Leptin Elicits In Vivo Eosinophil Migration and Activation: Key Role of Mast Cell-Derived PGD2.

Eosinophils are key regulators of adipose tissue homeostasis, thus characterization of adipose tissue-related molecular factors capable of regulating eosinophil activity is of great interest. Leptin is known to directly activate eosinophils in vitro, but leptin ability of inducing in vivo eosinophilic inflammatory response remains elusive. Here, we show that leptin elicits eosinophil influx as well as its activation, characterized by increased lipid body biogenesis and LTC4 synthesis. Such leptin-triggered eosinophilic inflammatory response was shown to be dependent on activation of the mTOR signaling pathway, since it was (i) inhibited by rapamycin pre-treatment and (ii) reduced in PI3K-deficient mice. Local infiltration of activated eosinophils within leptin-driven inflammatory site was preceded by increased levels of classical mast cell-derived molecules, including TNFα, CCL5 (RANTES), and PGD2. Thus, mice were pre-treated with a mast cell degranulating agent compound 48/80 which was capable to impair leptin-induced PGD2 release, as well as eosinophil recruitment and activation. In agreement with an indirect mast cell-driven phenomenon, eosinophil accumulation induced by leptin was abolished in TNFR-1 deficient and also in HQL-79-pretreated mice, but not in mice pretreated with neutralizing antibodies against CCL5, indicating that both typical mast cell-driven signals TNFα and PGD2, but not CCL5, contribute to leptin-induced eosinophil influx. Distinctly, leptin-induced eosinophil lipid body (lipid droplet) assembly and LTC4 synthesis appears to depend on both PGD2 and CCL5, since both HQL-79 and anti-CCL5 treatments were able to inhibit these eosinophil activation markers. Altogether, our data show that leptin triggers eosinophilic inflammation in vivo via an indirect mechanism dependent on activation of resident mast cell secretory activity and mediation by TNFα, CCL5, and specially PGD2.

Exploring the cyclooxygenase 2 (COX2)/15d-Δ(12,14)PGJ(2) system in hamster Sertoli cells: regulation by FSH/testosterone and relevance to glucose uptake.

We have previously described a stimulatory effect of testosterone on cyclooxygenase 2 (COX2) expression and prostaglandin (PG) synthesis, and the involvement of PGs in the modulation of testosterone production in Leydig cells of the seasonal breeder Syrian hamster. In this study, we investigated the existence of a COX2/PGs system in hamster Sertoli cells, its regulation by testosterone and FSH, and its effect on glucose uptake. COX2 expression was observed in Sertoli cells of both reproductively active and inactive adult hamsters. Testosterone and the plasma membrane-impermeable testosterone-BSA significantly induced COX2 expression, mitogen activated protein kinases 1/2 (MAPK1/2) phosphorylation and 15d-Δ(12,14)PGJ(2) production in Sertoli cells purified from photoperiodically regressed hamsters. These actions were abolished by the antiandrogen bicalutamide and by the inhibitor of MAPK kinase (MEK1/2) U0126, suggesting that testosterone exerts its stimulatory effect on COX2/PGs through a non-classical mechanism that involves the presence of androgen receptors and MAPK1/2 activation. FSH also stimulated COX2/PGs via MAPK1/2 phosphorylation. FSH and testosterone stimulate, whereas 15d-Δ(12,14)PGJ(2) via PPARγ inhibits, [2,6-(3)H]-2-deoxy-d-glucose ([(3)H]-2-DOG) uptake. Meloxicam, a selective COX2 inhibitor, further increases [(3)H]-2-DOG uptake in the presence of FSH or testosterone. Thus, in addition to their positive effect, FSH and testosterone may also exert an indirect negative regulation on glucose uptake which involves the COX2/15d-Δ(12,14)PGJ(2)/PPARγ system. Overall, these results demonstrate the presence of a COX2/PG system in hamster Sertoli cells which might act as a local modulator of FSH and testosterone actions.

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation.

PGD(2) is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD(2) was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1-5 µM) evoked PGD(2) synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 µM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 µM) or human eotaxin (6 nM) also enhanced HQL-79-sensitive PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.

Cutting edge: prostaglandin D2 enhances leukotriene C4 synthesis by eosinophils during allergic inflammation: synergistic in vivo role of endogenous eotaxin.

In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies-potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.