immunomodulatory activities.  The immunomodulatory effects of IFN-g are extensive and diverse.  In monocyte/ macrophages, IFN-g increases expression of class 1 MHC antigens; increases the production of IL-1, platelet-activating factor, H₂O₂, and pterin, protects monocytes against LAK cell-mediated lysis; and down-regulates IL-8 mRNA expression.  IFN-g selectively enhances IgG_2a secretion by LPS-stimulated B cell activation.  IFN-g has been shown to up-regulate ICAM-1, but not E-selectin or VCAM-1 expression on endothelial cells. IFN-g has also been reported to implicate in the development of a cholinergic phenotype in embryonic septal neurons.

IL-4 was initially characterized as a B cell stimulatory factor (BSF-1) for its crucial role in Th2 cell differentiation, B cell expansion and differentiation into plasma cells.   It was subsequently revealed that IL-4 is a pleiotropic cytokine with multiple immune response modulating functions on diverse cell types. IL-4 induces antibody class switching to IgE, up-regulates MHC class II production and plays a role in airway allergy.  IL-4 stimulates protective immunity against parasites.  It is also reported that IL-4 is implicated with metastasis.  IL- 4 is produced by naïve CD4⁺ T cells, Th2 cells, fetal thymocytes, CD8⁺ T cells, mast cells and basophils. IL- 4 exhibits approximately 25% amino acid sequence homology to IL-13 that shares a number of biological functions with IL-4.  Human IL-4 gene, composed of four exons and three introns, have been localized to a chromosome that contains tandem-arranged genes for IL-3, IL-5, IL-9, IL-13 and GM-CSF.

IL-13 is secreted mainly by activated T helper 2 cells.  IL-13 binds to IL-4Rα in addition to IL-13Rα1 and IL-13Rα2.  The function of IL-13 partially overlaps with IL-4.  Both IL-4 and IL-13 link to signal transducer and activator of transcription 6 (STAT6) through the receptor activation.  IL-13 can induce B-cell proliferation and IgE classs switching. However, IL-13 is less competent in this function comparing with IL-4.  Unlike IL-4, IL-13 does not play an important role in hematopoietic cell shift, such as naïve T helper differentiation to T helper 2.   IL-13 is also associated with many features of the allergic airway diseases, including mucus hypersecretion, goblet metaplasia, chemokine induction and recruitment of effector cells.  All of them contribute to airway obstruction.  Polymorphisms in IL-13 gene have been found to link to increased eosinophil count, serum total IgE and high risk of asthma. It is reported that IL-I3 signaling is significant in inducing physiological changes to non-immuno cells in parasitized organs. 

IL-17A is the proto-type of a newly discovered pro-inflammatory cytokine family which consists of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25) and IL-17F.   All the IL-17s have similar structure with four highly conserved cysteine residues and same 3 dimensional structures, which distinguish this family from other cytokines. IL-17 is produced by a group of CD4+ T help cells termed Th17 cells.  In addition to previous discovered Th1 and Th2 cells, the Th17 cells consist in the third group of T helper cells.  IL-23, a growth and stabilization factor was the first cytokine that was found to involve with the development of Th 17 cells.  Later on, other cytokines including TGF-beta, IL-6, and IL-21, were also found to contribute to the procedure.  The transcription factors implicated in the procedure were identified as STAT3, RORgt, and RORa.   IL-17, like IFN-g, is a potent mediator of delayed-type reaction.  IL-17 induces the secretion of many other pro-inflammatory cytokines and chemokines such as IL-6, IL-8, GM-CSF, G-CSF, TGF-beta, TNF-alpha, GRO-alpha, MCP-1, and stimulates the expression of NF-kappaB, mitogen activated protein kinase, and prostaglandings from many cell types.  By stimulating cytokine production and recruiting monocytes neutrophils to site of inflammation, the cytokine exerts its effect in response to tissue damage and invasion. 

IL-22 is produced by dendritic cells, T-cells and natural killer cells during bacterial infection, auto-immunity and tissue inflammation.  IL-22 acts upon innate immunity cells through its receptors expressed exclusively on these cells.  In CD4+ T helper cells, IL-22 expression has been found to be associated with Th17 and Th1.  Recently, an IL-22 expressing T helper cell subset (Th22) was characterized which is distinct from other T cells by co-expression of the chemokine receptor CCR6 and the skin-homing receptors CCR4 and CCR10.  IL-22 expression is elevated in psoriatic skin inflammation, atopic dermatitis, and inflammatory bowel disease.  In cutaneous T-cell lymphoma, IL-22 dominates the tumor microenvironment and STAT3 phosphorylation was observed.  IL-22 was also found to promote murine hepatocyte survival and ameliorate intestinal inflammation in mouse ulcerative colitis model.  A natural antagonist of IL-22, IL-22 binding protein, was found to down-regulate IL-22 function. 

Tumor Necrosis Factor alpha (TNF-a), also known as cachectin was initially named for its remarkable ability to cause hemorrhagic necrosis of tumors in mice.  The primary source of TNF-a is thought to be the monocyte/macrophage but various cell types are known to express this cytokine.  TNF-a is produced upon stimulation with cytokines such as IL-1, 1L-2, GM-CSF, TNF-a itself and with bacterial lipopolysaccharide.  Th1 cells secrete IFN-g, IL-2, IL-10, and TNF-alpha/beta.  Various pathological conditions are associated with the production of high levels of TNF-a.  These include septic shock syndrome, cachexia, autoimmune diseases, hepatitis, leukemia, myocardial ischaemia, organ transplantation rejection, multiple sclerosis, rheumatoid arthritis, and meningococcal septicemia.  TNF-a is a pleiotropic cytokine that can induce disease through TNF-a toxicity (tissue injury, catabolic illness, and mediating septic shock) and improve host defense mechanisms (stimulating inflammation and increasing immune cell function).  Since TNF-a has such a complex and important function in immune-response, studying TNF-a can help understanding its role in T cell differentiation.

This ELISA assay is a 3.5 hour solid phase immunoassay readily applicable to measure the levels of eight cytokines relevant to the differentiation of T helper cells in cell culture supernatant, and other biological fluids.  It showed no cross reactivity with other proteins.