Cytokines cHAPTER 12 characterized so far belong to one of four groups: the Cytokine bound hematopoietin family, the interferon family, the chemokine family, or the tumor necrosis factor family The structures of two members of the hematopoietin family, IL-2 and IL-4, are depicted in Figure 12-4. Although the amino acid sequences of these family members differ Add substrate considerably, all of them have a high degree of a-helical and measure structure and little or no B-sheet structure The molecules (a)Interleukin 2 36—42 strand 05 C-D Concentration of IL-12(pg/ml) FIGURE 12-3 ELISA assay of a cytokine. (a)The sample contain- ing the cytokine of interest is captured by specific antibody(blue) NHo COOH coated onto wells of a microtiter plate. A second specific antibody (blue), conjugated to an enzyme (E)such as horseradish peroxidase. forms a sandwich with the captured cytokine, immobilizing the en (b)Interleukin 4 yme in the microtiter well. A chromogenic substrate (S)is added. and the enzyme generates a color whose intensity is proportional to the amount of cytokine bound to the capture antibody. The optical density of this color produced by the unknown is compared with val ues on an appropriately determined standard curve. (b)The standard shown here is for human interleukin 12(IL-12). It is clear that assay is sufficiently sensitive to detect as little as 1 picogram of IL-12 /Part (b)courtesy of R&D Systems. cell lines whose growth depended on the presence of a partic lar cytokine provided researchers with the first simple assay systems. The derivation of monoclonal antibodies specific for each of the more important cytokines has made it possible to develop rapid quantitative immunoassays for each of them (Figure 12-3) FIGURE 12.4 Several representations of structures in the hemato. Cytokines Belong to Four Structural poietin family. (a) Left: Topographical representation of the primary Families structure of IL-2 showing a-helical regions(a and A-D)and connect ing chains of the molecule. Right: Proposed three-dimensional model Once the genes encoding various cytokines had been cloned, of IL-2. (b) Ribbon model of IL-4 deduced from x-ray crystallographic sufficient quantities of purified preparations became avail- analysis of the molecule In(a)and ( b)the a helices are shown in red able for detailed studies on their structure and function. and the B sheets in blue. The structures of other cytokines belonging to Cytokines generally have a molecular mass of less than the hematopoietin family are thought to be generally similar. /Part(b) 30 kDa. Structural studies have shown that the cytokines from/L Boulay and W.E. Paul, 1993, Curr Biol. 3: 573/cell lines whose growth depended on the presence of a partic￾ular cytokine provided researchers with the first simple assay systems. The derivation of monoclonal antibodies specific for each of the more important cytokines has made it possible to develop rapid quantitative immunoassays for each of them (Figure 12-3). Cytokines Belong to Four Structural Families Once the genes encoding various cytokines had been cloned, sufficient quantities of purified preparations became avail￾able for detailed studies on their structure and function. Cytokines generally have a molecular mass of less than 30 kDa. Structural studies have shown that the cytokines characterized so far belong to one of four groups: the hematopoietin family, the interferon family, the chemokine family, or the tumor necrosis factor family. The structures of two members of the hematopoietin family, IL-2 and IL-4, are depicted in Figure 12-4. Although the amino acid sequences of these family members differ considerably, all of them have a high degree of -helical structure and little or no -sheet structure. The molecules Cytokines CHAPTER 12 279 Add enzyme￾conjugated secondary antibody Cytokine bound to antibody coated onto well Add substrate and measure color S E E E E S (a) (b) Concentration of IL-12 (pg/ml) Optical density 10 1 0.1 0.01 0.1 1 10 100 α S S 105 C N 133 6 114 30 36 42 73 80 96 52 58 D A C B C 105 B–C loop β strand C–D loop A–B loop COOH NH2 A C B' B D α P65 C58 (a) Interleukin 2 (b) Interleukin 4 A B C 2 1 D FIGURE 12-3 ELISA assay of a cytokine. (a) The sample contain￾ing the cytokine of interest is captured by specific antibody (blue) coated onto wells of a microtiter plate. A second specific antibody (blue), conjugated to an enzyme (E) such as horseradish peroxidase, forms a sandwich with the captured cytokine, immobilizing the en￾zyme in the microtiter well. A chromogenic substrate (S) is added, and the enzyme generates a color whose intensity is proportional to the amount of cytokine bound to the capture antibody. The optical density of this color produced by the unknown is compared with val￾ues on an appropriately determined standard curve. (b) The standard curve shown here is for human interleukin 12 (IL-12). It is clear that this assay is sufficiently sensitive to detect as little as 1 picogram of IL-12. [Part (b) courtesy of R&D Systems.] FIGURE 12-4 Several representations of structures in the hemato￾poietin family. (a) Left: Topographical representation of the primary structure of IL-2 showing -helical regions ( and A–D) and connect￾ing chains of the molecule. Right: Proposed three-dimensional model of IL-2. (b) Ribbon model of IL-4 deduced from x-ray crystallographic analysis of the molecule. In (a) and (b) the helices are shown in red and the sheets in blue. The structures of other cytokines belonging to the hematopoietin family are thought to be generally similar. [Part (b) from J. L. Boulay and W. E. Paul, 1993, Curr. Biol. 3:573.]