Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle.

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.

beta(2)-adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells.

1. Airway smooth muscle (ASM) is a potential source of multiple pro-inflammatory cytokines during airway inflammation. beta-Adrenoceptor agonist hyporesponsiveness is a characteristic feature of asthma, and interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha are implicated in its cause. Here, the capacity of beta-adrenoceptor agonists to prevent release of GM-CSF, RANTES, eotaxin and IL-8, elicited by IL-1 beta or TNF alpha, was examined in human ASM cells. 2. Isoprenaline (approximately EC(50) 150 nM), a non-selective beta-adrenoceptor agonist, and salbutamol ( approximately EC(50) 25 nM), a selective beta(2)-adrenoceptor agonist, attenuated release of GM-CSF, RANTES and eotaxin, but not IL-8 (EC(50) >1 microM). The maximum extent of attenuation was RANTES > or = eotaxin > GM-CSF >> IL-8, and was prevented by either propranolol (1 microM), a non-selective beta-adrenoceptor antagonist, or ICI 118511 (IC(50) 15 nM), a selective beta(2)-adrenoceptor antagonist. 3. The cyclic AMP-elevating agents, dibutyryl cyclic AMP ( approximately EC(50) 135 microM), forskolin ( approximately EC(50) 530 nM) and cholera toxin ( approximately EC(50) 575 pg ml(-1)) abolished IL-1 beta-induced release of GM-CSF, RANTES and eotaxin, but not IL-8. 4. IL-1 beta (1 ng ml(-1)) attenuated early increases (up to 1 h) in cyclic AMP formation induced by salbutamol (1 microM), but not by forskolin (10 microM). The cyclo-oxygenase inhibitor, indomethacin (1 microM) prevented later increases (3 - 12 h) in IL-1 beta-stimulated cyclic AMP content, but did not prevent the attenuation by salbutamol of IL-1 beta-induced cytokine release. 5. We conclude in human ASM cells that activation of beta(2)-adrenoceptors and generation of cyclic AMP is negatively-linked to the release, elicited by IL-1 beta or TNF alpha, of eosinophil-activating cytokines such as GM-CSF, RANTES and eotaxin, but not IL-8.

Cultured human airway smooth muscle cells stimulated by interleukin-1beta enhance eosinophil survival.

Airway smooth muscle may be an important cellular source of proinflammatory mediators and cytokines and may participate directly in airway inflammation. In this study we have examined whether airway smooth muscle cells could contribute to mechanisms of eosinophil accumulation by prolonging their survival. To investigate this possibility, conditioned medium from human airway smooth muscle cells stimulated with interleukin (IL)-1beta was examined on the in vitro survival of highly purified human peripheral blood eosinophils. After 7 d, when cultured in control medium, less than 1 +/- 0.2% of the initial eosinophil population remained viable. In contrast, culture in medium conditioned for 96 h by human airway smooth muscle cells stimulated with IL-1beta (1 pg-100 ng/ml) resulted in a concentration-dependent increase in eosinophil survival. (The concentration that produced 50% of this effect was 0.03 ng/ml IL-1beta.) Maximum eosinophil survival occurred at 1 to 3 ng/ml IL-1beta. This effect was also time-dependent and was readily detected in airway smooth muscle cell-conditioned medium after just 3 h of stimulation with IL-1beta (1 ng/ml). It continued to increase before reaching a plateau around 24 h, with no decrease in activity for up to 120 h of stimulation. Conditioned medium from unstimulated airway smooth muscle cells did not enhance eosinophil survival. The survival-enhancing activity was completely inhibited (the concentration that inhibited 50% [IC50] was 6.9 microg/ml) by a polyclonal goat antihuman antibody to granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3-100 microg/ml), but antibodies (10-100 microg/ml) to IL-3 and IL-5, and a normal goat immunoglobulin G control had no effect on the eosinophil survival-enhancing activity. GM-CSF levels in culture medium from smooth muscle cells were markedly increased by IL-1beta and were maximum at 30 ng/ml (0.037 ng/ml/10(6) cells versus 3.561 ng/ml/10(6) cells, unstimulated versus 30 ng/ml IL-1beta). The IL-1 receptor antagonist inhibited both the production of GM-CSF (IC50 19. 1 ng/ml) and the eosinophil survival-enhancing (IC50 53.7 ng/ml) activity stimulated by IL-1beta. Release of GM-CSF elicited by IL-1beta was inhibited by dexamethasone but not by indomethacin. These data indicate that cultured human airway smooth muscle cells stimulated with IL-1beta support eosinophil survival through production of GM-CSF and thus may contribute to the local control of inflammatory cell accumulation in the airways.

Cyclic AMP-elevating agents prolong or inhibit eosinophil survival depending on prior exposure to GM-CSF.

1. Purified human eosinophils survived for up to 7 days when cultured in vitro in the presence of 1 ng ml-1 granulocyte-macrophage colony stimulating factor (GM-CSF) with a viability of 73%. In the absence of GM-CSF, eosinophil viability decreased after one day in culture, and only 4% of cells were viable by day 4. 2. Culture of eosinophils with cholera toxin produced a concentration-dependent decrease in GM-CSF-induced survival at 7 days (IC50 = 7 ng ml-1) which was associated with a 6 fold increase in the intracellular cyclic AMP concentration. This inhibition of cell survival could be prevented by the addition of the protein kinase A inhibitor, H89 (10(-6)M). 3. When eosinophils were cultured with dibutyryl cyclic AMP, there was a concentration-dependent inhibition of GM-CSF-induced survival at 7 days with an IC50 of 200 microM. The related cyclic nucleotide analogue, dibutyryl cyclic GMP did not inhibit GM-CSF-induced eosinophil survival over the same concentration range. 4. Culture of eosinophils with forskolin, or with the phosphodiesterase inhibitors, rolipram and SK&F94120, had no effect on GM-CSF-induced eosinophil survival at any concentration examined. 5. After 7 days' culture in the absence of GM-CSF, fractionation of eosinophil DNA on agarose gels demonstrated a 'ladder' pattern characteristic of apoptosis. GM-CSF prevented DNA fragmentation and this protection could be overcome by both cholera toxin and dibutyryl cyclic AMP. 6. GM-CSF did not affect intracellular cyclic AMP concentrations in unstimulated eosinophils or in cells stimulated by cholera toxin. Thus, GM-CSF does not apparently increase eosinophil survival by affecting cyclic AMP levels. 7. In the absence of GM-CSF both cholera toxin and dibutyryl cyclic AMP decreased the rate of eosinophil death, when compared to cells cultured with medium alone. The t1/2 values for cell death were 1.63 +/- 0.3, 2.46 +/- 0.3 and 4.62 +/- 1.0 days for cells cultured in the presence of medium, cholera toxin and dibutyryl cyclic AMP respectively. 8. In conclusion, cyclic AMP exerts opposing effects on eosinophil survival depending on prior exposure of the cells to GM-CSF.

Selective enhancement of GM-CSF, TNF-alpha, IL-1 beta and IL-8 production by monocytes and macrophages of asthmatic subjects.

Previous work has demonstrated an increase in the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by monocytes derived from asthmatic individuals. We have suggested that monocytes and macrophages enhance airways inflammation by augmented cytokine production. We tested this hypothesis by measuring the production of GM-CSF and macrophage-derived cytokines, namely interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8), from unstimulated and lipopolysaccharide (LPS)-stimulated peripheral blood monocytes and alveolar macrophages in 31 asthmatic and 11 normal, control subjects. The basal production of GM-CSF was four fold higher in the monocytes of asthmatic individuals, but there was no significant difference in the basal production of TNF-alpha, IL-1 beta and IL-8. After stimulation with LPS, asthmatic monocytes produced twofold more GM-CSF and fourfold more IL-1 beta than the monocytes from control subjects. Unstimulated macrophages from asthmatic subjects produced significantly less GM-CSF and TNF-alpha than macrophages from controls, and there was no difference in either IL-1 beta or IL-8 production. When stimulated by LPS, macrophages from asthmatic subjects produced twofold more GM-CSF, threefold more TNF-alpha and fourfold more IL-8. The levels of IL-8 produced by both monocytes and macrophages were at least 20 fold higher than those of the other cytokines measured. There is selectivity in the upregulation of cytokine production by monocytes and macrophages in asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of exogenous eicosapentaenoic acid on generation of leukotriene C4 and leukotriene C5 by calcium ionophore-activated human eosinophils in vitro.

Exogenous eicosapentaenoic acid (EPA) has been compared with exogenous arachidonic acid (AA) for its ability to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human eosinophils, and for its suitability as a parallel substrate in this pathway. Products were quantitated by specific RIA and tetraene and pentaene leukotrienes (LT) were separated by reverse-phase HPLC. Eosinophils were preincubated with control buffer, exogenous EPA or AA and stimulated optimally with 10 microM calcium ionophore (A23187) for 15 min. Mean generation of LTC4 in the absence of fatty acid was 6.0 = 1.1 ng/10(6) eosinophils (mean = SEM, n = 5). In the presence of EPA, the amount of LTC4 generated rose to peak at 16.5 +/- 1.9 ng/10(6) eosinophils at 10 micrograms/ml EPA and then fell to 8.3 +/- 3.1 ng/10(6) cells at 40 micrograms/ml EPA. The EPA derivative, LTC5 was first detectable at 5 micrograms/ml EPA with 4.8 +/- 1.2 ng/10(6) cells and gradually rose with increasing dose of EPA to be maximal at 40 micrograms/ml with 12.7 +/- 2.2 ng/10(6) cells. Identity of the LTC5 was confirmed by an identical retention time to synthetic LTC5 standard, immunoreactivity to a specific antibody against LTC4 and LTC5 and a typical UV absorbance spectrum. When eosinophils were preincubated with AA and similarly stimulated, LTC4 generation gradually increased from a baseline of 6.7 +/- 0.7 ng/10(6) cells in the absence of fatty acid to reach a maximum of 12.9 +/- 0.8 ng/10(6) cells at 40 micrograms/ml of AA. Total LTC generation was nearly twofold more with cells incubated with EPA than with cells incubated with AA (p < 0.05). Thus, EPA does not suppress LTC generation from eosinophils but stimulates it at lower doses and is a substrate for LTC5 generation.

Glucocorticoids inhibit granulocyte-macrophage colony-stimulating factor-1 and interleukin-5 enhanced in vitro survival of human eosinophils.

The effects of three corticosteroids, hydrocortisone, dexamethasone and methylprednisolone, on eosinophil survival enhanced by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant murine interleukin-5 (rmIL-5) have been studied. Eosinophils were incubated at a concentration of 5 x 10(5) cells/ml in the presence of different concentrations of the three steroids with either rhGM-CSF (1 ng/ml) or rmIL-5 (50 U/ml). The eosinophils were cultured in the presence of the same concentrations of rhGM-CSF and rmIL-5 alone as a positive control and medium alone as a negative control. Viability was assessed after 7 days by trypan blue exclusion. All three steroids inhibited rhGM-CSF-enhanced eosinophil survival in a dose-dependent manner; the dose of these drugs producing 50% inhibition (IC50) was greater than 1.0 x 10(-4) M, 6.5 x 10(-6) M and 1.8 x 10(-6) M for hydrocortisone, dexamethasone and methylprednisolone, respectively. When eosinophils were cultured with the same concentration of rhGM-CSF in the presence of two non-glucocorticoids, beta-oestradiol and testosterone, neither of these steroids inhibited eosinophil survival over the concentration range 1 x 10(-10) M to 1 x 10(-4) M (n = 5). Dexamethasone and methylprednisolone, but not hydrocortisone, also inhibited eosinophil survival induced by rmIL-5 in a dose-dependent manner. These results suggest one mechanism for the efficacy of corticosteroids against eosinophil-related disorders.

Identification of the major activity derived from cultured human peripheral blood mononuclear cells, which enhances eosinophil viability, as granulocyte macrophage colony-stimulating factor (GM-CSF).

Eosinophils (EOSs) cultured in the presence of 50% peripheral blood mononuclear cell (PBMC)-derived culture supernatants remained 67% +/- 7% (mean +/- SEM; n = 5) viable for 7 days. In the absence of PBMC supernatant, only 15% +/- 7% of cells remained viable for 7 days. PBMC supernatants from six atopic individuals, with eosinophilia, and six normal subjects, with no eosinophilia, were compared for EOS viability-enhancing activity with the same target EOSs. Optimal conditions for the production of viability-enhancing activity by mononuclear cells were established as a 24-hour culture period, with a concentration of 2 x 10(6) cells per milliliter. Comparison of monocyte-enriched and lymphocyte-enriched culture supernatants for the production of the EOS viability-enhancing activity indicated that both cell types released the factor. C-18 Sep-Pak separation of the PBMC culture supernatant yielded a major EOS viability-enhancing activity in the aqueous eluent, suggesting a hydrophilic molecule. This major activity was neutralized by a specific antibody to granulocyte/macrophage colony-stimulating factor but was unaffected by specific antibodies to interleukin-3 and interleukin-5. A second, minor viability-enhancing activity was observed in the 100% methanol fraction, indicating the presence of a more hydrophobic molecule. The supernatants from the PBMCs of the atopic individuals consistently enhanced EOS survival to a greater extent than supernatants from the PBMCs of the normal, nonatopic individuals.