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The invention of immunotherapy, such as immune checkpoint inhibitors (ICIs) for advanced-stage non-small cell lung cancer (NSCLC), has become a new standard of care for a defined group of NSCLC patients. However, the possible impacts of ICI interactions with analgesics for alleviating cancer-related pain are unclear and lack clinical evidence. Many studies have indicated that opioids detrimentally affect the immune system, possibly harming patients of ongoing immunotherapy. Opioids may repress the immune system in various ways, including impairing T cell function, upregulating immunosuppressor Treg cells, and interrupting intestinal microflora composition that disrupts the entire immune system. Furthermore, opioids can influence tumor progression and metastasis directly as opioid receptors are overexpressed in several types of NSCLC. In contrast, another analgesic acting on cyclooxygenase (COX) inhibition (i.e., NSAIDs) may be a candidate for adjuvant therapy since COX-2 is also expressed in the tumor cells of NSCLC patients. In addition, COX-2 is associated with tumor proliferation and metastasis. Therefore, both prospective and retrospective studies should confirm the advantages and disadvantages of the concurrent use of analgesics and ICIs in a clinical setting.

Pharmacology is one of the cornerstones in health sciences curricula as well as research-oriented biomedical programs in higher education. New educational insights and scientific developments in teaching and learning, as well as exciting discoveries in pharmacology research, must prompt pharmacology teachers to regularly rethink and adjust their teaching. Reflecting on pharmacology education, this paper touches upon some educational issues that might inspire readers of this journal who are involved in pharmacology teaching.

Toll-like receptors are critical structures in sensing the invading pathogens via conserved moieties termed pathogen associated molecular patterns and in directing the innate and adaptive immune responses. Studies have shown that Toll-like receptors are not limited to normal immune cells but are expressed on tumour cells as well, including those of lymphoid neoplasms particularly B-cell malignancies, multiple myeloma and chronic lymphocytic leukemia. Neoplastic plasma cells in multiple myeloma usually show a different pattern of Toll-like receptor expression compared to normal B cells. These receptors on multiple myeloma cells, have been indicated to have a role in their proliferation, differentiation and survival, probably through induction of autocrine IL-6 secretion, and in their immunomodulatory functions. Moreover, it is speculated that these molecules may contribute to osteolytic lesions through activation of osteoclasts, and to angiogenesis through induction of pro-angiogenic factors. Knowledge on Toll-like receptor signalling in the biology of malignant plasma cells or their cellular microenvironment may give new insights into pathogenesis of multiple myeloma and may open new avenues for the therapy of this disease.

Summary Background Mast cells are important effector cells in innate or acquired immunity that contribute to host defence. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive and pro-inflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor. Objective In this study, we investigated the effect of CSM on the allergic activation of mast cells through IgE and antigen. Methods Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The release of granules, production of leukotrienes, chemokines and cytokines was determined in the supernatants by ELISA. The effect of CSM exposure on intracellular signalling, especially the nuclear factor (NF)-κB and extracellular signal-regulated kinase (Erk)1/2 pathways, was analysed by Western blotting. Results CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells, activating transcription factor (ATF)-1 was phosphorylated after stimulation with IgE/Ag. LPS-activated mast cells were not influenced by CSM. Conclusion Our study suggests that exposure to cigarette smoke may lead to a reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial-derived LPS.

Allergies are the result of an inappropriate reaction against innocuous environmental proteins. The prevalence and severity of allergic diseases has increased dramatically during the last decade in developed countries. Allergen-specific T helper (Th) cells play a pivotal role in the pathogenesis of allergic hypersensitivity reactions. These Th cells activate a complex immune reaction that triggers the release of potent mediators and enhances the recruitment of inflammatory cells, which in turn elicit an inflammatory response that leads to the clinical symptoms of allergic disease. The current therapies for allergic diseases focus primarily on control of symptoms and suppression of inflammation, without affecting the underlying cause. However, the knowledge about the pathophysiology of allergic diseases has substantially increased, offering new opportunities for therapeutic intervention. In this review, we will focus on current insights into the mechanism of allergic reactions.

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.

Summary Background: The neurotrophin nerve growth factor (NGF) has been implicated as a mediator in allergic asthma. Direct evidence that inhibition of NGF-induced activation of neurotrophin receptors leads to improvement of airway symptoms is lacking. We therefore studied the effects of inhibitors of NGF signal transduction on the development of airway hyper-responsiveness (AHR) and pulmonary inflammation in a guinea-pig model for allergic asthma. Methods: Airway responsiveness to the contractile agonist histamine was measured in vivo in guinea-pigs that were sensitized and challenged with ovalbumin (OVA). Inflammatory cell influx and NGF levels were determined in bronchoalveolar lavage fluid (BALF). Substance P, a key mediator of inflammation, was measured in lung tissue by radioimmunoassay, while substance P immunoreactive neurons in nodose ganglia were measured by immunohistochemistry. Results: OVA challenge induced an AHR after 24 h in OVA-sensitized guinea-pigs. This coincided with an increase in the amount of NGF in BALF. Simultaneously, an increase in the percentage of substance P immunoreactive neurons in the nodose ganglia and an increase in the amount of substance P in lung tissue were found. We used tyrosine kinase inhibitors to block the signal transduction of the high-affinity NGF receptor, tyrosine kinase A (trkA). Treatment with the tyrosine kinase inhibitors (K252a or tyrphostin AG879) both inhibited the development of AHR, and prevented the increase in substance P in the nodose ganglia and lung tissue completely whereas both inhibitors had no effect on baseline airway resistance. Neither treatment with K252a or tyrphostin AG879 changed the influx of inflammatory cells in the BALF due to allergen challenge. Conclusions: We conclude that substance P plays a role in the induction of AHR in our model for allergic asthma which is most likely mediated by NGF. As both tyrosine kinase inhibitors AG879 and K252a show a similar inhibitory effect on airway function after allergen challenge, although both tyrosine kinase inhibitors exhibit different non-specific inhibitory effects on targets other than trkA tyrosine kinases, it is likely that the induction of substance P derived from sensory nerves is mediated by NGF via its high-affinity receptor trkA.

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-α (TNF-α) 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-α 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 κB (NF-κB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-κB pathway.

Elevated levels of nerve growth factor have been linked to the onset and persistence of many pain-related disorders and asthma. Described here are the design, expression, refolding, and purification of a monomeric (nonstrand-swapped) form of the binding domain of the nerve growth factor receptor, designated TrkAd5. We have shown that TrkAd5 produced recombinantly binds nerve growth factor with picomolar affinity. TrkAd5 has been characterized using a variety of biophysical and biochemical assays and is shown here to be stable in both plasma and urine. The palliative effects of TrkAd5 are demonstrated in animal models of inflammatory pain and allergic asthma. We conclude that TrkAd5 will prove effective in ameliorating both acute and chronic conditions where nerve growth factor acts as a mediator and suggest a role for its application in vivo as a novel therapeutic.

Objective Mast cells play pivotal roles in immediate-type and inflammatory allergic and nonallergic reactions. Cross-linking of the high-affinity receptor for IgE (FceRI) on mast cells activates a signaling pathway leading to Ca 2+ 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 β-hexosaminidase and leukotriene C 4 (LTC 4 ) 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.

Background We have previously demonstrated that the administration of nerve growth factor (NGF) to guinea-pigs results in airway hyper-responsiveness within 1 h. Objective In the present study we document the involvement of NGF in the acute allergic airway response. Methods Guinea-pigs that are sensitized to ovalbumin show an acute bronchoconstriction directly after challenge with ovalbumin. Results Intratracheal application of 10 µg of antibodies directed against NGF (anti-NGF) 1 h before the challenge reduces the acute severe bronchoconstriction to approximately 40% and the sustained bronchoconstriction to approximately 20% of the reaction in controls. This shows a high potency of anti-NGF in diminishing the direct bronchoconstriction. Inhibition of the tyrosine kinases of the tyrosine kinase receptor A, the high-affinity receptor for NGF, has no effect on the bronchoconstriction. Therefore, we postulate that the p75, the low-affinity receptor for neurotrophins, is responsible for the acute bronchoconstriction. Our findings suggest a role for NGF in the induction of the acute asthmatic reaction. Conclusion These findings offer a new potential therapeutic strategy for the treatment of allergic asthma.

Nerve growth factor induces an airway hyperresponsiveness in vivo in guinea-pigs, as we have shown previously. Since antagonizing the neurokinin-1 (NK1) receptor can prevent this NGF-induced airway hyperresponsiveness and since sensory nerves release tachykinins, we investigated the role of sensory nerves in the NGF-induced airway hyperresponsiveness. We used isolated tracheal rings from guinea-pigs to measure tracheal contractility. In these rings sensory nerve endings are present, but these endings lack any contact with their cell bodies. In this in vitro system, NGF dose-dependently induced a tracheal hyperresponsiveness to histamine. The NK1 receptor antagonist SR140333 could block the induction of tracheal hyperresponsiveness. To further investigate the involvement of sensory nerve endings we used the cannabinoid receptor 1 (CB1) agonist R-methanandamide to inhibit excitatory events at the nerve terminal. The CB1 receptor agonist was capable of blocking the tracheal hyperresponsiveness to NGF in the isolated system, as well as the airway hyperresponsiveness to NGF in vivo. This indicates that NGF can induce an increase in airway responsiveness in the absence of sensory nerve cell bodies. NGF may act by increasing substance P release from sensory nerve endings, without upregulation of substance P in the neurons. Substance P in its turn is responsible for the induction of the NGF-induced airway hyperresponsiveness. Keywords: Nerve growth factor, airway hyperresponsiveness, guinea-pig, sensory nerve ending, substance P, SR140333, methanandamide Introduction The neurotrophin NGF is a newly studied mediator in relation to allergic diseases (Braun et al., 1998; de vries et al., 1999; Virchow et al., 1998). Circulating NGF levels are increased in humans with rhinoconjunctivitis, urticaria-angioedema or asthma; NGF serum levels were particularly high in patients with allergic asthma (Bonini et al., 1996). Moreover, an increase in the neurotrophins NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in bronchoalveolar lavage fluids after allergen challenge is reported in patients with asthma (Virchow et al., 1998). In patients with allergic rhinitis a very fast increase in NGF in the nasal lavage fluids is found 10 min after allergen challenge (Sanico et al., 2000). Furthermore, allergen challenge induced an increase in NGF levels in bronchoalveolar lavage fluid of sensitized mice (Braun et al., 1998). Inflammatory mediators, including interleukin-1, interleukin-4, interleukin-5, tumour necrosis factor α, and interferon-γ have been shown to induce the release of NGF (Brodie, 1996; Brodie et al., 1998; Hattori et al., 1994; Yoshida et al., 1992). In addition to neurons, non-neuronal cells such as mast cells (Leon et al., 1994), fibroblasts (Hattori et al., 1994), T-cells (Lambiase et al., 1997; Moalem et al., 2000), eosinophils (Solomon et al., 1998) and lymphocytes (Barouch et al., 2000) are able to synthesize NGF. NGF shows various pathologic properties in inflammatory models, which could be interesting in relation to allergic asthma as well (reviewed in Braun et al., 1999; 2000). NGF affects immune cell activity, as it promotes inflammatory mediator release from basophils (Burgi et al., 1996), mast cells (Matsuda et al., 1988), T- and B-cells (Lambiase et al., 1997; Otten et al., 1989) and macrophages (Susaki et al., 1996). Furthermore, NGF induces antibody synthesis and secretion from B cells (Otten et al., 1989), attracts mast cells (Sawada et al., 2000), and induces differentiation of mast cells (Welker et al., 2000) as well as of monocytes (Ehrhard et al., 1993). Moreover, NGF induces a shift to the production of Th2 type cytokines in a model for multiple sclerosis (Villoslada et al., 2000), as well as in a mouse model for allergic asthma (Braun et al., 1998). Furthermore, NGF is able to sensitize neurons and it induces an enhanced production of substance P and other tachykinins in sensory nerves (Lindsay & Harmar, 1989). Tachykinins play a role in neurogenic inflammation and in the development of airway hyperresponsiveness and asthma (Lundberg, 1995). We showed that administration of NGF induced an airway hyperresponsiveness in guinea-pigs (de vries et al., 1999). The use of a NK1 receptor antagonist could prevent this hyperresponsiveness. This points to a role for substance P, the preferred ligand of the NK1 receptor (de vries et al., 1999). Furthermore, Hunter et al. (2000) showed that NGF induces an increase in substance P in guinea-pig airway sensory neurons. The present study focuses on the role of sensory nerves in NGF-induced airway hyperresponsiveness. Isolated tracheal rings containing sensory nerve endings, without any contact with their cell bodies, were used to elucidate the involvement of sensory nerve endings as opposed to the neuronal cell body.

The involvement of bradykinin in virus-induced airway hyperresponsiveness (AHR) in guinea pig airways in vivo was determined with the B(2)-receptor antagonist Hoe 140. The efficacy of Hoe 140 treatment was assessed through its effect on the bradykinin-induced (up to 2.5 microgram/100 g B.W. administered intravenously) decrease in blood pressure (BP). Hoe 140 (0.1 micromol/kg), administered subcutaneously twice a day for 5 d almost completely blocked bradykinin-induced changes in BP. Four days after parainfluenza-3 (PI-3) virus infection, guinea pigs showed AHR; excessive airway contraction was found with histamine-receptor stimulation. This hyperresponsiveness was completely inhibited by pretreatment with Hoe 140 (0.1 micromol/kg) administered subcutaneously twice a day for five consecutive days, starting 1 d before virus inoculation. Interestingly, nebulized delivery of bradykinin itself to captopril-treated animals induced an AHR comparable to that observed in virus-treated guinea pigs. Viral infection also caused influx of bronchoalveolar cells into the lungs. Both histologic examinations and lung lavage experiments showed that this cell influx could not be inhibited by pretreatment with Hoe 140. In summary, the results of the study show that bradykinin is involved in a cascade of events leading to AHR after a viral infection in guinea pigs, without affecting bronchoalveolar cell influx.

Leukotrienes represent a group of lipid mediators that play a very important role in a wide variety of pathological conditions. The presence of leukotrienes in inflammatory sites has been extensively documented, and accordingly research efforts have been directed towards the development of drugs that interfere with the formation or effects of leukotrienes. Although clinical application of such drugs has been disappointing in the past, recent discoveries of more potent and selective drugs seem to be promising. This review attempts to highlight some of these exciting developments.

Bronchial epithelial cells express the intercellular adhesion molecule-1 that mediates binding of activated neutrophils via interaction with Mac-1 and/or leukocyte function-associated antigen-1. In this study, we examined whether increased intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) affected neutrophil adhesion to the human bronchial epithelial cells. It was found that the N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated neutrophil adhesion was concentration dependently inhibited when the cAMP analogs dibutyryl adenosine 3',5'-cyclic monophosphate or 8-bromoadenosine 3',5'-cyclic monophosphate were present. The beta-adrenergic receptor agonists isoprenaline and salmeterol, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), were also able to inhibit the fMLP-stimulated adhesion of neutrophils to bronchial epithelial cells. These agonists in combination with IBMX significantly increased the intracellular cAMP level in both neutrophils and epithelial cells. Preincubation of neutrophils with the long-acting beta2-adrenergic receptor agonist salmeterol (in the presence of IBMX) inhibited their fMLP-stimulated adhesion to epithelial cells, whereas pretreatment of epithelial cells did not influence the adhesion process. When ethanol-fixed epithelium was used, salmeterol pretreatment also diminished the adhesion of stimulated neutrophils. Moreover, combinations of salmeterol or isoprenaline with IBMX inhibited fMLP-upregulated Mac-1 expression. Therefore, we conclude from these data that elevation of intracellular cAMP in the neutrophil inhibits stimulated neutrophil adhesion to bronchial epithelial cells via Mac-1.

It has become clear that the bronchial epithelium is not just a passive barrier but plays an active role in inflammation. It can produce several inflammatory mediators and does express cell adhesion molecules of which intercellular adhesion molecule (ICAM)-1 can be upregulated by cytokines like interferon (IFN)-gamma. In the present study, we analyzed in detail the interaction of neutrophils with human bronchial epithelial cells, both primary cultured cells and the bronchial epithelial cell line BEAS-2B. Confluent monolayers of epithelial cells were incubated with freshly isolated 51Cr-labeled neutrophils for 30 min at 37 degrees C; then the nonadherent cells were removed by washing gently. Stimulation of the epithelial cells with IFN-gamma or the combination of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) (which doubles the ICAM-1 expression) increased neutrophil adhesion. Activation of the neutrophils themselves with N-formylmethionyl-leucyl-phenylalanine (fMLP), platelet-activating factor, or TNF-alpha also caused a profound enhancement of the adhesion. A significant additional increase was found when the epithelial cells had been exposed to IFN-gamma and the neutrophils were stimulated with fMLP simultaneously. This effect was even more pronounced with epithelium preincubated with IFN-gamma and TNF-alpha. With the use of monoclonal antibodies against CD18 and ICAM-1, it was demonstrated that the increased adhesion was mainly mediated by the ICAM-1/beta 2-integrin interaction. This study highlights that both the activation state of the bronchial epithelial cells and the activation state of the neutrophils are critical for their interactive adhesion.

The effect of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid (13-HODE) on degranulation of human polymorphonuclear leukocytes (PMNs) was investigated by measuring the expression of CD11b and CD67 on the plasma membrane. 13-HODE (5 μM) by itself induced degranulation of PMNs, but to a lesser extent as compared to PAF and fMLP. In addition, 13-HODE was found to inhibit the PAF-induced degranulation whereas an additive effect on the fMLP-induced PMNs degranulation was observed. These results indicate that 13-HODE can play a modulatory role in degranulation of PMNs.

Objective The interaction of human B lymphocytes as recirculating cells with their microenvironment components including fibronectin is an instrumental process that directs their further responses in an inflammatory milieu or during their development in secondary lymphoid organs. Factors derived from extracellular environment, including those of pathogens, termed pathogen-associated molecular patterns , may have effects on this interaction, yet no study to date has addressed these effects. In this study, we explored the effect of Toll-like receptor 9 (TLR9) triggering on the interaction of normal B cells with fibronectin and collagen. Materials and Methods The synthetic analog of TLR9 ligand, CpG-C, was used for stimulating the cells. The expression pattern of very late antigen−4 integrin was studied by fluorescence-activated cell sorting and Western blotting experiments, and cell adhesion was analyzed by fluorometric adhesion assay. Results CpG at 0.5 μM upregulated fibronectin receptor (very late antigen−4) expression and cell adhesion, and increasing the CpG concentration did not have further effect. Blocking experiments with TLR9 signaling inhibitor, TTAGGG, anti-α4 antibody, and IκBα phosphorylation inhibitor, Bay 11-7082, confirmed that the CpG-induced induction level was TLR9 (partly), very late antigen−4, and nuclear factor−κB-mediated, respectively. Conclusions This study indicates that TLR9 triggering on B cells influences their interaction with extracellular matrix, which will be critical in modulating activation of these cells in conditions, such as infections, and gives a basic insight into the contribution of innate immunity elements in B-cell functional responses.

The influence of parainfluenza type 3 (PI-3) virus on the release of inflammatory mediators by guinea pig pulmonary alveolar macrophages (PAMs) was investigated in vitro. Direct application of PI-3 virus dose-dependently stimulated the generation of chemiluminescence by PAMs and induced aggregation of PAMs. No significant effects of PI-3 virus on the release of linoleic acid metabolites by PAMs were detected. However, an increased release of the arachidonic acid metabolite thromboxane B2 (TxB2) was observed when PAMs were stimulated with PI-3 virus. PAMs were also cultured for 2 h or 18 h in the presence of PI-3 virus or control medium. The production of reactive oxygen species and the release of fatty acid metabolites by these PAMs were determined upon stimulation with opsonized zymosan particles or phorbol myristate acetate. The amounts of hydrogen peroxide and superoxide produced did not differ between virus- and control medium-incubated PAMs. However, the PI-3 virus-treated PAMs generated twice as much chemiluminescence when compared to PAMs incubated with control medium. The 2-h incubation period with PI-3 virus also resulted in a decreased release of TxB2 from the PAMs upon zymosan stimulation. The changes in the production of reactive oxygen species and the release of TxB2 by PAMs could account for damage to the airways and bronchial hyperresponsiveness often seen after viral infection.

Epithelial damage in the airways is a feature often observed in patients with asthma and is probably caused by the interaction of epithelial cells with leukocytes. As adhesion molecules are thought to be important in this interaction, we analyzed the expression and modulation of adhesion molecules on primary cultured human bronchial epithelial cells and the bronchial epithelial cell lines BEAS-2B and NCI-H292. E-selectin, P-selectin, and VCAM-1 were absent under basal and stimulated conditions. The adhesion molecules ICAM-1 (CD54), LFA-3 (CD58), and CD44 (H-CAM) were expressed basally on primary cultured human bronchial epithelial cells and the BEAS-2B and NCI-H292 cell lines. CD44 and LFA-3 expression did not change after stimulation with IFN-gamma or TNF-alpha. In contrast, ICAM-1 expression on human bronchial epithelial cells and BEAS-2B cells could be increased by incubation with PMA, IFN-gamma, TNF-alpha, and especially with the combination of IFN-gamma and TNF-alpha. The maximal ICAM-1 expression on both epithelial cell types was obtained with the combination of TNF-alpha and IFN-gamma after 48 h of incubation. The NCI-H292 cell line was different in that it only showed increased ICAM-1 expression after stimulation with PMA and IFN-gamma and not by the combination of IFN-gamma and TNF-alpha or with TNF-alpha alone. In conclusion, the bronchial epithelial cells tested express several adhesion molecules, but only ICAM-1 expression was influenced by inflammatory cytokines.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are major drugs used in the treatment of inflammation and pain in a wide variety of disorders. The best-known mechanism of action of NSAIDs is the inhibition of prostaglandin synthesis as a result of their action on cyclooxygenase (COX) enzymes. However, data have been accumulating through the years indicating that NSAIDs also act on other targets in cell signaling. It has been established that NSAIDs induce anti-inflammatory effects independent of COX. Acetylsalicylic acid (ASA) and other inhibitors of COX induce severe bronchospasms and asthmatic attacks in a significant population of asthmatic patients. The etiology of ASA induced asthma is complex and not fully understood, but most evidence points towards an abnormality of arachidonic acid (AA) metabolism. Since doses of ASA necessary to treat chronic inflammatory diseases appeared much higher than those required to inhibit PG synthesis, COX-independent mechanisms of NSAIDs were postulated. Recently, we have shown that NSAIDs induced expression of heat shock proteins specially HSP70. Heat shock proteins (HSPs) are normal intracellular proteins that are produced in greater amounts when cells are subjected to stress or injury. Interestingly, a potential pathogenic role for heat shock proteins in diseases such as autoimmune disease, vascular disease has been reported. Because mast cells have been reported to play a role in the pathogenesis of ASA induced asthma, a link between heat shock proteins and this disease could postulated. In this review, an overview is given on aspirin-induced asthma and the cells and mediators that may play a role therein. Mast cell signaling with regard to interaction with NSAIDs and heat shock proteins (HSPs) and toll-like receptors (TLRs) is further highlighted.

Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

Non-stimulated guinea-pig pulmonary macrophages (PM) convert arachidonic acid to thromboxane B 2 and 12-hydroxy-heptadecatrienoic acid, whereas linoleic acid is metabolized to two hydroxy compounds, i.e. 9-hydroxy- and 13-hydroxy-octadecadiedonic acid. Coincubation of PM with immune serum (2% v/v) resulted in a profound reduction of the release of these products. This effect seemed to be due to an inhibitory action on cyclooxygenase activity. Control serum also possessed inhibitory properties towards the release of fatty acid metabolites, possibly due to an effect on phospholipase activity. Because of the radical scavenging properties of 9-hydroxy-octadecadienoic acid, the modulation of the release of this product may be an important determinant in macrophage function.

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.

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.

We have previously demonstrated that the administration of nerve growth factor (NGF) to guinea-pigs results in airway hyper-responsiveness within 1 h.In the present study we document the involvement of NGF in the acute allergic airway response.Guinea-pigs that are sensitized to ovalbumin show an acute bronchoconstriction directly after challenge with ovalbumin.Intratracheal application of 10 microg of antibodies directed against NGF (anti-NGF) 1 h before the challenge reduces the acute severe bronchoconstriction to approximately 40% and the sustained bronchoconstriction to approximately 20% of the reaction in controls. This shows a high potency of anti-NGF in diminishing the direct bronchoconstriction. Inhibition of the tyrosine kinases of the tyrosine kinase receptor A, the high-affinity receptor for NGF, has no effect on the bronchoconstriction. Therefore, we postulate that the p75, the low-affinity receptor for neurotrophins, is responsible for the acute bronchoconstriction. Our findings suggest a role for NGF in the induction of the acute asthmatic reaction.These findings offer a new potential therapeutic strategy for the treatment of allergic asthma.

To investigate the potential anti-inflammatory effects of sesame oil, which is present in the injectable gold preparation Auromyose, the synthesis of tumour necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) by in vitro stimulated blood cells was measured before, during and after 12 weeks of dietary supplementation with 18 g of sesame oil daily in 11 healthy male volunteers. Neither TNF-alpha, PGE2 nor LTB4 production levels showed statistically significant changes during the 12 weeks of dietary supplementation with sesame oil. These results do not suggest an anti-inflammatory effect of sesame oil as present in injectable gold preparations which are used in the treatment of rheumatoid arthritis.

Lipid mediators released by inflammatory and immune cells play an important role in inflammatory and immune processes. Most attention has been focussed on arachidonic-derived mediators, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Literature data, however, suggest that also metabolites of the unsaturated fatty acid linoleic acid may be important in this respect. We have studied the formation and release of 9-hydroxy- and 13-hydroxy-linoleic acid (9-HODE and 13-HODE) by enriched populations of human peripheral blood neutrophils, eosinophils, basophils, monocytes, and lymphocytes. We demonstrate that the eosinophil preferentially produces 13-HODE, whereas the other cell types produce equal amounts of 9-HODE and 13-NODE. The biological significance of these findings is discussed.

Preincubation of guinea pig tracheas with the nitric oxide synthase inhibitor, Nω-nitro- l -arginine methyl ester (L-NAME, 120 μM) resulted in a significant upward shift of the histamine concentration-response curve with a concomitant inhibition of prostaglandin E2 production. Preincubation of the preparations with a 5-lipoxygenase inhibitor (AA-861, 2-(12-hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-p-benoquinone) or a leukotriene C4,D4,E4 receptor antagonist (FPL 55712, sodium 7-{3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxy propoxy}-4-oxo-8-propyl-4H-1-benzopyran-2-carboxylate) totally blocked the L-NAME-induced tracheal hyperresponsiveness. A shift from cyclo-oxygenase to lipoxygenase products, in particular leukotrienes, is likely to be responsible for the L-NAME-induced tracheal hyperresponsiveness.

1. The effect of 13-hydroxylinoleic acid (13-HODE) on changes in blood pressure in the rat was measured. 2. 13-HODE (0.1 - 100 micrograms kg-1) had no direct effect on blood pressure in the rat and had no effect on histamine (0.1 - 1000 micrograms kg-1)-induced changes in blood pressure. In contrast, it was found that 13-HODE itself induced a decrease in diastolic arterial blood pressure when it was injected intravenously after either a single dose of histamine (10, 100 or 1000 micrograms kg-1) or after a dose-response curve of histamine (0.1 - 1000 micrograms kg-1). 3. This hypotensive effect of 13-HODE was not observed after administration of the endothelium-dependent vasodilator, acetylcholine (0.1 - 10 micrograms kg-1), the endothelium-independent vasodilator, sodium nitroprusside (0.1 - 100 micrograms kg-1) or the inflammatory mediator, leukotriene B4 (0.1 - 300 micrograms kg-1). However, prior injection of bradykinin (0.1 - 100 micrograms kg-1) allowed a dose-dependent hypotensive effect of 13-HODE to be revealed. 4. The hypotensive effect of 13-HODE after histamine and bradykinin could be inhibited by neonatal capsaicin treatment of the rats (50 mg kg-1, s.c. on day 1 and 2 after birth). 5. Ruthenium red (120 micrograms kg-1 min-1), an inhibitor of excitatory effects on sensory nerves, and the CGRP antagonist, CGRP8-37 (1-3 micrograms kg-1 min-1) also inhibited the hypotensive effect of 13-HODE. 6. It is concluded that the hypotensive effect of 13-HODE in the rat after histamine and bradykinin is due to the release of a CGRP-like substance from sensory nerves. These results highlight the possibility that endogenous 13-HODE could be involved in the neurogenic regulation of blood pressure.

The influence of 13-hydroxy-linoleic acid (13-HODE) on the pulmonary resistance and dynamic compliance of guinea pigs in vivo was determined. Intravenously administered histamine and methacholine caused dose-dependent increases in pulmonary resistance and dose-dependent decreases in dynamic compliance in anesthetized, spontaneously breathing guinea pigs. Inhalation of an aerosol containing 13-HODE (10 μmol/L) enhanced the increases in pulmonary resistance observed after administration of histamine or methacholine when compared with the respective control animals. The effect of 13-HODE on the increase in pulmonary resistance after administration of histamine was dose-dependent. An enhancement in the pulmonary resistance was already measured after treatment of guinea pigs with aerosols of solutions containing 0.1 μmol/L 13-HODE when compared with that of control animals. In contrast, the changes in dynamic compliance were not affected by 13-HODE. These results indicate that 13-HODE may play an important role in the induction of airway hyperresponsiveness in vivo when it is produced or released in significant amounts in the airways. (J Allergy Clin Immunol 1995;96:36-43.)

We studied the effect of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid (13-HODE) on the increase of the intracellular calcium concentration, [Ca2+]i, induced by platelet-activating factor (PAF), leukotriene B4 (LTB4), and formyl-Met-Leu-Phe (fMLP) in human polymorphonuclear leukocytes (PMNs). 13-HODE by itself did not change the [Ca2+]i of PMNs. However, when PMNs were preincubated with 13-HODE and subsequently stimulated with PAF, LTB4, or fMLP, an inhibition of the increase of the [Ca2+]i was observed. The PAF-induced calcium response was concentration-dependently inhibited between 0.1 and 5 μM 13-HODE. These results suggest that 13-HODE can affect the increase of the [Ca2+]i and hence can modulate activation of PMNs. J. Leukoc. Biol. 56: 200–202; 1994.

Intradermal 13-hydroxyoctadecadienoic acid (13-HODE; 10−11−10−9 mol/site) inhibited oedema formation induced by the neutrophil-dependent mediator leukotriene B4 (LTB4) in the presence of calcitonin gene-related peptide (CGRP; 10−11 mol/site) in rabbit skin. In contrast, the responses to the direct acting mediators bradykinin and histamine were unaffected by 13-HODE. 13-HODE failed to induce oedema formation in rabbit skin when injected alone or in the presence of the potent vasodilator CGRP. These results present a novel interaction between 13-HODE and LTB4 that could have important implications in the pathogenesis of inflammation.

Abstract Background: Mast cells are important effector cells in innate or acquired immunity that contribute to host defense. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive, and proinflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability, and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor. Objective: In this study, we investigated the effect of CSM on allergic activation of mast cells through IgE and antigen. Methods: Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The releases of granules, production of leukotrienes, chemokines and cytokines was determined in supernatants by ELISA. The effect of CSM exposure on intracellular signaling, especially the NF-κB and Erk1/2 pathways, was analyzed by Western blotting. Results: CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells ATF-1 was phosphorylated after stimulation with IgE/Ag. LPS activated mast cells were not influenced by CSM. Conclusion: Our study suggests that exposure to cigarette smoke may lead to a reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial derived LPS.

Linoleic acid can be converted into 9-hydroxy-linoleic acid (9-Hode) or 13-hydroxy-linoleic acid (13-HODE) by the same enzyme systems that metabolize arachidonic acid into biologically active mediators. We now investigated whether 13-HODE is of importance for the regulation of airway smooth muscle function

The presence of polymorphonuclear leukocytes (PMNs) within the airways is a characteristic feature of a variety of lung diseases. Pulmonary alveolar macrophages (PAMs) and epithelial cells release many different factors which contribute to the recruitment of inflammatory cells into infected airways. PAMs and tracheal epithelial cells are able to produce linoleic acid metabolites (9-HODE and 13-HODE) besides arachidonic acid metabolites. The objective of the present study was to determine whether 9-HODE and 13-HODE posses chemotactic activity for isolated PMNs. It was found that 9-HODE and 13-HODE induced a chemotactic response of both human and bovine PMNs in vitro . The HODEs evoked chemotaxis with a linear dose response from 10 −10 M to the same extent as the arachidonic acid metabolite 15-HETE. At 10 −8 M, 9-HODE and 13-HODE were approximately half as potent in inducing chemotaxis as compared to LTB 4 .

The influence of the linoleic acid metabolite 13-hydroxy-octadecadienoic acid (13-HODE) on guinea pig tracheal reactivity to both contractile and relaxant agonists was investigated in vitro. 13-HODE induced an increased contraction of tracheal rings to histamine, whereas methacholine responsiveness was not significantly affected. Relaxant responses to isoprenaline and prostaglandin E2 were not influenced either.