The effect of different corticosteroids and cyclosporin A on interleukin-4 and interleukin-5 release from murine TH2-type T cells.

By secretion of interleukin-4 and interleukin-5, TH2-type T cells are thought to play an important role in the pathogenesis of asthma. Corticosteroids are currently the most effective therapy available for asthma, but recently it has been demonstrated that cyclosporin A improves lung function in patients with severe corticosteroid-dependent asthma. In order to examine the effects of corticosteroids and cyclosporin A on anti-CD3-induced production of interleukin-4 and interleukin-5 we used the murine TH2-type cell clone D10.G4.1. Interleukin-4/interleukin-5 release was inhibited by all drugs tested with the following IC50 values (nmol/l) for interleukin-4 and interleukin-5, respectively: budesonide (0.32/0.22), beclomethasone (0.65/0.33), dexamethasone (4.70/3.52), 6 alpha-methyl-prednisolone (24.04/17.02), hydrocortisone (34.27/22.55), and cyclosporin A (72.59/242.21). In conclusion, corticosteroids exert strong inhibitory effects on cytokine production by TH2-cells, which may explain, at least partly, its clinical efficacy in asthma. Cyclosporin A also showed a concentration-dependent inhibition; however, in relation to corticosteroids the inhibitory activity of cyclosporin A was found to be weaker.

Characterization of platelet-activating factor induced superoxide anion generation by guinea-pig alveolar macrophages.

Guinea-pig alveolar macrophages (AM) exhibited a concentration dependent superoxide anion (.O2-) generation as measured by means of lucigenin-chemiluminescence (CL) in response to platelet-activating factor (PAF) in the range of 1 nM to 100 nM. PAF effects on .O2(-)-generation were specific to the form of PAF that is biologically active in most systems; lyso-PAF had no effect. The CL-response was inhibited following incubation with EDTA (IC50: 859 microM), the intracellular Ca(2+)-antagonist TMB-8 (IC50: 73 microM), or the calmodulin antagonists W-7 (IC50: 13 microM) and trifluoperazine (IC50: 14 microM) indicating the involvement of Ca2+ in the signal transduction pathway. Increasing the intracellular cAMP-levels by PGE2 or forskolin resulted in an inhibition of the CL-response, whereas the beta-adrenoceptor agonist salbutamol showed no effect. On the other hand phosphodiesterase inhibition by IBMX (10 microM: 25%) or zardaverine (10 microM: 29%) also resulted in a transient inhibition of the CL-response. Protein kinase C (PKC) seemed not to be involved in the signal transduction, since the PKC-inhibitors staurosporine, H-7 and D-sphingosine were inactive. In contrast, the PAF receptor antagonists WEB-2086 (IC50: 700 nM) and WEB-2170 (IC50: 176 nM) exerted a strong inhibitory activity. The antiallergic/antiasthmatic drug azelastine also reduced the PAF induced CL-response (IC50: 12 microM), whereas the other antiallergic/antiasthmatic compounds showed almost no inhibition.

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine.

The effect of azelastine, an orally effective antiasthmatic antiallergic drug on the generation of oxygen-derived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC30 = 3.9 microM) and AM (IC30 = 9.8 microM). In AM zymosan-induced CL was inhibited 21.7% by 10 microM azelastine, whereas in PMNL it remained unchanged up to 10 microM azelastine. Azelastine has a significantly stronger inhibitory effect (IC30 = 4.2 microM) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibits oxygen-derived free radical generation by interaction with protein kinase C.

Effect of phosphodiesterase inhibition on IL-4 and IL-5 production of the murine TH2-type T cell clone D10.G4.1.

The effect of various phosphodiesterase (PDE) inhibitors on anti-CD3 induced interleukin-(IL)-4 and IL-5 production of the murine T helper cell clone of type 2 phenotype D10.G4.1 (D10) has been investigated in vitro. D10 cells were incubated in the presence of drugs and anti-CD3 mAb for 16 h before measurement of cytokines in the cell supernatants by ELISA. Whereas all PDE inhibitors tested exerted minimal effects on anti-CD3 induced IL-4 production, a marked increase in IL-5 production by the non-selective PDE inhibitors IBMX, theophylline and enprofylline was observed. The action of these non-selective PDE inhibitors was mimicked by the PDE IV-selective inhibitor rolipram and in part by the PDE III-selective inhibitors motapizone and milrinone, whereas the PDE V-selective inhibitor zaprinast was inactive. Rolipram and motapizone enhanced IL-5 production in a synergistic fashion. In support of the functional importance of PDE III and IV for IL-5 synthesis in intact murine D10 cells, we have found PDE III and IV to be the predominant isoenzyme activities in corresponding cell lysates. The stimulatory effect of rolipram on IL-5 production was almost totally reversed by the protein kinase A inhibitor KT-5720. In addition, the membrane-permeable cAMP analogue 8-bromo-cAMP mimicked the stimulatory effect of PDE inhibitors on IL-5 production while leaving IL-4 levels unaffected. Both results support the view that the action of the PDE inhibitors on murine D10 cells is mediated via an elevation of intracellular cAMP.

Histamine increases anti-CD3 induced IL-5 production of TH2-type T cells via histamine H2-receptors.

Besides its proinflammatory functions histamine released from basophils and mast cells during immediate-type hypersensitivity reactions is known to inhibit several lymphocyte functions like IL-2 and gamma-IFN production. Recently, it has been shown that T helper cells of type 2 phenotype (TH2) represent the T cell fraction which may play a pivotal role in the promotion of the allergic inflammatory eosinophilic late-phase reaction by secretion of cytokines, especially IL-4 and IL-5. We have investigated the effect of histamine on anti-CD3 induced IL-4 and IL-5 production by TH2 cells. Histamine in concentrations between 10(-7) and 10(-5) mol/l concentration-dependently increased anti-CD3 induced IL-5 production up to 120%, whereas IL-4 production was not affected. The activity of histamine in increasing IL-5 production was mimicked by the H2-receptor agonist dimaprit. Histamine induced increase in IL-5 production was inhibited by histamine H2-receptor antagonists, but remained unaffected by H1- or H3-receptor antagonists. Administration of forskolin which directly stimulates the production of cAMP, the second messenger of the H2-receptor, also resulted in an increase in anti-CD3 induced IL-5 production. These results indicate that the histamine-mediated increase in anti-CD3 induced IL-5 production is mediated via H2-receptors. Consequently, histamine released from mast cells and basophils during the early-phase allergic reaction may act as an important stimulatory signal for the initiation of the allergic inflammatory late-phase reaction by increasing local IL-5 production of allergen triggered TH2 cells.

Effect of corticosteroids, cyclosporin A, and methotrexate on cytokine release from monocytes and T-cell subsets.

Corticosteroids are the most effective drugs in the management of asthma. However, because of their known side effects and the existence of corticosteroid-resistant patients, there is a need for substitute medications in asthma therapy. Using cell lines, in the present study, the two corticosteroids dexamethasone (Dex), and beclomethasone (Bec), as well as the immunosuppressant cyclosporin A (CsA), and the antimetabolic drug methotrexate (Mtx) were examined in their effect on release of immunoreactive IL-1 beta, IL-2, IL-4, IL-5, and IL-8. THP-1 cells served as a test model for monocytes secreting IL-1 beta and IL-8 upon stimulation by lipopolysaccharide. Jurkat cells were used as a test model for TH1-type T-cells and were stimulated for IL-2 release with a combination of phytohemagglutinin and phorbol myristate acetate. Representing TH2-type T-cells, D10.G4.1 cells challenged by anti-CD3-mAb produced IL-4, and IL-5. Considerable qualitative and quantitative differences in the relative efficacy of the test compounds were found. Following IC50 values (nmol/l) of the test compounds were estimated (IL-1 beta/IL-8/IL-2/IL-4/IL-5): Dex (10.8/35.7/ > 10,000.0/5.1/4.1), Bec (30.9/102.2/8591.4/0.6/0.4), and CsA (318.7/6211.2/2.3/68.2/237.9). Mtx in concentrations up to 10,000.0 nmol/l was completely inactive. It can be concluded that corticosteroids show another inhibition pattern than CsA: corticosteroids affect mainly TH2-type T-cells, while CsA primarily inhibits the TH1-type T-cell response.