Charles I. Ezeamuzie

In vitro and in vivo anti-inflammatory effects of andrographolide

Andrographolide - the major active principle isolated from the plant Andrographis paniculata, has been shown to possess a strong anti-inflammatory activity. The possibility that the drug may affect asthmatic inflammation, through inhibition of the relevant inflammatory cytokines, has not been explored. The purpose of this study was, firstly, to investigate the ability of andrographolide to inhibit the release of inflammatory cytokines in vitro in a model of non-specific inflammation and subsequently to determine whether such effect can also be exerted in vivo in allergic lung inflammation. LPS-induced TNF-alpha and GM-CSF release from mouse peritoneal macrophages was inhibited by andrographolide in a concentration-dependent manner. The concentration of the drug producing 50% inhibition was 0.6 microM for TNF-alpha and 3.3 microM for GM-CSF. The maximal inhibition achieved (at 50 microM) was 77% and 94%, respectively, for the two cytokines. The drug was as efficacious as dexamethasone, but about 8-12 times less potent. The drug also suppressed LPS-induced expression of mRNA for the two cytokines, suggesting that this effect may contribute to the mechanism underlying its anti-inflammatory effects. In the in vivo study, intra-peritoneal treatment of ovalbumin-immunized and nasally-challenged mice with andrographolide significantly inhibited the elevation of bronchoalveolar fluid (BAF) levels of TNF-alpha and GM-CSF in a dose-dependent manner, with 30 mg/kg producing an inhibition of 92% and 65% of the cytokines, respectively) and almost completely abolishing the accumulation of lymphocytes and eosinophils. These results provide evidence that andrographolide is an effective anti-inflammatory drug that is active in vitro and in vivo, and affects both non-specific as well as antigen/antibody-dependent lung inflammation. Thus, andrographolide has the potential to be used in a variety of inflammatory conditions, including allergic lung inflammation.

Adenosine A3 receptors on human eosinophils mediate inhibition of degranulation and superoxide anion release

The role of adenosine A3 receptors on human eosinophil degranulation and superoxide anion (O2−) release was studied in vitro using the complement fragment C5a as the main stimulus and employing a number of selective agonists and antagonists. In the presence of cytochalasin B (CB), C5a induced a dose‐dependent release of the granular eosinophil peroxidase (EPO), but not O2−, whereas in the absence of CB O2−, but not EPO, was released. C5a‐induced EPO release was inhibited dose‐dependently by the selective A3 agonist N6‐(3‐iodobenzyl)‐5′‐N‐methylcarbamoyladenosine (IB‐MECA) and to a lesser extent by the less‐selective N6‐2‐(4‐amino‐3‐iodophenyl) ethyladenosine (APNEA). The IC50 (95% CI) for IB‐MECA was 6.8 μM (3.1–12.0 μM). At concentrations up to 100 μM, neither adenosine nor the selective A1 agonist N‐cyclopentyladenosine (CPA) and the selective A2 agonist 2‐[[2‐[4‐(2‐carboxyethyl)phenyl]ethyl]amino]‐N‐ethylcarboxamidoadenosine (CGS 21680) had any significant effect. The inhibitory effect of IB‐MECA was almost completely abolished by pre‐treatment with 1 μM of the selective A3 antagonist 9‐chloro‐2‐(2‐furyl)‐5‐phenylactylamino[1,2,4]triazolo[1,5‐c]quinazoline (MRS 1220), but not the selective A1 antagonist 1,3‐dipropyly‐8‐cyclopentylxanthine (DPCPX) or the selective A2 antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX). IB‐MECA also significantly inhibited C5a‐induced O2− release with IC50 (95% CI) of 9.5 μM (4.6–13.1 μM) whereas adenosine and the A1 agonist CPA potentiated this effect at low concentrations. The potentiation appeared to be a result of their direct O2− release from these cells, probably mediated via A1 receptors. The inhibition by IB‐MECA was selectively reversed by MRS 1220. These results show that the A3 receptors on human eosinophils mediate inhibition of both degranulation and O2− release and suggest a therapeutic potential for A3 agonists in diseases such as asthma in which activated eosinophils are involved.

Effects of some anti-asthma drugs on human eosinophil superoxide anions release and degranulation

Background: Eosinophil infiltration of bronchial tissues and subsequent release of inflammatory mediators by them are the hallmarks of bronchial asthma but it has not yet been clarified whether anti-asthma drugs affect these cells directly. In this study, we investigated the direct effects of 8 clinically used anti-asthma drugs [salbutamol, salmeterol, theophylline, denbufylline, disodium cromoglycate (DSCG), azelastine, ketotifen and dexamethasone] on superoxide anions (O–2) and eosinophil peroxidase (EPO) release from human blood eosinophils in vitro. Methods: Highly purified eosinophils were stimulated for O–2 release with platelet-activating factor (PAF) or interleukin-5 (IL-5), while for EPO release complement fragment (C5a) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) was employed. Generated products were assayed by standard techniques. Results: All the drugs, except ketotifen and dexamethasone, inhibited PAF-induced O–2 release in a dose-dependent manner. The IC50 values were 0.7, 5.8, 330, 3,500, 4,200 and 6,250 nM for DSCG, denbufylline, salmeterol, azelastine, salbutamol and theophylline, respectively. On IL-5-induced release, the effects were similar except that salbutamol completely failed to inhibit the release induced by this stimulus. In contrast, EPO release was generally poorly inhibited, especially when the release was induced by C5a. Only theophylline and azelastine (both at 10–4 M or more) were able to inhibit EPO release by both C5a and FMLP. Salbutamol and, to a lesser extent, salmeterol inhibited FMLP-, but not C5a-induced EPO release, while all the other drugs tested were inactive. Conclusions: The results show that some of the anti-asthma drugs, but not all, do exert direct effects on human blood eosinophils but these effects may be stimulus-dependent and by far more pronounced against O–2 release than against degranulation.

Histamine H2 receptors mediate the inhibitory effect of histamine on human eosinophil degranulation

The effect of histamine on human eosinophil degranulation and the receptor mediating such effect were studied in vitro using the complement C5a‐mediated eosinophil peroxidase (EPO) release model. Following pre‐treatment with 5 μg ml−1 cytochalasin B(CB), C5a induced a concentration‐dependent release of EPO from eosinophils isolated from healthy donors. Histamine (0.1–50 μM), but not L‐histidine, inhibited concentration‐dependently C5a‐induced EPO release with IC50 (95% CI) of 0.6 μM (0.3–1.2 μM) and maximal inhibition of ∼60%. A similar effect was seen with the selective H2 agonists dimaprit (IC50 (95% CI)=6.9 μM (3.2–10.6 μM)) and amthamine (IC50 (95% CI)=0.4 μM (0.2–0.7 μM)). Neither the selective H1 agonist 6‐(2‐(4‐imidazolyl)ethylamino)‐N‐(4‐trifluoromethylphenyl) heptanecarboxamide(HTMT), nor the selective H3 agonists imetit (up to 100 μM) had any significant effect. The inhibition by histamine was reversed by cimetidine (0.1–30 μM) and other H2 antagonists, but not the H1 antagonist mepyramine (1.0–100 μM), nor the H3 antagonist thioperamide (1.0–100 μM). Cimetidine (1–30 μM) shifted to the right the dimaprit log dose‐response curve, producing a pA2 value of 5.9 and Schilds plot slope of 0.98, thus confirming simple competitive antagonism. Histamine (10–100 μM) increased intracellular level of adenosine 3′,5′‐cyclic monophosphate, which was completely abolished by cimetidine (30 μM), but not mepyramine or thioperamide. The cyclic AMP analogue – dibutyryl cyclic AMP – also inhibited degranulation (IC50 ∼300 μM). The cyclic AMP phosphodiesterase(PDE) IV inhibitor rolipram (10 μM) synergistically enhanced the inhibition of EPO release by histamine. These results suggest that histamine, via stimulation of H2 receptors and a consequent elevation of intracellular levels of cyclic AMP, inhibits human eosinophil degranulation.

Positive coupling of atypical adenosine A3 receptors on human eosinophils to adenylyl cyclase

Adenosine A(3) receptors are reported to couple negatively to adenylyl cyclase (AC) but their mediation of anti-inflammatory effects in human eosinophils prompted us to investigate their coupling to AC. The A(3)-selective agonists IB-MECA and Cl-IB-MECA evoked a concentration-dependent generation of cAMP (EC(50), 3.2 and 1.8 microM, respectively) and were more potent than the A(2A) agonist CGS 21680 (EC(50)=15.4 microM) and adenosine (EC(50)=19.2 microM). The cAMP response was additive to that produced by forskolin (10 microM). The effect of IB-MECA was insensitive to A(1) and A(2A) receptor antagonists, but was antagonized by the A(3)-selective antagonist MRS 1220 (0.1-2.5 microM) in a competitive manner. The estimated K(B) of 190 nM was, however, atypical. The cyclo-oxygenase inhibitor, indomethacin, had no effect on the cAMP response. A general inverse relationship between cAMP generation and inhibition of degranulation was seen. We conclude that in human eosinophils, an atypical form of A(3) receptors positively coupled to AC may exist. The resulting cAMP generation may underlie the anti-inflammatory actions of A(3) agonists in eosinophils.

Anti-tussive and bronchodilator mechanisms of action for the enaminone E121

AIMS In this study, we investigated whether the enaminone, E121, has anti-tussive effects in a guinea pig model of cough, and if so, whether this effect is mediated via a central or peripheral site of action. We also assessed whether E121 has bronchodilator effects and the molecular mechanisms underlying any anti-tussive and/or bronchodilator effects. MAIN METHODS Whole body plethysmography was used to assess both cough and airway obstruction. A stereotaxic apparatus was used to administer drugs intracerebroventricularly (i.c.v.). Effects of E121 were examined in vitro on contractile effects in guinea pig bronchioles. KEY FINDINGS Pre-treatment of animals with E121 resulted in a significant inhibition in the citric acid-induced cough and airway obstruction compared to vehicle-pretreated animals. The K(ATP) antagonist, glibenclamide, significantly inhibited the anti-tussive and bronchoprotective effects of E121. Also, intra-tracheal administration of E121 resulted in a significant inhibition of both the citric acid-induced cough response and airway obstruction compared to vehicle-pretreated animals. By contrast, i.c.v. administration had no effect. Finally, E121 significantly inhibited carbachol-induced airway smooth muscle contractions, an effect that was reduced by both glibenclamide and propranolol. Interestingly, E121 enhanced histamine-induced cAMP release in human eosinophils although it did not directly elevate cAMP levels. SIGNIFICANCE The enaminone, E121, has anti-tussive and bronchodilatory effects and is topically, but not centrally, active. The anti-tussive mechanism of action of E121 seems to be K(ATP) channel dependent, whereas its bronchodilatory effects appear to be mediated via activation of both K(ATP) channels and β(2) receptors. Therefore, E121 may potentially represent a novel therapy for cough, particularly cough associated with airway obstruction.

Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production

The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)‐stimulated degranulation of highly purified human eosinophils using the specific PKC activator – phorbol 12‐myristate 13‐acetate (PMA). C5a‐induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration‐dependent manner by PMA (IC50: 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31‐8220 (1 μM). In the presence of phosphodiesterase inhibitor rolipram (5 μM), PMA stimulated a pronounced concentration‐dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC50: 55 nM vs 22.5 μM). The inactive PMA analogue, 4α‐PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31‐8220 (1 μM) and the selective inhibitor of the novel PKCδ, rottlerin (1–3 μM), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01–0.1 μM). Western blot analysis revealed the presence of six PKC isoforms (α, βI, βII, δ, ι and ζ) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA‐induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCδ isoform, can stimulate cAMP production, and that this may be the basis for its potent anti‐degranulatory effect.

Interactions between Theophylline and Salbutamol on Cytokine Release in Human Monocytes

The combination of β2-adrenoceptor agonists (β2-agonists) with inhaled steroids has become the standard treatment for mild to moderate asthma. Theophylline has also been combined successfully with inhaled steroids. However, the possible interaction between theophylline and β2-agonists, with regard to their anti-inflammatory effects, has not been clarified. The aim of this study was to investigate the in vitro interaction between theophylline and salbutamol on cytokine generation from human monocytes and compare it with a similar interaction between dexamethasone and salbutamol. Purified monocytes from normal donors were pretreated with the drugs (alone or in combination) and stimulated with lipopolysaccharide for 24 h. Released tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and their corresponding mRNA expressions, were determined and analyzed. Salbutamol (≥ 0.1 μM) significantly inhibited the release of TNF-α, but also significantly enhanced that of IL-6. In contrast, theophylline (50 μM) and dexamethasone (0.1 μM) strongly inhibited the generation of both cytokines. It is noteworthy that when the drugs were used in combination the effects of theophylline and salbutamol were additive in inhibiting TNF-α release, but theophylline blocked the IL-6-enhancing effect of salbutamol. A similar effect was seen when dexamethasone was combined with salbutamol. These results show that β2-agonists have opposing effects on the generation of TNF-α and IL-6, but that when they were combined with clinically relevant concentrations of theophylline, theophylline, like dexamethasone, was capable of augmenting the anti-inflammatory effects of the β2-agonists while at the same time preventing their proinflammatory effect. Thus, theophylline may have a potentially useful steroid-sparing effect.

Low-affinity IgE receptor (FcεRII)-mediated activation of human monocytes by both monomeric IgE and IgE/anti-IgE immune complex

Monocytes and macrophages of individuals with allergic diseases express increased levels of the low-affinity IgE receptors (FcepsilonRII or CD23) on their surfaces. The cross-linking of CD23-bound IgE antibody by allergen activates the cells to release inflammatory mediators. In mast cells, the binding of IgE to the high-affinity IgE receptors (FcepsilonRI) has recently been shown to activate these cells independent of allergen. It has not been determined if such is true of the binding of IgE to the low-affinity receptors. The purpose of this study was, therefore, to determine whether monomeric IgE alone can activate CD23-bearing human monocytes and how this may relate to the activation by IgE/anti-IgE immune complex. Purified monocytes, cultured for 48 h with IL-4 to up-regulate CD23 were sensitized with human myeloma IgE and further cultured for 24 h with or without anti-human IgE antibody. The release of cytokines TNF-alpha and MIP-1alpha (as an index of activation) was determined by enzyme immunoassay. Results showed that in IL-4-treated/CD23-bearing monocytes, sensitization with IgE alone caused a release of TNF-alpha and MIP-1alpha. The addition of anti-IgE antibody to cross-link the bound IgE resulted in the enhancement of the response. Such activation by monomeric IgE and IgE/anti-IgE immune complex was blocked with an anti-CD23 antibody, confirming the specific involvement of CD23 molecules. Neither of the activation modalities elevated intracellular cAMP, contrary to previous report. These results show for the first time, that in CD23-bearing monocytes, IgE sensitization alone can activate monocytes, and that ligation of such IgE by anti-IgE antibody only enhances the response. These observations have implications for the understanding of the pathophysiology of IgE-dependent inflammation accompanying many allergic diseases.

Protein Kinase C- and Reactive Oxygen Species-Dependent Stimulation of Intracellular cAMP in Human Eosinophils

Objective: The objective of this study was to investigate the role of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the signalling pathway of the novel protein kinase C (PKC)- and reactive oxygen species (ROS)-dependent stimulation of intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) production in human eosinophils. Materials and Methods: Immunomagnetically purified human eosinophils were stimulated in vitro with a PKC activator, phorbol myristate acetate (PMA), and the cAMP response in the presence of a phosphodiesterase inhibitor, rolipram, was determined. The role of ERK1/2 phosphorylation was investigated using specific inhibitors and Western blot analysis. Results: The PMA-stimulated eosinophils responded with a profound increase in intracellular levels of cAMP that was dependent on both PKC and ROS, as confirmed by the use of specific inhibitors: Ro 31-8220 for PKC and diphenyleneiodonium (DPI) for the ROS-generating enzyme NADPH oxidase. Pre-treatment of cells with the ERK1/2 inhibitor PD 98059, but not the p38-MAPK inhibitor SB203580, nor the PI3 kinase inhibitor, wortmannin, abolished the response. PMA treatment induced the phosphorylation of ERK1/2 with a time course that is consistent with a role in the cAMP response. The ERK1/2 phosphorylation was abolished by the ERK1/2 inhibitor PD 98059 and the PKC inhibitor Ro 31-8220, but not the NADPH oxidase inhibitor DPI. Conclusion: These results reveal the involvement of ERK1/2 in the signalling mechanism of PMA-stimulated, PKC- and ROS-dependent stimulation of cAMP production in human eosinophils, and show that ERK1/2 phosphorylation is upstream of ROS production in the signalling pathway.