Production of IL12 and its regulation

Although human IL-12 was originally discovered in the supernatants of transformed human B cell lines, normal B cells (human or mouse) were not found to synthesize significant amounts of IL-12 (murine B cells stimulated by bacterial DNA may be an exception). The main producers of IL-12 belong to the macrophage/dendritic cell (DC) lineage. It is important to note that the capacity of macrophages to synthesize IL-12 depends on their functional stage (Table 2). Neutrophils, upon stimulation by lipo-polysaccharide (LPS), were also found to produce IL-12, although less efficiently than monocytes. Furthermore, Langerhans cells, microglia cells, astrocytes, connective tissue type mast cells and keratinocytes could be identified as IL-12 producers.

Regulation of IL-12 synthesis is complex because it is controlled by two independent genes which have to be coexpressed in the same cell. There arc indications that mRNA expression for the two chains does not always correlate. The p40 chain is generally-produced in excess over the IL-12 heterodimer. The ratio of p40 to IL-12 can vary from 5:1 to 100:1, depending on the cell type and the stimulus. This may not be trivial because in the case of murine macrophages the IL-12 antagonist, II.-12(p40)., could be detected among the excess p40 produced. It is obvious that this complicates the determination of IL-12 considerably (see below).

The stimuli leading to IL-12 production have been mainly studied with macrophages/DCs (Figure li. With respect to the function of IL-12 as a mediator of innate resistance and a link to specific immunity, it is essential that this cytokine (together with other cytokines such as tumor necrosis factor a (TNFa], IL-1, etc.) is produced upon infection/interaction of macrophages with gram-negative as well as gram-positive bacteria on their products (e.g. LPS and others). Furthermore, infection with certain parasites and viruses can induce IL-12 synthesis. A second pathway leading to IL-12 production in macrophages/DCs is initiated by ligation of CD40, which occurs under physiological conditions by interaction with activated, CD40L-expressing T cells of the Th1 subtype. No IL-12 is produced upon interaction of macrophages/DCs with TH2 cells. This is probably due to the release of IL-10 which inhibits IL-12 production (see below). This pathway of IL-12 production is important to maintain and perhaps further expand an already existing Tn l-type response.

Some of the modulators of IL-12 production and their effects are listed in Table 2. It should be noted that IL-10 and transforming growth factor (3 (TGF(3) not only inhibit IL-12 synthesis but also its biological

Table 1 Some structural characteristics of IL-12 and its receptor

Interleukin 12 (IL-12)

Human (h) and murine (m) IL-12 are both 70-75 kDa proteins composed of two polypeptide chains, IL-12p40 and IL-12p35, linked by one disulfide bridge. Murine IL-12 can function on human cells but human IL-12 does not function on mouse cells hlL-12p40: -40 kDa, 306 amino acids calculated mol. wt 34699, 10 Cys: 8 in intrachain disulfide linkages. 1 Cys forms a bridge to IL-12p35, 1 Cys modified by thioglycolic acid or by cysteine, 4 potential N-glycosylation sites, 10% carbohydrate Amino acid sequence shows homology to IL-6R a chain Encoded on chromsome 5q31-q33

hlL-12p35: -35 kDa, 197 amino acids, calculated mol. wt 22500, 7 Cys: 6 in intrachain disulfide linkages, 1 Cys forms bridge to IL-12p40, 3 potential N-glycosylation sites, 20% carbohydrate Amino acid sequence shows homology to IL-6 and G-CSF Encoded on chromosome 3p12-q13.2

mlL-12-p40: -40 kDa, 70% cDNA sequence and 70% amino acids sequence identity with hlL-12p40

Encoded on chromosome 11

mlL-12p35: -35 kDa, 67% cDNA sequence, 60% amino acids sequence identity with hlL-12p35

Encoded on chromosome 6(3) Interleukin 12 receptor (IL-12R)

Two receptor chains IL-12RJJ! and IL-12Rp2 have been identified and cloned hlL-12HfV -100 kDa, type I membrane protein, 638 amino acids calculated mol. wt 70400, extracellular portion

516 amino acids, transmembrane region 31 amino acids, intracellular portion 91 amino acids. hlL-12Rp2: -130 kDa, type I membrane protein, 835 amino acids, calculated mol. wt 94 059, extracellular portion

595 amino acids, transmembrane portion 24 amino acids, intracellular portion 216 amino acids Both (J, and B2 chains are members of the cytokine receptor superfamily and are related to gp130 of the IL-6R and to receptors of LIF and G-CSF. Coexpression of IL-12R0, and p2 confers IL-12 responsiveness and results in binding sites of Ka 50 pM and of Kd 5-10 nM; expression of either chain alone results in binding sites of Ka 2 to >50 nM.

mlL-12Rf3,: 54% amino acid sequence identity with hlL-12R(l,

The cDNA of mlL-12R(5, was reported to occur in 3 additional splice variants (|}la, p,b and (l.J mlL-12R(32: 68% amino acid sequence identity with hlL-12R(S

h + mlL-12R: Expressed on 'resting' NK cells but not on resting T and B cells, Upregulation upon activation on T

cells and NK cells. Expression on activated B cells is not yet firmly established IL-12 (pP40)z: a competitive inhibitor of IL-12

IL-12(p40)2 is a disulfide-linked homodimer that can be secreted in addition to the IL-12p40 monomer by cells producing IL-12p40

hlL-12(p40)2: Binds to the high affinity hlL-12R with 5- to 10-fold lower affinity than hlL-12 and is therefore not a potent inhibitor of hlL-12

mlL-12(p40)2: Binds to the high affinity mlL-12R with similar affinity as mlL-12 itself but without stimulation of the cells. mlL-12(p40)2 is therefore a potent competitive inhibitor of mlL-12

mlL-12p40: Binds with 25- to 50-fold lower affinity to the IL-12R than mlL-12(p40)2

(monomer)

activities on T cells. Furthermore, the role of CD46, which is a receptor for measles virus and a binding protein for C3b/C4b, is remarkable because it selectively inhibits the synthesis of IL-12 but not that of TNFa and IL-6 by macrophages.

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