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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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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 .

Vaccination of guinea pigs with Haemophilus influenzae leads to an impairment of beta-adrenoceptor function in lung. We have used an autoradiographic technique to study the distribution of changes in lung beta-adrenoceptor density. H. influenzae induced a decrease in beta-adrenoceptors in peripheral lung membranes of 22 +/- 5% (mean +/- S.E.M., n = 7), while the affinity of binding was unaffected. Tracheal beta-adrenoceptor binding was not influenced by H. influenzae. Autoradiography revealed a 27% reduction in beta-adrenergic binding sites on alveolar septa. Bronchial epithelial beta-adrenoceptors were decreased for 36%, and vascular smooth muscle and endothelial beta-adrenoceptors were also reduced. beta-Adrenoceptors on airway smooth muscle were unaffected. H. influenzae affected both the beta 1- and beta 2-subtypes of receptors. It is concluded that in this animal model of atopy beta-adrenoceptors may be decreased on several different cell types within the lungs, which may influence overall airway and vascular reactivity.

1. Pretreatment of guinea-pigs with endotoxin (1 mg kg-1 b.w., i.p., 4 days before the experiments) results in respiratory airway hyperreactivity in vitro. Dose-response curves with either arecoline or histamine on isolated tracheae from these animals display increased maximal contractions, and decreased EC50 values. 2. Tracheae denuded of epithelium respond with a similar hyperreactivity to histamine as observed in preparations from endotoxin pretreated animals. Removal of the epithelial layer of tracheae from endotoxin pretreated guinea-pigs did not additionally affect the histamine dose-response curve. 3. The cyclo-oxygenase inhibitor indomethacin (10 microM) induces histamine hyperreactivity which is equal in intact and epithelium-denuded tracheae from saline or endotoxin pretreated guinea-pigs. Similar results are obtained with the combined lipoxygenase/cyclo-oxygenase inhibitor nordihydroguaiaretic acid (10 microM). 4. Histamine (0.1 mM) induces an increase in prostaglandin E2 (PGE2) formation by the tracheal spiral in vitro, which is reduced by 34% by endotoxin pretreatment, and by about 60% following epithelium removal irrespective of endotoxin pretreatment. 5. Arachidonic acid (AA, 22 microM) stimulation of the guinea-pig trachea in vitro induces a relaxation, and an increase in PGE2 production. In preparations lacking the epithelium, AA induces a contraction which coincides with a 60% reduced increase in PGE2 formation. These effects are not altered by endotoxin pretreatment. 6. It is concluded that the endotoxin-induced respiratory airway hyperreactivity may be caused by a disturbed ability of epithelial cells to synthesize PGE2. The decreased formation of this prostaglandin is rather the consequence of a diminished liberation of AA from the phospholipid stores than a dysfunction of the cyclo-oxygenase enzyme.

Previously we showed that guinea pig alveolar macrophages (AMs) incubated with serum obtained from Haemophilus influenzae -treated animals had detrimental effects on airway smooth muscle β-adrenergic receptor function. In the present study the influence of H. influenzae treatment on several functions of guinea pig AMs was examined. Sera obtained from animals 4 days after intraperitoneal administration of H. influenzae or from control guinea pigs possessed similar opsonic capacities. No effects of these sera on hydrogen peroxide release by AMs were observed as compared to the basal hydrogen peroxide release of AMs. Interestingly, stimulation of AMs with serum from control animals resulted in a diminished cyclo-oxygenase product formation, which was potentiated after incubating AMs with serum from H. influenzae -treated guinea pigs. No differences in phagocytic activity of AMs isolated from control or H. influenzae -treated animals were observed. When AMs were incubated with phorbol myristate acetate or zymosan, the cells produced superoxide anion and released hydrogen peroxide. However, the amounts of superoxide and hydrogen peroxide released did not differ between AMs isolated from control or H. influenzae -treated animals.

In the guinea-pig intraperitoneal administration of the Gram-negative bacterium Haemophilus influenzae induces a decrease of beta-adrenoceptor number and results in impairment of beta-adrenoceptor function in the peripheral and central airways, respectively. In the present study, the time-course of these events was studied and compared with changes in catecholamines in plasma, in organs involved in immunoregulation (spleen, thymus), and in the heart and the lung. The number of beta-adrenoceptor binding sites in peripheral lung tissue and beta-adrenoceptor function in isolated tracheal spirals were significantly decreased 3 and 4 days after administration of H. influenzae (24-33%). No significant changes were observed at day 1 and day 8. The effects on tracheal beta-adrenergic receptor function were characterized by a decrease of maximal relaxation only, whereas EC50-values were not affected. These data are indicative of an effect on the functional coupling of the receptors to the biochemical events leading to smooth muscle relaxation. No changes were observed in catecholamine concentrations in the lung, heart, and the thymus after H. influenzae-treatment. Plasma noradrenaline, though, was significantly increased at day 1 after H. influenzae. At day 8 plasma noradrenaline had returned to control levels. Interestingly, the effect on spleen noradrenaline was opposite to the effect seen in plasma. A significant decrease in spleen noradrenaline was observed after H. influenzae at days 1, 3, and 8, with a maximum of 42% at day 1. It is suggested that the decrease in spleen noradrenaline may have a causal relationship with the changes in lung beta-adrenoceptors.