Search

Chronic obstructive pulmonary disease is a major health problem and will become the third largest cause of death in the world by 2020. It is currently believed that an exaggerated inflammatory response to inhaled irritants, in particular, cigarette smoke (CS), causes the progressive airflow limitation, in which macrophages and neutrophils are attracted by chemokines, leading to oxidative stress, emphysema, small airways fibrosis, and mucus hypersecretion. Smoking is also associated with an increase in mast cell numbers in bronchial mucosa. This study was conducted to determine the direct effects of CS on mast cell function, using murine bone marrow-derived mast cells (BMMC) as an in vitro model. BMMC were cultured from BALB/cBy mice for 3 weeks. Cells were treated with CS medium (CSM) for 30 min or 16 h. The effects of CSM on mast cell degranulation and chemokine production were measured. Moreover, we investigated the effect of CSM on IkappaB-alpha degradation and p38, Erk1/2, p65, and CREB expression by Western blotting. We found that CSM stimulated the release of chemokines in a noncytotoxic manner but did not induce mast cell degranulation. CSM induced phosphorylation of Erk1/2, p38, and CREB and increased translocation of p65 without degradation of IkappaB-alpha NF-kappaB in mast cells. The induction of chemokine production by CSM in mast cells could promote and prolong the inflammatory process. Our observations suggest that mast cells may contribute to the pathogenesis of emphysema through a direct effect of CS on the production of proinflammatory chemokines.

Cigarette smoke is the major risk factor for the development of chronic obstructive pulmonary disease (COPD). Macrophages are suggested to orchestrate the chronic inflammatory response and tissue destruction associated with COPD by secreting interleukin (IL)-8, a major neutrophil chemoattractant. The combination of inhaled corticosteroids and long-acting beta(2)-adrenoceptor agonists are increasingly used as maintenance therapy in patients with COPD. The aim of this study was to determine whether combined fluticasone propionate, a corticosteroid, and salmeterol, a long-acting beta(2)-adrenoceptor agonist, can suppress IL-8 production by human macrophages. To mimic resident macrophages in the lung, human monocytes were cultured for 5 days in medium containing Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF). In human Monocyte-Derived Macrophages, we found that cigarette smoke medium strongly enhanced IL-8 release in a time- and concentration-dependent manner. IL-8 release by cigarette smoke was significantly suppressed in a concentration-dependent manner by fluticasone and salmeterol. Coincubation of the drugs potentiated the inhibitory effect on cigarette smoke medium-induced IL-8 production and longer preincubation times resulted in more IL-8 inhibition. Interestingly, preincubation of cells with suboptimal concentration of salmeterol for 4 h before fluticasone administration for 30 min potentiates the inhibitory effect of fluticasone on IL-8 release. In conclusion, combination therapy may provide benefits over monotherapy for the treatment of COPD patients.

Immunoglobulin (Ig) E-dependent activation of mast cells is central to the allergic response. The engagement of IgE-occupied receptors initiates a series of molecular events that causes the release of preformed, and de novo synthesis of, allergic mediators. Cysteinyl leukotrienes are able to contract airway smooth muscle and increase mucus secretion and vascular permeability and recruit eosinophils. Mast cells have also recently been recognized as active participants in innate immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat-shock proteins (HSPs). We previously demonstrated that treatment of mast cells with heat shock or acetylsalicylic acid results in an increase of TNF-alpha and IL-6 release. This effect was paralleled by expression of HSP70. In the current study, we further investigated the effects of heat shock and acetylsalicylic acid on the activation of mast cells and the release of cysteinyl leukotrienes. In mouse mast cells, derived from a culture of bone marrow cells, responsiveness to heat shock, acetylsalicylic acid and exogenous or endogenous HSP70 was monitored by measuring leukotriene C4 release. We show that after heat shock treatment and exposure to acetylsalicylic acid leukotriene production was increased. Moreover, exogenous rHSP70 also induced leukotriene production. Because it has been reported that leukotriene production in mast cells may be mediated by Toll like receptor (TLR) activation, and HSP70 also activates TLRs signaling, we further explored these issues by using mast cells that are not able to produce HSP70, i.e. heat shock factor-1 (HSF-1) knockout cells. We found that in HSF-1 knockout bone marrow derived mast cells, heat shock and acetylsalicylic acid failed to induce release of leukotrienes. Moreover, in wild type cells the surface expression of TLR4 was attenuated, whereas the intracellular expression was up-regulated. We conclude that heat shock and acetylsalicylic acid induce the production and release of heat shock proteins from mast cells, which in turn stimulate leukotriene synthesis through activation of TLR4.

Recently it has been shown that mast cells, through release of their pro-inflammatory mediators, are involved in aspirin attacks in aspirin-sensitive patients. To date, little information is available concerning 8-isoprostane (8-epi-prostaglandin F) production by mast cells. Therefore, we examined whether exposure of mast cells to aspirin can lead to isoprostane production. In this study we show that in mast cells, IgE and antigen stimulates an intracellular oxidative burst inducing H(2)O(2) and 8-epi-PGF production. Moreover, we show that exposure of mast cells to aspirin directly induces the production of 8-epi-PGF. Our study suggests that production of 8-epi-PGF by mast cells could contribute to the inflammatory response in e.g. aspirin-sensitive asthma patients.

Background: The major risk factor for the development of COPD is cigarette smoking. Smoking causes activation of resident cells and the recruitment of inflammatory cells into the lungs, which leads to release of pro-inflammatory cytokines, chemotactic factors, oxygen radicals and proteases. In the present study evidence is found for a new cellular mechanism that refers to a link between smoking and inflammation in lungs., Methods: Employing human monocyte-derived macrophages, different techniques including FACS analysis, Cytometric Bead Array Assay and ELISA were achieved to evaluate the effects of CS on pro-inflammatory cytokine secretion including IL-8. Then, Toll-like receptor neutralization was performed to study the involvement of Toll-like receptor-4 in IL-8 production. Finally, signaling pathways in macrophages after exposure to CS medium were investigated performing ELISA and Western analysis., Results: We demonstrate that especially human monocytes are sensitive to produce IL-8 upon cigarette smoke stimulation compared to lymphocytes or neutrophils. Moreover, monocyte-derived macrophages produce high amounts of the cytokine. The IL-8 production is dependent on Toll-like receptor 4 stimulation and LPS is not involved. Further research resolved the cellular mechanism by which cigarette smoke induces cytokine production in monocyte-derived macrophages. Cigarette smoke causes subsequently a concentration-dependent phosphorylation of IRAK and degradation of TRAF6. Moreover, IkappaBalpha was phosphorylated which suggests involvement of NF-kappaB. In addition, NFkappaB-inhibitor blocked cigarette smoke-induced IL-8 production., Conclusion: These findings link cigarette smoke to inflammation and lead to new insights/therapeutic strategies in the pathogenesis of lung emphysema.

Objective: It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid, ibuprofen, and indomethacin, induce anti-inflammatory effects through inhibition of cyclooxygenase enzyme activity. However, it has also been established that a variety of their anti-inflammatory effects are independent of cyclooxygenase. In the search for alternative modes of action, it was found that NSAIDs share some cellular effects with heat shock treatment. This prompted us to investigate whether NSAIDs modulate production of proinflammatory cytokines by mast cells through the heat shock response., Materials and Methods: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to heat shock and NSAIDs was monitored by measuring tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production and signaling pathways., Results: In bone marrow-derived mast cells (BMMC), we found that heat shock and a number of NSAIDs induced heat shock protein 70 (HSP70), which was closely paralleled with inhibition of IL-6 and TNF-alpha production. Surprisingly, in BMMC from HSF-1(-/-)mice, heat shock and selected NSAIDs were still able to suppress cytokine production in the absence of HSP70 induction., Conclusion: In this article, we provide evidence that inhibition of release of proinflammatory cytokines by NSAIDs and heat shock may be attributed to inhibition of the inhibitory nuclear factor kappaB (NF-kappaB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-kappaB pathway.

Objective: Mast cells are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat shock proteins (HSPs). It has been reported that HSPs are capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system. In the current study, we explored whether the stress response induces HSPs and affects the signaling pathways of mast cells., Methods: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to exogenous and endogenous HSP70 was monitored by measuring cytokine release., Results: Using BMMC, we show that treatment with heat shock or acetylsalicylic acid results in a selective induction of HSPs, and leads to release of HSP70 into the extracellular environment. The release of HSP70 from mast cells may be of functional importance. We found that after induction of HSP70, the production of TNF-alpha and IL-6 was increased. In a number of experiments, we demonstrated that exogenous/secreted HSP70 is most likely responsible for the activation of mast cells to produce cytokines. Extracellular HSP70 induced production of TNF-alpha and IL-6 through the activation of the TLR4 receptor pathway, which was evidenced by an abrogation of the response in mast cells cultured from TLR4(null) or HSF-1(-/-) mice., Conclusion: Our experiments suggest that stress conditions can induce pro-inflammatory cytokine production by mast cells through an autocrine or paracrine stimulation of TLR receptors after a heat shock response. The recognition that heat shock proteins induce mast cell activation suggests an involvement of these cells in the immunological processes induced by heat shock response.

Objective: Mast cells play pivotal roles in immediate-type and inflammatory allergic and nonallergic reactions. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) on mast cells activates a signaling pathway leading to Ca2+ mobilization and is followed by degranulation and the release of histamine and other preformed mediators, as well as de novo synthesis of arachidonic acid metabolites. In a previous study, we have demonstrated that heat shock activates heat shock transcription factor-1 (HSF-1), induces heat shock protein 70 (HSP70), and suppresses cytokine production in bone marrow-derived mast cells (BMMC). In this study, we further investigated the effects of heat shock on the activation of mast cells and the release of mast cell mediators., Methods: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-)mice, responsiveness to heat shock was monitored by measuring beta-hexosaminidase and leukotriene C4 (LTC4) release., Results: Using BMMC, we found that heat shock inhibits degranulation of BMMC without affecting leukotriene production. To further elucidate the mechanism of suppression of degranulation, we studied the effects of heat shock on the regulation of signal transduction in more detail. We found that heat shock inhibits calcium mobilization and tyrosine phosphorylation of Syk and SHIP upon IgE receptor activation, but increases the phosphorylation of SHP-1 and -2. Moreover, our results revealed that suppression of tyrosine phosphorylation of Syk and SHIP coincided with an increased tyrosine phosphatase activity., Conclusion: The inhibitory action of heat shock toward mast cell degranulation is likely due to shifting the balance between kinase and phosphatase activity.

Several studies have demonstrated that nonsteroidal anti-inflammatory drugs, such as acetylsalicylic acid (ASA), can have inhibitory or enhancing effects on inflammatory cell function. These effects seem independent of cyclooxygenase activity and prostaglandin synthesis inhibition. Here, we examined the effect of ASA on bone marrow-derived mast cells in more detail. ASA blocked the expression of cyclooxygenase-2, the production of tumor necrosis factor-alpha and interleukin-6, and the release of granule mediators from mast cells in a concentration-dependent fashion. Concomitantly, ASA inhibited nuclear factor (NF)-kappaB activity, as well as the phosphorylation and breakdown of the inhibitory protein IkappaB-alpha. We thus propose that the anti-inflammatory effects of ASA in mast cells are due to suppression of IkappaB kinase activity, thereby inhibiting subsequent phosphorylation and degradation of IkappaB-alpha, activation of NF-kappaB, and transcription of proinflammatory cytokines. The inhibition of BMMC degranulation was independent of NF-kappaB activation, however. Interestingly, the expression of cyclooxygenase-2 was not inhibited at 1mM ASA, but was even enhanced significantly. The latter might contribute to the adverse effects of ASA in ASA-sensitive asthmatics.