T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4

Abstract

Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE-stimulated hMCs elaborated minimal cys-LT (0.1 +/- 0.1 ng/10(6) hMCs) and abundant prostaglandin (PG)D(2) (16.2 +/- 10.3 ng/10(6) hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE-dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD(2) (by 2. 5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE-mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD(2) generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C(4) synthase (LTC(4)S) mRNA within 6 h, and increased the expression of LTC(4)S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC(4)S biosynthetic pathway (induction of LTC(4)S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.

Figure 1

Effect of IL-4 priming of hMCs on IgE-mediated histamine release and eicosanoid generation as measured by ELISA. hMCs were maintained in SCF and IgE for 5 d with and without 10 ng/ml of IL-4. Results depict percent histamine release, cys-LT generation_, and PGD₂ production after 5 d of priming and passive sensitization followed by activation with anti-IgE (black bars) or treatment with buffer alone (hatched bars). Results are the mean ± SEM of four experiments and the IL-4 effect was significant for histamine (P = 0.0006) and cys-LT (P = 0.03). Ctl., control.

Figure 2

Effect of IL-3 or IL-5 treatment during IL-4 priming of hMCs for IgE-mediated release of histamine and generation of cys-LTs and PGD₂, as measured by ELISA. hMCs were activated with anti-IgE (black bars) or buffer (hatched bars) after 5 d of passive sensitization and priming with IL-4 in the presence of SCF with or without the addition of IL-3 or IL-5. Results are mean ± SEM for five (for SCF plus IL-4 and SCF plus IL-4 plus IL-3) or three (SCF plus IL-4 plus IL-5) experiments. Ctl., control.

Figure 4

Dose dependence and kinetics for the IL-4–mediated upregulation of LTC₄S protein expression. (a) Dose-dependent effects at 5 d of IL-4, from 0.1 to 50 ng/ml, on LTC₄S protein with a constant concentration of SCF (100 ng/ml), as demonstrated by SDS-PAGE immunoblot. Single lanes containing lysates from 10⁵ hMCs treated for 5 d with SCF alone, and with SCF plus IL-13 (10 ng/ml), are included. (b) Kinetic effects of IL-4 (10 ng/ml) on LTC₄S protein in hMCs incubated for 0 to 5 d with SCF (100 ng/ml) to maintain viability. The displayed blots are representative of three experiments each.

Figure 3

Effect of IL-4 priming on 5-LO/LTC₄S pathway protein expression by hMCs. (a) SDS-PAGE immunoblot was performed with lysates from hMCs (10⁵/lane), treated for 5 d with SCF with or without IL-4, with polyclonal Abs specific for cPLA₂, 5-LO, FLAP, and LTC₄S, as specified in Materials and Methods. The displayed blot is a single experiment representative of three experiments. (b) Quantitative densitometry revealed a fivefold increase in LTC₄S signal after IL-4 treatment for 5 d (n = 11, P = 0.0005).

Figure 6

IL-4–mediated upregulation of steady-state LTC₄S mRNA. (a) RNA blot analysis depicting hybridization signals for LTC₄S mRNA in samples of total RNA (14 μg/lane) extracted from hMCs that were harvested at the indicated times after being transferred to fresh SCF-containing medium with (+) or without IL-4. (b) Ribosomal 18S RNA signal in the corresponding lanes. Results are representative of two experiments performed.

Figure 5

IL-4–mediated upregulation of LTC₄S biosynthetic activity. LTA₄ methyl ester and reduced glutathione were provided as substrates to the sonicated lysates from 2 × 10⁵ hMCs for each condition. Sonicates were prepared from hMCs harvested from their original medium containing SCF, IL-6, and IL-10 at the start of the experiment (day 0), and again after these hMCs were treated with SCF alone (100 ng/ml) or with SCF and IL-4 (10 ng/ml) for the indicated times. Products were resolved by RP-HPLC and quantitated based on the internal standard PGB₂ as indicated in Materials and Methods. Results are mean ± SEM for four experiments.

Figure 7

Effect of IL-3 or IL-5 priming on 5-LO/LTC₄S pathway protein expression by hMCs. SDS-PAGE immunoblot was performed with lysates from hMCs (10⁵/lane), treated for 5 d with SCF (100 ng/ml) with or without IL-3 or IL-5 (5 ng/ml each), using polyclonal Abs specific for cPLA₂, 5-LO, FLAP, and LTC₄S as specified in Materials and Methods. The LTC₄S signal from hMCs primed for 5 d with IL-4 is included for comparison. The displayed blot is a single experiment representative of three experiments.

Figure 8

Effects of IL-3 and IL-5 on immunolocalization of 5-LO in hMCs. hMCs were maintained for 5 d in SCF alone (a–d), with 10 ng/ml of IL-4 (e and f), with IL-4 plus 5 ng/ml of IL-3 (g and h), or IL-4 plus 5 ng/ml of IL-5 (i and j). Images on the left are photographed through a 40× objective, whereas those on the right are photographed at 100× for nuclear detail. Identical fields of hMCs are photographed under blue fluorescence (a, c, e, g, and i) to show location of nuclei as identified by Hoechst staining, and under green fluorescence (FITC) to demonstrate localization of 5-LO immunoreactivity in the same cells (d, f, h, and j). FITC staining with a preimmune rabbit IgG (b) is included as a specificity control. The images are taken from a single experiment representative of three performed, for which the mean data are presented in the text.