Method Booklet 7. Cytokine. Bioactivity Methods

Transcription

1 Method Booklet 7 Cytokine Bioactivity Methods

2 Cytokine Bioactivity Methods Table of Contents I. Introduction General Review Cytokine Structure Cytokine Actions Cytokine Receptors Cytokine Facts Production of Cytokines Handling of Cytokines II. Bioassays Proliferation Cytotoxicity Cytostasis Chemotaxis Calcium Flux III. Appendix Viable Cell Counting Recent Reviews Websites Summary of Products Technical/Customer Services: (USA) (Europe) Technical/Customer Services: (USA) (Europe) 3

3 Introduction Cytokines are a diverse and unique category of protein mediators that are secreted largely by CD4+ T cells and macrophages. They are produced during the activation and effector phases of innate and specific immunity. Cytokines play important roles in the development, function and control of the cells of the immune system as well as many other systems. They are potent molecules that can cause changes in cell proliferation, differentiation and migration. Like hormones, cytokines initiate action by binding to specific receptors on the cell surface of target cells. However, unlike hormones, cytokines are usually made by a variety of cells, rather than in a gland, and act locally (autocrine or paracrine) instead of distally (endocrine). Cytokines may be pleiotropic (one cytokine, multiple effects), redundant (multiple cytokines, one effect) and antagonistic (one cytokine inhibits another cytokine). See Table 1. Cytokine actions may be grouped into five broad areas: Development of cellular and humoral immune responses Induction of inflammation Regulation of hematopoiesis Control of cellular proliferation and differentiation Induction of would healing Predicting the action(s) of a cytokine is difficult since a cytokine is not alone in the host system. There may be synergistic or antagonistic actions between different cytokines to produce an unexpected event and/or a cytokine may start a cascade of cytokine production in which the later cytokines affect the earlier cytokine. Cytokines do not have "antigen specificity". However, since cytokine receptors are generally expressed only after a cell is activated by antigen, the resulting immune response is antigen specific. Secreted primarily from leukocytes, cytokines stimulate the humoral and cellular immune responses, as well as the activation of phagocytic cells. Cytokines can also be expressed on the cell membrane while others may be stored in extracellular matrix. Cytokines are typically produced during a short period of time after cell activation in the context of immune reactions or pathological or inflammatory processes. Interleukins are cytokines that are secreted from leukocytes. Cytokines that are secreted from lymphocytes are also termed lymphokines, while those secreted by monocytes or macrophages are termed monokines. The term interleukin derives from the fact that these cytokines are secreted from leukocytes, but also affect the cellular responses of leukocytes. More specifically, interleukins are growth factors targeted to cells of hematopoietic origin. Interferons are a family of cytokines initially thought to be involved in antiviral responses. However, they are also potent immunomodulators. They can promote or inhibit the synthesis of antibodies by activated B-cells and also activate macrophages, natural killer cells, and T-cells. They also possess direct antiproliferative activities and are cytostatic or cytotoxic for a number of different tumor types. Chemokines are a family of pro-flammatory activation inducible cytokines. These proteins are mainly chemotactic for various cell types. The chemokine name is derived from (chemo)tactic cyto(kine). Chemokines are multipotent cytokines that localize and enhance inflammation by induced chemotaxis and cell activation of inflammatory cells at sites of inflammation. Chemokines are also essential mediators of normal leukocyte trafficking. Colony Stimulating Factors (CSFs) are cytokines that stimulate the proliferation of specific pluripotent stem cells of the bone marrow in adults. Granulocyte-CSF (G-CSF) is specific for the proliferation and differentiation of hemapoietic progenitor cells committed to the granulocyte lineage. Macrophage-CSF (M-CSF) influences the proliferation and differentiation of stem cells into macrophages but mainly the growth and differentiation of monocytes. Granulocyte-macrophage- CSF (GM-CSF) is responsible for the growth and development of granulocytes and macrophage progenitor cells. The Tumor Necrosis Factor (TNF) family consists of two molecular species, TNF-α and TNF-β. TNF-α induces expression of other autocrine growth factors, increases cellular responsiveness to growth factors and induces signaling pathways that lead to proliferation. TNF-α also induces expression of a number of nuclear proto-oncogenes as well as other interleukins. TNF-β is characterized by its ability to kill a number of different cell types as well as the ability to induce terminal differentiation in others. The induction of TNF-β results from elevations of IL-2 as well as the interaction of antigen with T-cell receptors. Originally, the cytokines were named according to their function (eg. T cell growth factor, now called IL-2), but with the pleiotropy of cytokines observed, this made functional specific names confusing. Historically, cytokines have been ordered into five different protein families. Main classes of cytokines include: Interleukins - regulate interactions between leukocytes Interferons - response to viral infections (α, β, γ, ω) Chemokines - chemotaxis and inflammation (eg. IL-8) Colony Stimulating Factors - allow cells to grow (eg. GM-CSF) Tumor necrosis factors - derived from macrophages, anti-tumor activity 4 tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 5

4 Table 1 A limited sample of cytokines listed with their primary activity and primary source. Interleukin Primary Activity Principal source IL-1-α and -β Enhance the activation of T cells Macrophages and other antigen in response to antigen presenting cells (APCs) IL-2 Proliferation of B cells and activated Activated Th1 cells, NK cells T cells, NK function IL-3 Growth of hematopoietic progenitor cells Activated T cells IL-4 B cell proliferation, eosinophil and mast Th2 and mast cells cell growth and function, IgE and class II MHC expression on B cells, inhibition of monokine production IL-5 Eosinophil growth and function Th2 and mast cells IL-6 Acute phase response, B cell proliferation, Activated Th2 cells, APCs, and thrombopoiesis, synergistic with IL-1 and TNF other somatic cells on T cells IL-7 T and B lymphopoiesis Thymic and marrow stromal cells IL-8 Chemoattractant for neutrophils and T cells Macrophages, other somatic cells IL-9 Hematopoietic and thymopoietic effects T cells IL-10 Inhibits cytokine production, promotes B cell Activated Th2 cells, CD8+ T and proliferation and antibody production, suppress B cells, macrophages cellular immunity, mast cell growth IL-11 Synergistic hemotopoietic and thrombopoietic Stromal cells effects IL-12 Proliferation of NK cells, IFN-γ production, B cells, macrophages promotes cell-mediated immune functions IL-13 IL-4-like activity Th2 cells IL-14 Induces B-cell proliferation, inhibits Ig secretion, T cells selectively expands some B-cell populations IL-15 T cell growth factor with similar T cell T cells activities to IL-2 IL-16 CD4+ T lymphocyte attractant CD8+ T cells IL-17 Stimulate epithelial, endothelial and fibroblastic Activated memory B cells cells to secrete IL-6, IL-8, GM-CSF IL-18 Activates IFN-γ by spleen cells, enhances NK Liver cell cytotoxicity, augments GM-CSF production and decreases IL-10 production Interferons Primary Activity Principal source IFN-α and -β Antiviral effects, induction of class I MHC on Macrophages, neutrophils and somatic cells, activation of NK cells and some somatic cells macrophages IFN-γ Induces class I MHC on all somatic cells, induces Activated Th1 and NK cells class II MHC on APCs and somatic cells, activates macrophages, neutrophils, NK cells, promotes cellmediated immunity, antiviral effects Colony Primary Activity Principal source Stimulating Factor G-CSF Stimulate proliferation of bone marrow stem Activated monocytes, cells of granulocyte lineage macrophages and neutrophils M-CSF Stimulate proliferation of bone marrow cells of Monocytes, granulocytes, macrophage lineage endothelial cells, fibroblasts GM-CSF Stimulate proliferation of bone marrow stem cells Activated T cells and of both granulocyte and macrophage lineage macrophages Tumor Necrosis Factors Primary Activity Principal source TNF-α Induce expression of other autocrine growth Activated macrophages factors, of nuclear proto-oncogenes, and induces signaling pathways that lead to proliferation TNF-β Kill or induce terminal differentiation of various Cytotoxic T cells cell types Cytokine Structure Structurally, early members of the cytokine family shared a common up-up-down-down four-helix bundle topology, initially observed in porcine growth hormone. Today five different structural families of cytokines can be generated based on X-ray and NMR studies, gene organization, chromosomal location and receptor usage. Table 2 illustrates representative members of these families. Table 2 Family Member Receptor Type Alpha helix Long chain 4 helix bundle IL-3, -6, -7, G-CSF Class I Short chain 4 helix bundle IL-2, -4, -5, -13, G-CSF, GM-CSF Class I M-CSF The Ig superfamily IFN-α Class II Inter-subunit 4 helix bundle IL-10 Class II IFN-β, IFN-γ Class II Beta sheet Beta-sandwich (jelly roll motif) TNF-α, TNF-β TNF-R Beta-Trifoil IL-1α, IL-1β, IL-18 The Ig superfamily Chemokine (triple-stranded, IL-8, and others 7-transmembrane antiparallel β sheet) 6-protein coupled receptors 6 tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 7

5 Cytokine Actions As described above there are many cytokines, and these can have multiple actions. It is easiest to classify these molecules according to their general actions. Four broad groups can be identified: A. Proinflammatory Cytokines (TNF, Interleukin-1, and Interleukin-6) These cytokines are central in helping to initiate and potentiate any inflammatory response, with tumor necrosis factor alpha and interleukin-1 being major examples. They are secreted by almost any cell and are expressed early in the inflammatory response. The effects include local changes, resulting in mobilization of antigen presenting cells and activation of endothelial cells to express adhesion molecules. This results in recruitment of inflammatory cells. Interleukin-1 and TNF also act systemically to induce the acute phase reaction, including fever. B. Chemokines (Interleukin-8 and many others) Interleukin-8 is the prototype member of this family but many others exist. These cytokines are important in inflammatory cell recruitment and chemotaxis. Several classes can be recognized due to differences in the amino acid structure. Different chemokines recruit different classes of inflammatory cells, eg. neutrophils, lymphocytes or macrophages. They form a net over endothelial cells and are important in triggering adhesion molecules on the surface of leukocytes. Chemokines also produce a chemotactic gradient for inflammatory cells to follow. C. Hematopoiesis Colony stimulating factors and some interleukins (eg. Interleukin-3) are crucial for the development of inflammatory cells from the bone marrow precursors. A number of colony stimulating factors are known. However, other cytokines are also important, and different cytokines are able to induce specific differentiation from the hemapoietic stem cells and partly committed progenitors. D. Immunomodulatory Cytokines (IL-2, IL-4, IL-5, IFN-γ and others) Cytokines are particularly important in the development of T and B lymphocyte responses. Cytokines are important both for the activation of lymphocytes and also in determining the type of immune response that develops. In the initial stage of activation, interleukin-2 is important. Following the initial stimulation of the T helper lymphocytes by the antigen presenting cells, interleukin-2 is secreted and acts on the cell to activate it. This then leads to increased interleukin-2 production, which acts on other helper cells and also other lymphocytes to activate them. The type of immune response that develops is also governed by the cytokines that are secreted. Cytokines secreted by CD4 + T-cells have been divided into two broad groups: T helper 1 (Th1) and T helper 2 (Th2) types. Th1 cells have a role in cellular immunity, while Th2 cells are involved in humoral immunity. IL -2, IL-12 and IFN-γ are produced by Th1 cells and are referred to as Type I cytokines, while Th2 cells produce IL-4, IL-5, IL-10 and IL-13 (Type 2) cytokines. It should be noted that there is some overlap, with cytokines such as IL-3 and TNF-α being included in both groups. Type I Cytokines The synthesis and secretion of IL-2 represents the early consequences of antigen or mitogeninduced activation of mature resting T cells. IL-2 interaction with its high-affinity receptors promotes clonal expansion of the effector T-cell population originally activated by antigen. IL-12 directly induces initial Th1 development and the production of IFN-γ secreting Th1 cells. From a humoral immunity standpoint, recombinant IL-12 has been shown to be a suppressor of IL-4 induced IgE production. Also, IL-12 has been hypothesized to play an early role in hematopoiesis. IFN-γ controls the class of antibody produced in B cells, up-regulates class I and II MHC complex antigens, and increases the efficiency of macrophage-mediated killing of intracellular parasites. Type II Cytokines IL-4 secretion by Th2 cells elicits humoral response of selective production of IgG, IgE and IgA isotypes. On B-lymphocytes IL-4 regulates the expression of surface antigens, resulting in the enhancement of the antigen-presenting capacity of B cells. IL-5 has a major role in the host as an eosinophil hematopoietic growth factor. IL-5 also can modify basophil function and induce basophilic differentiation. With effects on both eosinophil and basophil function, IL-5 plays a role in the pathogenic inflammatory responses of hypersensitivity and other diseases associated with eosinophil infiltration. IL-5 also induces stimulated B cells to differentiate into Ig-secreting cells. IL-10 has a broad range of activity on a variety of cell types including both immunosuppressive and immunostimulatory effects. IL-10 produced in Th2 cells inhibits the production of cytokines, especially IFN-γ by Th1 cells responding to antigen. Also, IL-10 is a potent down regulator of cell mediated immune response which results in potent anti-inflammatory activities. IL-13 is a pleiotropic cytokine produced by activated Th2 cells. In humans, IL-13 brings about changes in the morphology and phenotypes of monocytes by inducing expression of the IgE receptor and upregulating expression of MHC class II. Also, IL-13 is involved in IgE switching and the induction of IL-4 independent IgG 4 and IgE synthesis in the presence of T cells. Cytokine Receptors The ability of cytokines to influence the course of cell growth and differentiation depends on their recognition and binding to specific receptors. These receptors are cell surface molecules which transduce cytokine binding into intra-cytoplasmic signals that trigger developmental processes within the cell (see Table 3). The binding of their cognate ligands activates many cytokine receptors. This ligation results in the formation of an active multi-subunit complex that frequently results in oligomerization of the ligand-binding subunit. This in turn leads to activation of intracellular signals. 8 tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 9

6 Table 3: Common cytokines, target cells and receptor subunits Cytokines Target Cell Receptor GM-CSF Monocyte, Macrophage, Mast Cell, T-cell (T-helper 2) GM-CSFR-α, β chain M-CSF Monocyte, Macrophage CSF-1R IFN-α T-helper 1, B-cell, Monocyte, Macrophage IFN-αRI, IFN-αRII IFN-β T-helper 1, B-cell, Monocyte, Macrophage IFN-αRI, IFN-αRII IFN-γ Natural Killer, T-cell (T-suppressor, T-helper 1), IFN-γR B-cell, Monocyte, Macrophage IL-1 (α & β) Monocyte, Macrophage, T-helper 2, Neutrophil (beta only) IL-1RI, IL-1RII, IL-1RIII IL-2 T-cell (T-helper 1, T-helper 2, T-suppressor), IL-2Rα, IL-2Rβ, B-cell, Monocyte, Macrophage IL-2Rγ chain IL-3 Mast Cell, T-cell (T-helper 1, T-helper 2), IL-3Rα, β chain Monocyte, Macrophage IL-4 Basophil, Mast Cell, T-cell (T-helper 2, T-suppressor), B-cell IL-4Rα, IL-2Rγ chain IL-5 B-cell, Mast Cell, T-cell (T-helper 2?), Monocyte, Macrophage IL-5Rα, β chain IL-6 Neutrophil, Monocyte, Macrophage, Mast Cell, B-cell, T-cell? IL-6R, gp130 IL-7 T-helper 1, T-helper 2, B-cell IL-7R, IL-2Rγ chain IL-8 Neutrophil, Monocyte, Macrophage IL-8R-HA, IL-8-LA IL-9 Monocyte, Macrophage, T-cell (T-helper 1, T-helper 2) IL-9R, IL-2Rγ chain IL-10 Monocyte, Macrophage, B-cell, T-helper 1, T-helper 2? IL-10Rα, IL-10Rβ IL-11 B-Cell, Hematopoietic stem cell IL-11R, gp130 IL-12 Monocyte, Macrophage, T-helper 1, B-cell IL-12Rβ 1, IL-12Rβ 2, IL-2Rγ chain IL-13 Basophil, Mast Cell, T-cell (T-helper 1?, T-helper2) IL-13Rα1, IL-13Rα2, IL-4Rα, IL-2Rγ chain IL-14 B-cell, Mast Cell, T-cell? IL-14R IL-15 NK cells, Monocyte, Macrophage, T-helper 1, IL-15Rα, IL-2Rβ, T-helper 2 IL-2Rγ chain IL-16 Eosinophil, Mast Cell, T-cell (T-suppressor, T-helper?) CD4 IL-17 Monocyte, Macrophage, B-cell, T-helper 1, fibroblasts IL-17R IL-18 Monocyte, Macrophage IL-18Rα, IL-18Rγ,? (subunit not identified) TNF-α Neutrophil, Natural Killer, Monocyte, Macrophage, CD120a, CD120b T-cell (T-suppressor, T-helper 1, T-helper 2), B-cell TNF-β B-cell, T-cell (T-suppressor, T-helper 1, CD120a, CD120b T-helper 2), Monocyte, Macrophage A relatively simple signal transduction model arose during the mid-1990s, which included a cytokine/interferon receptor with associated JAK kinases and downstream STAT transcription factors. The JAK kinases would be activated upon ligand-induced receptor oligomerization. They would phosphorylate STAT molecules on tyrosine residues, which would allow the STAT molecules to dimerize, promote nuclear translocation, increase their DNA binding activity and regulate gene expression. Many cytokine receptors were shown to utilize this basic signal transduction model (eg. IL-2R, IL-3R, IL-5R, Epo-R, GM-CSFR and others). cytokine binds to its ligand on a target cell. BioSource International provides reagents to study intracellular cytokine pathways (please see Appendix). Table 4 Cytokine Receptors Pathways GM-CSF GM-CSFRα, β chain JAK1,2/STAT5; Ras/Raf/MAPK IL-1 IL-1RI, IL-1RII, IL-1RIII PLC; Src; MAPK IL-2 IL-2Rα, IL-2Rβ, γ chain JAK1, Pyk2/JAK3/STAT3,5; Ras/Raf/MAPK; PKB/AKT IL-3 IL-3Rα, β chain JAK1,2/STAT5; Src/STAT3; Ras/Raf/MAPK; AKT IL-4 IL-4Rα, γ chain JAK1,3, STAT3,5,6;IRS;MAPK IL-5 IL-5Rα, β chain JAK2/STAT1,5; Ras/Raf/MAPK IL-6 IL-6R, gp130 JAK1,2, STAT1,3; Ras/Raf/MAPK IL-7 IL-7R, γ chain JAK1,3/STAT3,5; Ras/Raf/MAPK IL-9 IL-9R, γ chain JAK1,3/STAT1,3,5 IL-10 IL-10Rα, IL-10Rβ JAK1, STAT1,3 IL-12 IL-12Rβ1, IL-12Rβ2, γ chain JAK2, STAT3,4 IL-13 IL-13Rα1, IL-13Rα2, IL-4Rα, STAT3,5,6 γ chain IL-15 IL-15Rα, IL-2Rβ, γ chain JAK1,3/STAT3,5; MAPK IL-16 CD4 PKC; SAPK; MAPK IL-17 IL-17R JAKs, STAT1,2,3,4; JNK; Ras/Raf/MAPK IL-18 IL-18Rα, IL-18Rγ,? STAT4; JNK; MAPK TNF-α CD120a, CD120b TRADD/RIP/TRAF/MAPK/NFkB; Pyk2 TNF-β CD120a, CD120b TRADD; Pyk2 IFN-α IFN-αRI, IFN-αRII JAK1, Fyn, ERK2/STAT1; STAT2,4; IRS-1 IFN-β IFN-αRI, IFN-αRII JAK1, ERK2/STAT1 IFN-γ IFN-γR JAK1, JAK2/Pyk2/ERK2/STAT1 M-CSF CSF-1R Src/STAT3; Pyk2/PI3-kinase; PKC/Paxillin Later, alternate pathways linking cytokine-ligand binding to signal transduction were described. A common intermediate pathway initiating from cytokine receptors is the Ras/Raf/MEK/ERK (MAPK) cascade, which can result in the phosphorylation and activation of additional downstream kinases and transcription factors such as p90 Rsk, CREB, Elk and Egr-1. From the cloning of mammalian and non-mammalian cytokine receptors, notable amino acid homologies and conservation of characteristic sequence motifs have been determined. These determinations have allowed the grouping of the cytokine receptors into a gene family and subfamilies. Cytokine receptors are represented structurally in Figure 1. The Ig superfamily: IL-1, IL-18, M-CSF Class I (hematopoietins): IL-2, 3, 4, 5, 6, 7, 9, 11, 12, 13, 15, GM-CSF, G-CSF; contain both a CCCC- and a Trp-Ser-X-Trp-Ser (WSXWS)-conserved motifs; Class II (IFN): IFN-α, -β, -γ; contain the CCCC-conserved motif; TNF-R: TNF-α, -β, CD40, NGF, Fas; contain repeats of C1, C3, C2; The following table gives examples of major signalling molecules involved when a particular 10 tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 11

7 Figure 1 Members of the Class I receptor family lack an intrinsic tyrosine kinase activity. Upon binding of a ligand to the extracellular domain of the Class I receptors, the receptor molecules form homo tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 13

8 dimers or heterodimers (in some cases trimers) and the intracellular receptor domains become associated with a variety of signaling molecules, in particular JAK and STAT. The conserved extracellular domain of these receptors has a length of approximately 200 amino acids which contain four positionally conserved cysteine residues in the amino-terminal region and a Try-Ser-X-Trp-Ser motif (WSXWS) located proximal to the transmembrane domain. The four cysteines appear to be critical to the maintenance of the structural and functional integrity of the receptors. The WSXWS consensus sequence is thought to serve as a recognition site for functional protein-protein interaction of cytokine receptors. The absence of intrinsic protein tyrosine kinase activity is a notable feature of the intracellular domain of Class I receptors. The cytoplasmic domains of the family members are less well conserved, but some sequence similarities have been reported. Box 1 and Box 2 are two short stretches of conserved amino acid residues close to the membrane-spanning region of the receptor molecules. Some members of the Class I family also contain another conserved region, Box 3, in the middle of the cytoplasmic domain. Cytokine Receptors - Shared Subunits Common IL-3R β subunit IL-3α β β β 5α GMα Common gp130 subunit IL-6α gp gp gp 11α LIF Increasingly it has been found that the Class I receptors are multicomponent molecules with one component (usually the α component) being important in the specificity of cytokine binding and the second (or occasionally third) component then passing on the signal to the cell. Often this second component is shared between several cytokine receptors. If the γ chain is congenitally absent, as in the disease Severe Combined Immunodeficiency (SCID), then the immune function is severely impaired as none of these cytokine receptors can function. See Figure 2 and Table 3 for examples of the members of the Class I family which share either of three different signal transducing receptor components, gp130, common IL-3 beta subunit, or the gamma subunit of the IL-2 receptor. Thus the differential expression of receptor components and signaling molecules, and the selective recruitment of different intracellular signaling components explain some of the overlapping biological activities of the corresponding cytokine ligands. Members of the Class II cytokine receptors are only distantly related to members of the Class I receptors. Class II receptors are multimeric receptors composed of heterologous subunits. The extracellular domains share structural similarities in their ligand binding domain. Several conserved intracellular motifs have been described, and these probably function as binding sites for the intracellular effector proteins JAK and STAT. Many of the cytokine receptors exist in membrane-bound and soluble forms. The soluble receptor forms may arise from proteolytic cleavage of transmembrane receptors or by utilizing spliced receptor mrnas. These soluble receptors may act as inhibitors of cytokine activities. Figure 2 IL-2α Common IL-2Rγ chain (γ) γ γ γ γ γ 15α 4α Since cytokines are present in the body of humans and other animals in nano- to pico-molar concentrations, natural sources of significant quantities of the proteins are not readily available, nor are cytokines simple to purify. Large amounts of purified cytokines are required, however, for the enormous amount of research being conducted on these molecules. Recombinant technology has filled this demand very nicely. The recombinant versions of most cytokines compare very well to their natural counterparts in terms of biological activity. For example, cytokines have been successfully expressed in E. coli, baculovirus, yeast, NSO and CHO mammalian cells. The choice of the expression system is determined by the nature of the protein structure and biological activity, i.e., glycosylation requirements for activity will rule out E. coli as an expression system. When working with recombinant proteins, it is important to understand the basic character of the protein as a chemical molecule and as an immune modulator. Below is a brief review of the Interleukin proteins, the TNF family, the IFN family and the CSF family. The size and nature of the protein is stated along with the main cellular source. In addition, the main biological activity and an appropriate cell line for biological testing is given. This is not meant to be a comprehensive review. For detailed cytokine information, see the appendix section of this booklet for websites and appropriate review references. 7α 9α 14 tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 15

9 Cytokine Facts: Granulocyte-macrophage colony stimulating factor (GM-CSF): 127 AA (14 kda) monomeric protein with two glycosylation sites. Glycosylation is not required for activity, but the glycosylated form is more active in vivo. GM-CSF initiates many biological responses. For example, GM-CSF is secreted by T-cells and macrophages following mitogen or antigen stimulation. GM-CSF is required for the growth and development of granulocyte and macrophage progenitor cells. In addition, it stimulates myeloblasts and monoblasts and initiates irreversible differentiation of these cells and induces chemotaxis of eosinophils. Human GM-CSF activity is measured by the proliferation of the TF-1 cell line and mouse GM-CSF is assayed by measuring the proliferation of the cell line MC/9. Interferon-alpha (IFN-α): AA (19-26 kda) size protein may be observed since there are 23 different variants of IFN-α. There are two disulfide bonds that are required for full activity. Glycosylation is observed with some isoforms. IFN-α is produced by monocytes, lymphoblastoid cells and fibroblasts. IFN-α is a strong antiviral, antiparasitic, antiproliferative agent. Cell lines to assay the cytopathic activity of IFN-α include MDBK and WISH. Interferon-beta (IFN-β): 166 AA (20 kda) glycoprotein containing a single disulfide bond that is required for biological activity. IFN-β is produced by fibroblasts and some endothelial cell types. IFN-β is involved in humoral immune response regulation and is an antiviral agent. IFN-β is also antiproliferative against a number of tumor cell lines. The biological activity of IFN-β can be measured by detecting the inhibition of GM-CSF proliferation of TF-1 cells. Interferon-gamma (IFN-γ): 146 AA (~17 kda) dimeric protein that contains two cysteines that are not involved in disulfide bonding. Glycosylation is not required for biological activity. IFN-γ is produced mainly by T-cells and NK cells activated by antigens and mitogens. IFN-γ is involved in many biological processes. For example, IFN-γ is antiviral, antiparasitic and inhibits the proliferation of cells. In macrophages IFN-γ induces the secretion of TNF-α and stimulates the release of reactive oxygen species. It is also involved with bone growth and inhibits bone resorption. Human IFN-γ activity is measured by detecting the cytostasis of the WiDr cell line and mouse or rat IFN-γ activity is measured by detecting the cytostasis of the cell line Wehi279. Interleukin-1 (IL-1): either a 269 AA or a 271 AA (17 kda) protein with no disulfide bonds. There are two distinct molecular forms of IL-1 (IL-1α and IL-β) derived from two different genes. These two proteins are 26% homologous at the amino acid level and bind to the same receptor. The predominant function of IL-1 is to enhance the activation of T-cells in response to antigen and APCs. The IL-1s are secreted primarily by macrophages but also neutrophils, endothelial cells, smooth muscle cells and B- and T-cells. IL-1 bioactivity is measured in a proliferation assay using the cell line D10S. The cell line may be utilized to assay human, swine, rat and mouse IL-1α or IL-1β. Interleukin-2 (IL-2): 133 AA (15 kda) protein with a single disulfide bond that is critical for activity. IL-2 is glycosylated but this is not required for biological activity. IL-2 is produced by CD4 positive T- cells following activation by mitogens or allogens. Transformed B-cells, T-cells, Leukemia cells and NK cells also secrete IL-2. IL-2 causes proliferation of T-cells and is a central regulator of immune responses. There are many cell lines available to assay the biological activity of IL-2. Mouse CTLL cells are used to determine the proliferative activity of recombinant IL-2. This cell line will react to human, mouse, swine and rat IL-2. Interleukin-3 (IL-3): 133 AA (~15-17 kda) protein with a single disulfide bond. IL-3 is glycosylated but this is not required for biological activity. IL-3 is mainly produced by antigen or mitogen activated T-cells. IL-3 provides the cytokine connection between the immune system and the hematopoietic system. For example, IL-3 supports the proliferation and development of almost all types of hematopoietic progenitor cells and can act as a chemoattractant for eosinophils. There are many cell lines available to assay the biological activity of IL-3. Mouse NFS60 cells are used to determine the proliferative activity of recombinant mouse IL-3 and human TF-1 cells to assay human IL-3. Interleukin-4 (IL-4): 129 AA (~20 kda) protein with three disulfide bonds that are critical for biological activity. IL-4 is glycosylated but this is not required for biological activity. IL-4 is mainly produced by activated CD4 positive Th2 helper cells. IL-4 promotes the proliferation and differentiation of activated B-cells. In addition, IL-4 up-regulates class II MHC antigen expression and IgE receptors. There are many cell lines available to assay the biological activity of IL-4. Mouse MC/9 cells are used to determine the proliferative activity of recombinant mouse IL-4 and human TF-1 cells to assay human IL-4. Rat IL-4 activity is either verified by measuring IL-4 induced MHC antigen expression on rat splenocytes by flow cytometry or by measuring the proliferation of the intestine endothelial cell line IEC-6. Interleukin-5 (IL-5): 113 AA for the murine protein and 115 AA for the human. The biologically active form is a disulfide linked n-glycosylated homodimer (glycosylation is not required for activity). IL-5 is mainly produced by T-cells and promotes the growth and differentiation of eosinophils. Cell lines useful in IL-5 assays include B13, BCL1, T88-M, TALL-103 and TF-1 cells. TF-1 cells are used in a proliferation assay to measure IL-5 activity. Interleukin-6 (IL-6): 185 AA protein with disulfide bonds that are critical for activity. IL-6 is glycosylated but this is not required for biological activity. IL-6 is secreted by many cell types, but is mainly produced by stimulated monocytes, fibroblasts and epithelial cells. IL-6 is an important regulator of acute phase reaction. It is also a B-cell differentiation factor and an activator of T-cells. There are many cell lines available to assay the biological activity of IL-6. Mouse B9 cells are used to determine the proliferation activity of recombinant IL-6. The cell line will react to human, mouse, swine and rat IL-6. Interleukin-7 (IL-7): 152 AA for the human protein and 129 AA for the murine homologue. The disulfide bonds are required for biological activity. IL-7 is secreted from adherent bone marrow stromal cells and thymic cells. IL-7 stimulates the proliferation of pre-b and pro B-cells. IL-7 also supports the maturation of megakaryocytes. Mouse 2E8 cells are used in a proliferation assay to determine the activity of recombinant IL-7. The cell line will react to human and mouse IL-7. Interleukin-8 (IL-8): 72 AA (8 kda) non-glycosylated protein. IL-8 has two disulfide bonds and is a member of the CXC chemokine family. IL-8 is produced by stimulated monocytes. It is also produced by macrophages, fibroblasts, endothelial cells, keratinocytes, melanocytes, hepatocytes and a number of tumor cell lines. IL-8 is chemotactic for all known types of migratory immune cells. IL-8 also inhibits the adhesion of leukocytes to activated endothelial cells. The biological activity of IL-8 is assayed by neutrophil chemotaxis tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 17

10 Interleukin-9 (IL-9): 144 AA (14 kda) extensively glycosylated protein with 5 disulfide bonds. IL-9 is secreted from mitogen or antigen stimulated CD4 positive T helper cells. IL-9 is a cofactor in many immune processes. IL-9 stimulates the proliferation of T helper cells. IL-9 also induces the secretion of IL-6 in mast cells. Human IL-9 is assayed detecting the proliferation of the cell line MO7E and murine IL-9 is assayed with TS1.C3. Interleukin-10 (IL-10): 160 AA homodimeric protein with 2 disulfide bonds per monomer. IL-10 is secreted from activated CD8 positive T cells, B-cell lymphomas, LPS activated monocytes and mast cells. IL-10 inhibits the synthesis of a number of cytokines, such as, TNF-α, IL-1 and IL-6 from macrophages and IL-2, IFN-γ, and TNF-β from Th1 cells. Human and mouse IL-10 is assayed by measuring the increased proliferation of the cell line MC/9 co-stimulated with a low dose of m IL-4. Rat IL- 10 may either be assayed by measuring the inhibition of TNF-α production from LPS stimulated rat peripheral macrophages or utilizing the mast cell line, D36, in a proliferation assay. Interleukin-11 (IL-11): 179 AA (~23 kda) non-glycosylated protein with no cysteine residues. IL-11is mainly produced by bone marrow stromal cells and also mesenchymal cells. IL-11 is a cofactor in many immune responses and in cell differentiation. For example, IL-11 works with IL-3 in stimulating the generation of megakaryocyte colonies. In addition, IL-11 prevents cell death by apoptosis and inhibits the differentiation of preadipocytes. Human and mouse IL-11 is assayed by measuring the proliferation of the cell lines, TF-1, 7TD1 or T1165. Interleukin-12 (IL-12): 503 AA (~70 kda) heterodimeric protein comprised of a p40 and p35 subunit. The subunits are linked by disulfide bonds that are critical for biological activity. In addition, the p40 subunit contains 10 cysteine residues and the p35 subunit contains 7. This tertiary structure requires insect, yeast or mammalian expression for active recombinant protein. The primary source of IL-12 is dendritic cells, peripheral lymphocytes, and B cells. IL-12 induces the secretion of IFN-γ, IL-2 and TNF-α and stimulates the proliferation of PHA activated lymphocytes. Human IL-12 is assayed by measuring the proliferation of PHA activated peripheral lymphocytes and mouse IL-12 is by measuring the increased production of IFN-γ from mouse splenocytes. Interleukin-13 (IL-13): 111 AA (~12 kda) protein with 2 disulfide bonds that are critical for biological activity. IL-13 is mainly produced by activated Th2 cells. IL-13 down regulates macrophage produced cytokines such as TNF-α, IL-1, IL-6, IL-8 and IL-12 in response to LPS challenge or IFN-γ. IL- 13 induces monocyte differentiation and also induces B-cell differentiation and proliferation. TF-1 cells are used to assay human IL-13. Interleukin-14 (IL-14): also known as High molecular weight B-cell growth factor. The factor is kda and is secreted from T-cells. IL-14 functions as a mitogen for activated B-cells. Interleukin-15 (IL-15): 114 AA (~13 kda) protein with disulfide bonds that are critical for activity. IL-15 is glycosylated but this is not required for biological activity. T-cells are a major source of IL-15. In addition, IL-15 is secreted by astrocytes and microglia after stimulation with IL-1β, IFN-γ or TNF-α. IL-15 activity resembles some biological activities attributed to IL-2. IL-15 induces proliferation of PMA activated peripheral blood mononuclear cells. IL-15 also functions as a maturation factor for NK cells. Mouse CTLL cells are used to determine the proliferative activity of recombinant IL-15. The cell line will respond to human and mouse IL-15. Interleukin-16 (IL-16): 121 AA (~13 kda) which is known to be active as a tetramer. The major cellular sources is the epithelium, although mast cells, CD8 positive cells, CD4 cells and cosinophils are also sources. IL-16 stimulates a migratory response in CD4 positive cells such as lymphocytes, monocytes and eosinophils. IL-16 activity is measured by detecting the chemotaxis of CD4 positive T lymphocytes. Interleukin-17 (IL-17): 272 AA (~31 kda) glycosylated homodimer. CD4 positive T-cells are a major source of IL-17. IL-17 stimulates epithelial, endothelial or fibroblastic cells to secrete IL-6, IL-8 and G- CSF. IL-17 also increases the expression of ICAM-1 in fibroblasts. IL-17 activity is measured by detecting IL-6 production in foreskin fibroblasts. Interleukin-18 (IL-18): 158 AA (~18 kda). Liver cells are a major source of IL-18. IL-18 induces IFN-γ in activated peripheral blood lymphocytes. Human IL-18 activity is measured by detecting IFN-γ secretion from CD3 stimulated PBMCs and mouse IL-18 activity is measured by detecting IFN-γ secretion in ConA stimulated murine lymph node cells. Tumor necrosis factor-alpha (TNF-α): 157 AA (17kDa) protein with a single disulfide bond that is not necessary for activity. TNF is active as a homotrimer. TNF-α is secreted by macrophages,monocytes, neutrophils, T-cells and NK cells following LPS challenge. TNF causes cytolysis and cytostasis of many tumor cell lines. TNF has a wide spectrum of activities, including chemotaxis of neutrophils, alteration of the endothelium, inhibition of anticoagulatory mechanisms, and promotion of angiogenesis. TNF-α activity is measured by detecting the cytolysis of the murine cell line L929. This cell line is appropriate for testing human, mouse and rat TNF-α. Swine TNF-α is tested with PK-15 cells. Tumor necrosis factor-beta (TNF-β): 171 AA (17kDa) protein that forms heterodimers with LT-beta that anchors the complex in the cell membrane. TNF-β is produced by mitogen activated T-lymphocytes and leukocytes. Additional cell sources include fibroblasts, astrocytes, myeloma cells, endothelial cells, epithelial cells and certain transformed cell lines. TNF-β induces the synthesis of GM-CSF, G-CSF and IL-1. It is a cytolytic factor many tumor cell lines and induces reactive oxygen species in neutrophils. TNF-β activity is measured by detecting the cytolysis of the murine cell line L tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 19

11 Recombinant Proteins Manufactured at BioSource International Authenticity and Expression: Recombinant protein product quality is monitored throughout the development process. The cytokine gene is first cloned from appropriate cdna libraries and then sequenced and compared to published DNA sequences in the NIH GenBank database to confirm the authenticity of the sequence. After reviewing the current literature regarding the protein and similar proteins, an expression system is chosen that will most likely result in a biologically active protein. Properties that are examined include: non-covalent and covalent structure, cysteine residue number, glycosylation requirements, and secondary protein structure. Many of the recombinant proteins manufactured at BioSource are expressed in E. coli due the ease of genetic manipulation, availability of optimized expression plasmids, and ease of growth. Once the protein has been expressed, purified, and re-folded if necessary, the authenticity of the protein is confirmed again utilizing biological assays and immunological methods such as ELISA and Western blot. Refolding and Biological Activity: When using bacterial expression systems, often the over-expressed protein may aggregate into inclusion bodies. These inclusion bodies are highly insoluble and must be solubilized in a strong denaturant before further purification. Once solubilized the now denatured protein must be refolded to its natural native configuration, purified to remove extraneous bacterial proteins and renatured failure sequences, before bioactivity is evaluated. The goal of the refolding/purification process is to end with a homogeneous solution of bioactive protein, i.e., only one form of the protein, such as a trimer for TNF-α. At BioSource International, we have developed proprietary refolding/purification protocols that result in active, stable, low endotoxin and homogeneous protein solutions. BioSource in-house bioassay testing includes: cell proliferation, cytostasis, cytotoxicity, chemotaxis, calcium flux, secondary cytokine up-regulation and induction of surface antigen expression assays. Please see Chapter II for detailed protocols. Purity: Purification levels are measured by SDS-PAGE with either coomassie or silver staining. When necessary, homogeneity is confirmed by size exclusion chromatography or RP-HPLC. All preparations are sterile filtered prior to processing and lyophilization. Endotoxin levels are monitored by the gel clot LAL method. Lyophilization and QC: After purification optimal processing buffers are selected to ensure that maximum activity is retained after lyophilization. The proteins are lyophilized without carrier protein for maximum flexibility at the research level. The products are processed in aseptic environments to ensure high quality, low endotoxin products. Final products are tested for: Biological activity utilizing frequently cited cellular activity assays for testing. Structural homogeneity as determined by denaturing PAGE, native protein electrophoresis, absorbance profiles, and general chromatography. Freeze/Thaw stability as determined by recovery of biological activity after multiple freeze thaw cycles. Low Endotoxin levels- recombinant proteins are manufactured to have less than 0.1 ng endotoxin per microgram protein as determined by the LAL gel clot method. Helpful Hints for Handling Cytokines: Everything needed to be successful with a BioSource International, Inc. recombinant protein is on the data sheet. Read this carefully and follow the recommended reconstitution suggestions for the protein. The buffer in which the protein was originally lyophilized was chosen because it is the optimal buffer for the stability and resuspension of the lyophilized protein. When diluting below 100 µg/ml, add carrier protein. Before opening a vial of protein, centrifuge the tube. This will move the protein to the bottom of the vial and result in a mass accurate product. (During lyophilization and shipping the protein may have moved and become adherent to the top of the vial.) Recombinant protein stability varies from protein to protein. Refer to the product insert for each protein for exact recommendations. In general, however, there are some common guidelines. Lyophilized products are stable at +4 to 20 C. Limit freeze thaw cycles by aliquoting the protein immediately following the initial reconstitution. The aliquoted samples should be stored in the 20 C. Only use polypropylene vials or tubes except with TGF-beta. (TGF-beta is sold in glass bottles and should only be stored in silanized glass). Note the ED 50 range given in the package insert. This number is generated from calculating the concentration required for 50% of the cells to respond. This number is easily converted into units by the following equation: 1 x 10 6 / ED 50 (ng/ml) = specific activity. The activity reported in the package inserts is an average of 6 samples run in duplicate after various manipulations, i.e., multiple freeze thaws. Thus, the numbers are typical for most laboratory settings tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 21

12 Table 5 The following are examples of each category of bioassays that are used at BioSource International, Inc. to validate recombinant cytokines. Cytokines Assay Type Cell Line Species/Type Reference GM-CSF Proliferation TF-1, h/ erythroleuk, Kitamura, T., et al., (1989) J Cell Physiol 140: MC/9 m/ mast cell Zhao, M., et al., (1993) J Neuroimmunol 43: M-CSF Proliferation m bone marrow Heyworth, C.M. and Spooncer, E. (1993) Haemopoiesis: a practical approach p IRL Press, Oxford University Press. IFN-alpha Viral Inhibition b MDBK Rubenstein, S., et al., (1981) J Virol 37: IFN-beta Viral Inhibition b MDBK Rubenstein, S., et al., (1981) J Virol 37: IFN-gamma Cytostasis WiDr h/ adenocarcinoma Pfizenmaier, K., et al., (1985) Cancer Res 45: IL-1 (alpha and beta) Proliferation D10S m/ T helper Orrencole, S.F. and Dinarello, C.A. (1989) Cytokine 1: IL-2 Proliferation CTLL-2 m/ Tcell Giglia, J.S., et al., (1985) Cancer Res 45(10): IL-3 Proliferation TF-1, h/ erythroleuk, Kitamura, T., et al., (1989) J Cell Physiol 140: MNFS-60 m/ myelog leuk Weinstein, Y., et al., (1986) Proc Natl Acad Sci 83: IL-4 Proliferation TF-1, h/ erythroleuk, Kitamura, T., et al., (1991) Int Immunol 3(6): MC/9 m/ mast cell Thompson Snipes, L. (1991) J Exp Med 173: IL-5 Proliferation TF-1 h/ erythroleuk Kitamura, T., et al., (1991) Int Immunol 3(6): IL-6 Proliferation B9 m/ B hybridoma Braciak, T., et al., (1993) J Immunol 151(10): IL-7 Proliferation 2 E 8 m/ B lymphoma Ishihara, K., et al., (1991) Dev Immunol 1(3): IL-8 Chemotaxis h neutrophils Schroeder, J., et al., (1987) J Immunol 139:3474. IL-9 Proliferation TF-1 h/ erythroleuk Kitamura, T., et al., (1991) Int Immunol 3(6): IL-10 Proliferation D36, m/ Mast cell, Schlaak, J., et al., (1994) J Immunol Meth 168: MC/9 m/ mast cell Thompson Snipes, L. (1991) J Exp Med 173: IL-11 Proliferation 7TD1 m/ B hybridoma Van Snick, J., et al. (1986) Proc. Natl. Acad. Sci. USA 83: IL-12 Proliferation mouse splenocytes Mattner, F., et al., (1993) Eur J Immunol 23:2202. IL-13 Proliferation TF-1 h/ erythroleuk McKenzie, A.N.J. (1993) Proc Natl Acad Sci USA 90: IL-15 Proliferation CTLL-2 m/ Tcell Giglia, J.S., et al., (1985) Cancer Res 45(10): IL-16 Chemotaxis stim h lymphocytes Loetscher, M., et al., (1993) J. Exp. Med. 184:963 IL-17 Proliferation neonatal h dermal fibroblasts Fossiez, F. (1996) Journal of Exp. Med. 183: IL-18 Proliferation stim h T-cells Yoshimoto, T., et al., (1997) Proc Natl Acad Sci U S A 94(8): TNF-alpha Cytotoxicity L929, m / fibroblast, Flick, D.A. and Gifford, G.E. (1984) J Immunol Meth 68(1-2): PK-15 p/ kidney Bertoni, G., et al., (1993) J Immunol Meth 160(2): TNF-beta Cytotoxicity L929 m/ fibroblast Flick, D.A. and Gifford, G.E. (1984) J Immunol Meth 68(1-2): Bioassays Bioassays exploit the many different biological activities of cytokines which include cell proliferation induced by cytokines, chemotaxis, cytotoxicity, capacity to induce colony formation, cellular degranulation, or the induction of the secretion of further cytokines or other compounds. These assays frequently involve the use of primary cell cultures and established cell lines that depend upon the presence of (a) particular cytokine(s) for their growth or survival or that respond to a given cytokine. It should be noted that the reliability of an indicator cell line is an important factor associated with the successful performance of bioassays and it should be kept in mind that overall sensitivities of cell lines may be highly variable when maintained in continuous cell culture. Generally biological responses induced by cytokines show saturation kinetics which can be used to quantitate their amounts from dose-response curves. It should be noted that cytokine assays frequently measure only one single aspect of the many biological activities of a given cytokine. Potential complex interactions among cytokines must also be considered when interpreting data obtained from cytokine bioassays. Different cytokines may act through the same receptor and/or receptor signaling components and therefore induce similar biological responses that may only differ in the doses required for half-maximum effects. If available, specific monoclonal antibodies against cytokines can be used to confirm that the signal in the assay is due to the correct cytokine. Bacterial endotoxins are known to be good inducers of cytokines and hence their presence in test samples to be assayed may lead to false results as bacterial endotoxins trigger cells to produce cytokines during incubation. Another drawback of bioassays may be the use of supernatants or sera or other body fluids because biologically active cytokines bound to the cell surface cannot be detected in this way. In addition, biological fluids may contain soluble receptors interfering with the assays in a variety of ways. Different quantitative results may be obtained also when biological fluids are assayed by bioassay and immunoassays. This is thought to be related in some cases to the presence of different antigenic forms (including complexes with soluble receptors or monomeric forms of cytokines) that cannot be detected by bioassay. Some bioassays do not differentiate between different molecular species of cytokines or unrelated factors binding to the same cell surface receptor. IL1-α and IL-1β (see: IL-1), for example, are structurally unrelated factors that show almost identical biological activities because they bind to the same receptor. Table 5 provides a list of assay types, cell lines and references which are routinely used in the biological testing of cytokines. This is not a complete list and more detailed information may be found in appropriate review references and the web sites listed in the appendix section of this booklet. Cytokine assays employed at BioSource International, Inc. fall into five basic categories: Proliferation Cytotoxicity Cytostasis Chemotaxis Calcium flux 22 tech.support@biosource.com (USA) tech.support@biosource.be (Europe) Technical/Customer Services: (USA) (Europe) 23