Human Lung Fragments Research Articles

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.

What role do coagulation disorders play in the pathogenesis of leptospirosis?

Leptospirosis is a zoonosis of worldwide distribution, spread by the urine of infected animals. It is a major public health problem, especially in developing countries, where circumstances for transmission are most favourable. The clinical picture varies from mild disease to a severe illness with haemostatic derangements and multiorgan failure eventually leading to death. Although the haemorrhagic complications of severe disease are serious, the pathophysiology is scarcely elucidated. The complex mechanisms involved in inflammation-induced coagulation activation are extensively studied in various infectious diseases, i.e. Gram-negative sepsis. Tissue factor-mediated coagulation activation, impairment of anticoagulant and fibrinolytic pathways in close concert with the cytokine network are thought to be important. But for human leptospirosis, data are limited. Because of the growing interest in this field, the impact of leptospirosis, and the availability of new therapeutic strategies, we reviewed the evidence regarding the role of coagulation in leptospirosis and provide suggestions for future research.

Response to C3a, mast cells, and asthma

This is in response to a “Letter to the Editor (1)” by Dr. Bradding regarding our manuscript entitled “Airway smooth muscle cells enhance C3a-induced mast cell degranulation following cell-cell contact” that was published in your journal recently (2). Dr. Bradding contends that our manuscript, which utilizes a C3a-responsive human mast cell line, is misleading because “it ignores wealth of consistent literature demonstrating that human lung mast cells and human lung fragments do not exhibit any response to C3a or C5a.” We disagree with this view for the reasons discussed below. Although studies performed in the 1980s and 1990s, showed that dispersed human lung mast cells are unresponsive to C3a and C5a (3–5), these studies did not characterize the expression of C3a receptor in mast cells and failed to consider the possibility that lung tissue may contain a subpopulation of mast cells that could be responsive to anaphylatoxins. Human mast cells (MC) consist of a mixture of at least two cell types that can be distinguished based on the protease content of their granules. It is well documented that MCTC cells (tryptaseand chymase-positive) that are found predominantly in the skin are responsive to C3a and C5a whereas MCT cells (tryptase positive) present in other tissues are unresponsive to these anaphylatoxins (6). The previous demonstration that dispersed lung mast cells did not respond to C3a or C5a probably reflects the fact that 90% of these cells are MCT cells (7). Indeed, a recent report by Oskeritzian et al. (8) showed that MCTC cells purified from a mixture of human lung mast cells are highly responsive to C5a for histamine release and leukotriene C4 generation, mediators that promote airway hyperreactivity. Most importantly, mast cells that infiltrate into the airway smooth muscle in asthma are positive for both tryptase and chymase (9). Given that MCTC cells are also responsive to C3a for mediator release (10, 11), we argue that a C3a responsive human mast cell line is an appropriate in vitro cellular model to delineate the signaling pathways involved in mast cell-airway smooth cell interaction relevant to asthma. REFERENCES

Contribution of Anaphylatoxins to Allergic Inflammation in Human Lungs

The contribution of complement activation to allergic asthma remains controversial. In order to elucidate the role played by the complement split products, anaphylatoxins C3a and C5a, we evaluated their effects on production of cysteinyl-leukotrienes (cysLTs) by human lung fragments following an anaphylactic reaction. The lung tissues obtained from two patients with lung cancer showed C5aR-, C5L2R-, and C3aR-mRNA expression. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). The co-addition of human C5a at doses of 0.1 to 10 ng/ml to the anti-IgE antibody potentiated cysLT production. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1,000 ng/ml seemed to increase cysLT production, but not to any significant extent. A novel C5a receptor complementary peptide, acetylated peptide A, dose-dependently inhibited cysLT production by the human lung fragments following the anaphylactic reaction in the presence of 1 ng/ml C5a. However, this peptide did not inhibit cysLT production in the presence of 100 ng/ml C3a. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lung tissues and contributes to allergic inflammation in disorders such as asthma, thus acetylated peptide A may be useful for suppressing allergic inflammation in the lungs.

Role of protein kinase A in the inhibition of human mast cell histamine release by β-adrenergic receptor agonists

ABSTRACT Background Although β-adrenergic receptor agonists inhibit antigen-induced release of histamine, leukotrienes and prostaglandin D 2 from human lung fragments, dispersed human lung mast cells and human skin mast cells, subcellular mechanisms for the inhibition of histamine release by β-adrenergic receptor agonists are not well delineated. The aim of the present study was to investigate the inhibitory mechanisms of β-adrenergic receptor agonists for human mast cell histamine release using human cultured mast cells. Methods Human mast cells were obtained by culturing umbilical cord blood CD34 + cells in the presence of stem cell factor and interleukin-6. Cultured mast cells were sensitized with human myeloma IgE and stimulated with antihuman IgE. Results Stimulation of mast cells induced the elevation of intracellular cytosolic free Ca 2 + concentrations ([Ca 2 +] i ) and the translocation of protein kinase C (PKC) from the cytosol to the cell membrane, followed by the release of stored histamine. Isoproterenol, salbutamol and dibutyryl cAMP inhibited both the histamine release and PKC translocation, whereas they failed to affect the elevation of [Ca 2 +] i . H-89, a protein kinase A (PKA) inhibitor, abrogated the inhibition. Conclusions The present results suggest that PKA activation induced by β-adrenergic receptor agonists plays a crucial role in inhibiting IgE-mediated histamine release from human cultured mast cells through suppressing PKC translocation.

Interleukin-8 plays a significant role in IgE-mediated lung inflammation.

Interleukin (IL)-8 is a potentially important cytokine in allergic respiratory responses since it is released by many resident lung cells, and it is a potent granulocyte chemoattractant. Therefore, we induced an immunoglobulin (Ig)E-mediated response in human lung samples and studied whether IL-8 was produced in sufficient quantities to promote human neutrophil and eosinophil migration across naked filters and endothelial and pulmonary epithelial monolayers cultured on these filters. Fresh human lung fragments from 16 thoracotomy specimens were treated with either a 1:100 dilution of anti-IgE or buffer (control) for 30 min. All anti-IgE treated lung samples had significant release of histamine and neutrophil and eosinophil chemotactic activity. Fourteen of the 16 lung samples had a significant increase in IL-8 subsequent to anti-IgE treatment (p<0.01). Anti-IL-8 antibody (4 microg x mL[-1]) inhibited 42% and 53% of neutrophil and eosinophil chemotactic activity respectively, contained in supernatants from anti-IgE-treated lung samples. Finally, we found that IL-8 at a concentration near that measured after anti-IgE treatment of lung samples (2,000 pg x mL[-1]) induced neutrophil and eosinophil migration through naked filters and endothelial and pulmonary epithelial cell monolayers. Thus, human lung IgE-mediated responses in vitro results in the rapid release of interleukin-8 in amounts sufficient to affect a biological response, granulocyte transcellular migration, indicating that interleukin-8 may play a significant role in allergic respiratory diseases.

The effect of selective phosphodiesterase 3 and 4 isoenzyme inhibitors and established anti-asthma drugs on inflammatory cell activation.

1. This study aimed to evaluate the effects of phosphodiesterase (PDE) inhibitors and currently prescribed anti-asthma drugs for their ability to inhibit inflammatory cell activation in vitro. 2. Alveolar macrophages and eosinophils were isolated from the bronchoalveolar lavage (BAL) fluid of ovalbumin (Ovalb)-sensitized guinea-pigs. Opsonized zymosan (OZ) and PAF stimulated leukotriene B4 (LTB4) release from eosinophils was measured by radioimmunoassay. Ovalb-induced superoxide generation was measured by reduction of cytochrome C. 3. Monocytes were separated from human peripheral venous blood and mast cells were dispersed from human lung fragments. Lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) release from monocytes was measured by ELISA and anti-IgE stimulated histamine release from mast cells was measured by a radioenzymatic method. 4. The beta 2 agonist, salbutamol inhibited TNF-alpha release from monocytes and histamine release from mast cells whilst having no effect on eosinophil-derived LTB4 release or macrophage superoxide generation. 5. The PDE 3 inhibitor, milrinone produced a concentration-related inhibition of TNF-alpha release from monocytes which achieved statistical significance at 10(-5) M but inhibited LTB4 release from eosinophils and superoxide generation from macrophages only at the highest concentration (10(-3) M) examined. Milrinone had no effect on histamine release from mast cells. 6. The selective PDE 4 inhibitors, denbufylline and rolipram and the corticosteroid, beclomethasone produced a concentration-related inhibition of LTB4 release from eosinophils, TNF-alpha release from monocytes and superoxide generation from alveolar macrophages whilst having no effect on histamine release from mast cells. 7. The mixed PDE 3/4 inhibitor, benzafentrine produced a concentration-related inhibition of LTB4 release from eosinophils, TNF-alpha release from monocytes, superoxide generation from alveolar macrophages and histamine release from mast cells. 8. In conclusion these data clearly show that both established anti-asthma medication as well as PDE inhibitors have the potential to inhibit inflammatory cell activation in vitro but that the anti-secretory actions of beta 2 agonists, corticosteroids and PDE inhibitors are distinct.

424 Propranolol enhances histamine and interleukin-5 generation from anti-IgE challenged human lung fragments: evidence for inverse agonist effect

424 Propranolol enhances histamine and interleukin-5 generation from anti-IgE challenged human lung fragments: evidence for inverse agonist effect

707 Activated human lung fragments and human mast cells synthesize IL-13

707 Activated human lung fragments and human mast cells synthesize IL-13

Development of markers for human basophils and mast cells

Development of markers for human basophils and mast cells

Antiinflammatory steroids inhibit granulocyte/macrophage colony-stimulating factor production by human lung tissue.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor which has been shown to induce proliferation and activation of inflammatory cells, and may play a role in allergic diseases and experimental allergic reactions. Since little is known about the involvement of cytokines in allergic inflammation in the lung, we investigated whether human lung fragments produce GM-CSF in vitro. The present studies demonstrate that human lung fragments produce GM-CSF in vitro and that glucocorticoids are potent inhibitors of this cytokine production. Human lung was cut into fragments, rinsed, and cultured in 60-mm tissue culture plates containing 50 mg of tissue in RPMI 1640 with antibiotics in the presence or absence of a variety of steroids for 18 h. Lung fragments were rinsed and then incubated for an additional 4 h. Supernatants were harvested and analyzed for GM-CSF activity using the GM-CSF/interleukin (IL)-3 responsive M-07e human leukemic cell line. Steroids alone had no effect on M-07e proliferation. Human lung fragments produced 32.1 +/- 11.8 ng of GM-CSF equivalents per gram wet weight of tissue during the 4 h incubation (mean +/- S.E.M., n = 5, range 9.2-74.2). While specific antisera against human GM-CSF neutralized 96.8 +/- 2.8% (n = 5) of the activity, anti-IL-3 antibody had no effect, suggesting most or all of this activity was GM-CSF. Treatment of lung fragments in vitro for 18 h with hydrocortisone (HC) inhibited the production of GM-CSF dose-dependently. Maximal inhibition of GM-CSF production was 72.8 +/- 4.0% at a concentration of 10(-6) M hydrocortisone (n = 5), and the molar concentration of HC that inhibited of GM-CSF production by lung tissue by 50% (IC50) was approximately 4.5 x 10(-7) M. Kinetic studies revealed that a 6 h preincubation with the drug was required for 50% inhibition of GM-CSF production. HC and other glucocorticoids, at a concentration of 0.1 microM, demonstrated significant inhibition of GM-CSF release. Based on the rank order of potency of several glucocorticoids, and the fact that nonglucocorticoid steroids including testosterone and beta-estradiol (0.1 microM) had no effect, we suggest that this is a specific receptor-mediated effect. We conclude that human lung produces GM-CSF in vitro and that antiinflammatory steroids are potent and effective inhibitors of the production of this cytokine. This may contribute to the therapeutic efficacy of these drugs in pulmonary diseases.

Formoterol on airway smooth muscle and human lung mast cells: a comparison with salbutamol and salmeterol

Formoterol, like salbutamol and salmeterol, relaxed isolated preparations of guinea-pig trachea and human bronchus, and inhibited antigen-induced mediator release from human lung fragments in a concentration-related fashion. In each case, these actions were mediated through β 2-adrenoceptors, with formoterol being 50–120-fold more potent than salbutamol, and 2–27-fold more potent than salmeterol. The duration of action of formoterol was longer than that of salbutamol in all preparations, but was markedly shorter than that of salmeterol, whose actions persisted for many hours despite continuous or extensive washing of the tissues. In conscious guinea-pigs, inhaled formoterol, salbutamol and salmeterol all caused dose-related inhibition of histamine-induced bronchoconstriction. Formoterol was again more potent (10–20-fold) than either salbutamol or salmeterol. However, while the actions of a threshold-effective dose of formoterol persisted for less than 3 h, somewhat longer than those of salbutamol (< 1.5 h), an equivalent dose of salmeterol was active for at least 6 h. Therefore, while formoterol is a potent β 2-adrenoceptor agonist in vitro and in vivo, and is consistently longer-acting than salbutamol, its duration of action is markedly shorter than that of salmeterol.

Mechanism of occupational asthma due to Western red cedar (Thuja plicata)

Occupational asthma due to Western red cedar is the most common form of occupational asthma in the Pacific Northwest and affects 4-13.5% of the exposed population. It has been shown to be caused by plicatic acid, a low molecular weight compound present uniquely in the wood. The mechanism of asthma induced by plicatic acid is not known, as specific IgG antibodies were found only in about 20% of patients. Sera from patients with red cedar asthma failed to passively sensitize human lung fragments of human basophils. Basophils from patients with this disease released histamine when challenged directly with plicatic acid in a specific manner. Immunologic mechanisms other than Type I hypersensitivity reaction are likely to be involved.

Immunologic studies of the mechanisms of occupational asthma caused by western red cedar

Background: Occupational asthma caused by western red cedar (Thuja plicata) is a common problem in sawmill industries. The objective of this study was to examine the cellular and immunologic mechanisms of western red cedar asthma (WRCA) more closely. Methods: Bronchial biopsy specimens, bronchoalveolar lavage (BAL) mast cells and peripheral blood basophils from patients with WRCA, patients with atopic asthma, and nonatopic control subjects were challenged in vitro with plicatic acid (PA), PA-human serum albumin conjugate (PA-HSA), grass pollen, or calcium ionophore. Results: PA (100 μg/ml) released histamine from the basophils of 9 of 11 patients with WRCA, 1 of 7 patients with atopic asthma, and 2 of 7 normal subjects. PA triggered histamine release from 10 of 11 bronchial biopsy specimens and 8 of 8 BAL samples from patients with WRCA. Interestingly, PA released histamine from BAL cells and bronchial biopsy specimens from 3 of 7 normal subjects but in none of the patients with atopic asthma. PA-HSA-induced histamine release from basophils and biopsy specimens was confined to patients with WRCA. PA-specific IgE was not detectable in serum from most patients with WRCA, and their serum did not transfer PA sensitivity to human lung fragments or lactate-stripped basophils. After pretreatment with anti-IgE in the absence of calcium, basophils from 14 subjects with WRCA still responded to PA (mean 64% to 67% of pretreatment response), whereas responses to grass pollen or anti-IgE were abolished. Conclusions: This study confirms that PA releases histamine from bronchial mast cells of most patients with WRCA but not from those of patients with atopic asthma. The PA response of some normal subjects suggests that PA may have both specific and nonspecific actions on mast cells and basophils, whereas the serologic studies indicate histamine release in WRCA cannot simply be attributed to PA-specific IgE.

Human lung mast cells and pulmonary macrophages produce tumor necrosis factor-alpha in sensitized lung tissue after IgE receptor triggering.

Tumor necrosis factor (TNF) is considered to play a key role in the pathogenesis of allergic disorders. We examined TNF production in human lung fragments after IgE receptor triggering at mRNA and protein levels. IgE receptor triggering was performed by sensitizing lung fragments with monoclonal human IgE and then exposing them to anti-human IgE antibody. Cytotoxic activity against L929 cells appeared in the culture supernatant of lung fragments 2 h after IgE receptor triggering and increased for up to 4 h. This cytotoxic activity was completely neutralized by anti-human TNF antibody. Northern blot analysis demonstrated that 1.8-kb TNF mRNA transcripts in sensitized lung fragments were expressed as early as 1 h after IgE receptor triggering and continued up to 4 h. Immunohistochemical analysis revealed TNF localization in tissue mast cells, alveolar macrophages, tissue macrophages, and bronchial epithelial cells. Double staining with anti-TNF antibody and alcian blue clearly identified that lung mast cells are one of the TNF-positive cell types in the pulmonary tissue. With immunoelectron microscopy, TNF immunoreactivity was detected in the rough endoplasmic reticulum and the perinuclear spaces in tissue macrophages, and in the cytosol and the perinuclear spaces in bronchial epithelial cells. In addition, IgE was detected on the cell surface of mast cells, tissue macrophages, and alveolar macrophages. These results suggest that TNF is released from mast cells and pulmonary macrophages through IgE receptor triggering and may play a key role in the allergic reaction in human airway.

LY 186655, a phosphodiesterase inhibitor, inhibits histamine release from human basophils, lung and skin fragments

LY 186655 (Tibenelast, Lilly) is a new phosphodiesterase inhibitor, not derived from the xanthine, possessing bronchodilating activity in animals. The aim of this work was to study the effect of LY 186655 and theophylline on histamine release from human leukocytes, skin and lung fragments. Histamine was measured using a spectrofluorometric method. Both drugs (3 × 10 −5 −3 × 10 −3M) exhibited a dose-dependent inhibition on anti-IgE (1/2000)-induced histamine release from human leukocytes. At 3 × 10 −3M, theophylline was significantly more effective than LY 186655 (mean inhibition 94 and 42%, respectively). On lung fragments, theophylline and LY 186655 (3 × 10 −5 −3 × 10 −3M) caused strong and comparable inhibitory effects on anti-IgE (1/500)-induced histamine release with a mean inhibition reaching maximally 65%. Histamine release induced by compound 48/80 (1 mg/ml) on sliced human foreskin was reduced with both drugs (3 × 10 −3M) by about 37%. We conclude that LY 186655 inhibits in vitro immunological histamine release from human lung and cutaneous mast cells as well as basophils with a similar pattern of activity to theophylline.

Effect of AS-35 on Agonist-Induced Contractions and the Resting Tonus of Airway Smooth Muscles and the In Vitro Release of Chemical Mediators from Passively Sensitized Lung Fragments from Humans and Guinea Pigs

Effects of 9-[(4-acetyl-3-hydroxy-2-n-propylphenoxy)methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido [1,2-a]pyrimidin-4-one (AS-35) on the resting tonus or contractions induced by agonists, such as leuko-triene (LT) D4 and specific antigen of isolated guinea pig tracheas or human bronchi, and the in vitro anaphylactic release of histamine and LTs from human lung fragments were investigated and compared with the effects of FPL 55712 and disodium cromoglycate. AS-35 as well as FPL 55712 did not affect the contractions induced by acetylcholine and histamine of the isolated guinea pig trachea. However, the compound at relatively low concentrations obviously inhibited contractions induced by LTD4, and the antagonistic activity was stronger than that of FPL 55712. Treatment of the isolated human bronchus with AS-35 tended to induce the inhibition of both LTD4- and antigen-induced contractions and the relaxation of the resting tonus in a concentration-dependent manner. The inhibitory potency at 10−6g/mi was slightly stronger than that of FPL 55712, but this was not statistically significant. The anaphylactic release of histamine and LTs from the lung fragments appeared to be inhibited by the treatment with AS-35 5 min prior to the antigen challenge. From these results, it is suggested that AS-35 is effective against allergic asthma through antagonism towards peptide-LTs released anaphylactically in addition to inhibition of the chemical mediator release.

O-194 - Influence of islet-activating protein (IAP) on anaphylactic chemical mediator release from human lung fragments and mast cells

O-194 - Influence of islet-activating protein (IAP) on anaphylactic chemical mediator release from human lung fragments and mast cells

Effect of NZ-107, a newly synthesized pyridazinone derivative, on antigen-induced contraction of human bronchial strips and histamine release from human lung fragments or leukocytes.

The effects of a newly synthesized pyridazinone derivative, NZ-107, 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone, and two well-known antiasthmatic drugs, amlexanox (orally active disodium cromoglycate-like drug) and disodium cromoglycate (DSCG) on antigen-, histamine- and leukotriene C4 (LTC4)-induced constriction of isolated human tracheal muscle, and histamine release from human lung tissues and leukocytes were investigated in vitro. In some experiments, salbutamol was used as a reference drug. NZ-107 inhibited antigen-, histamine- and LTC4-induced contraction of tracheal muscle. Amlexanox and DSCG did not affect the contractile response of tracheal muscle caused by each stimulant. Salbutamol inhibited antigen-induced contraction of tracheal muscle. NZ-107, amlexanox, DSCG and salbutamol clearly inhibited the antigen-induced release of histamine and LTC4 from human lung tissue. The antigen-induced histamine release from atopic human leukocytes was inhibited by NZ-107 and amlexanox, but not by DSCG. Pretreatment with IL-3 did not alter antigen-induced contraction of tracheal muscle and histamine release from lung tissue, but antigen- or calcium ionophore A 23187-induced histamine release from leukocytes was clearly enhanced. Amlexanox inhibited the IL-3-induced enhancement of histamine release from leukocytes in the case of both stimuli, but NZ-107 and DSCG had no effect. These data suggest that NZ-107 has potent anti-allergic actions based on the inhibition of antigen-induced contraction of human tracheal muscle and mediator release from human lung tissue and leukocytes.

Effects of adenosine on histamine release from human lung fragments.

The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A23187 or with concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyloxy]-phenyl]-1, 3-dipropylxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence of XAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a site antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.