Are cysteinyl leukotrienes involved in allergic responses in human skin?

BACKGROUND: Cysteinyl leukotrienes have been suggested to be involved in producing the symptoms of both the early and late phases of the allergic response in the lung and other tissues. OBJECTIVE: To use scanning laser Doppler imaging, microdialysis and immunocytochemistry to explore the mediator and cellular mechanisms of the dermal allergic response. METHODS: Thirteen atopic volunteers received intradermal injections into the forearm of grass pollen or D. pteronyssinus extract. Changes in dermal blood flow up to 8 h were monitored by scanning laser Doppler imaging. The release of histamine, PGD2 and LTC4/D4/E4 was assessed by dermal microdialysis. Skin biopsies were taken at 6 h to determine numbers of mast cells, eosinophils, basophils, Langerhans' cells, and monocytes/macrophages, and the expression of COX-1, COX-2, 5-LO and FLAP. RESULTS: Allergen provocation produced an immediate weal and flare response followed by an erythematous induration peaking at 6 h. During the first hour, c. 84 pmoles of histamine and c. 0.3 pmoles of PGD2 were recovered by microdialysis (both P < 0.001) but LTC4/D4/E4 was undetectable. No histamine, PGD2 or LTC4/D4/E4 was detectable at later times. Immunocytochemical examination of biopsies taken at 8 h showed increased numbers of eosinophils and basophils and in COX-2, 5-LO and FLAP, but not COX-1. Expression of 5-LO and FLAP was associated primarily with eosinophils. CONCLUSIONS: These findings suggest that inflammatory cells recruited to the site of allergen injection are not activated to release detectable amounts of cysteinyl leukotrienes. Hence, it is unlikely that the late-phase erythematous induration is mediated by this autocoid.     

Expression of 5-lipoxygenase and cyclooxygenase pathway enzymes in nasal polyps of patients with aspirin-intolerant asthma

In aspirin-intolerant subjects, adverse bronchial and nasal reactions to cyclooxygenase (COX) inhibitors are associated with over-production of cysteinyl-leukotrienes (cys-LTs) generated by the 5-lipoxygenase (5-LO) pathway. In the bronchi of patients with aspirin-intolerant asthma, we previously linked cys-LT over-production and aspirin hyper-reactivity with elevated immunoexpression in eosinophils of the terminal enzyme for cys-LT production, LTC4 synthase. We investigated whether this anomaly also occurs in the nasal airways of these patients. Immunohistochemical expression of 5-LO and COX pathway proteins was quantified in nasal polyps from 12 patients with aspirin-intolerant asthma and 13 with aspirin-tolerant asthma. In the mucosa of polyps from aspirin-intolerant asthmatic patients, cells immunopositive for LTC4 synthase were four-fold more numerous than in aspirin-tolerant asthmatic patients (p=0.04). There were also three-fold more cells expressing 5-LO (p=0.037), with no differences in 5-LO activating protein (FLAP), COX-1 or COX-2. LTC4 synthase-positive cell counts correlated exclusively with mucosal eosinophils (r=0.94, p<0.001, n=25). Co-localisation confirmed that five-fold higher eosinophil counts (p=0.007) accounted for the increased LTC4 synthase expression in polyps from aspirin-intolerant asthmatic patients, with no alterations in mast cells or macrophages. Within the epithelium, increased counts of eosinophils (p=0.006), macrophages (p=0.097), and mast cells (p=0.034) in aspirin-intolerant asthmatic polyps were associated only with 2.5-fold increased 5-LO-positive cells (p<0.05), while the other enzymes were not different. Our results indicate that a marked over-representation of LTC4 synthase in mucosal eosinophils is closely linked to aspirin intolerance in the nasal airway, as in the bronchial airways.     

Expression of cysteinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal allergic rhinitis

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are bioactive lipids that have been shown to contribute to allergic and inflammatory diseases. Eosinophils and mast cells have the capacity to produce large amounts of CysLTs after allergic or non-allergic stimulation. Molecular identification of both the synthetic and signalling proteins in the CysLT pathway allows the investigation of expression of the CysLT enzymes and receptors in active allergic rhinitis. OBJECTIVE: We examined the expression of the proteins involved in the synthesis of CysLTs and the cysteinyl leukotriene-1 (CysLT1) and cysteinyl leukotriene-2 (CysLT2) receptors in inflammatory cells from patients with active seasonal allergic rhinitis. METHODS: Nasal lavage samples were obtained from patients during active seasonal allergic rhinitis. Specific cellular immunocytochemical techniques were used to detect the cysteinyl leukotriene synthetic proteins, namely 5-lipoxygenase (5-LO), 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase (LTC4S). In situ hybridization and immunocytochemical techniques were used to identify the mRNA and proteins for the CysLT1 and CysLT2 receptors. RESULTS: 5-LO, FLAP and LTC4S, and the CysLT1 and CysLT2 receptors were expressed in the majority of eosinophils and in subsets of mast cells and mononuclear cells. 5-LO, FLAP and the CysLT1 receptor, but not LTC4S or the CysLT2 receptor, were expressed in a subset of nasal neutrophils. CONCLUSIONS: Our study demonstrates the presence of CysLT pathway proteins in key allergic and inflammatory cells from the upper airway of patients with active seasonal allergic rhinitis. Our expression data highlight the potential of CysLT-modifying agents to treat both upper and lower airway symptoms in patients suffering from allergic rhinitis and asthma.     

The cellular biology of eosinophil eicosanoid formation and function

Eosinophils are capable of generating eicosanoid derivatives of arachidonic acid by means of cyclooxygenase and the 5- and 15-lipoxygenase (LO) pathways. Moreover, eosinophils, because of their expression of leukotriene (LT) C(4) synthase, are a major source of 5-LO-derived cysteinyl LTs, which are potent paracrine mediators of bronchial obstruction and inflammation pertinent to asthma. The regulation of eicosanoid formation within eosinophils involves activation of key enzymes at specific intracellular sites. Calcium ionophore-elicited translocation of 5-LO to the membranes of the nuclear envelope is associated with LTC(4) formation. In addition, lipid bodies, the formation of which is initiated by specific receptor-mediated signaling pathways, are sites of cyclooxygenase- and LO-pathway eicosanoid formation. Newly formed LTC(4) can be immunolocalized at perinuclear membranes in ionophore-activated eosinophils and at lipid bodies in CCR3 agonist (eg, eotaxin) chemokine-stimulated eosinophils. The local generation of eicosanoids at distinct sites within eosinophils may be important for the roles of these eicosanoids, both as paracrine mediators pertinent to inflammation and as intracrine signal-transducing mediators that help regulate cellular responses of eosinophils.     

Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium

BACKGROUND: Symptoms of allergic rhinitis are accompanied by infiltration of the nasal mucosa with inflammatory cells, predominantly eosinophils and metachromatic cells (basophils and mast cells). Specific immunotherapy (IT) reduces mucosal eosinophilia and numbers of metachromatic cells in the epithelium. A specific marker distinguishing basophils from mast cells was recently developed. OBJECTIVES: The basophil-specific monoclonal antibody 2D7 was used to determine the influence of subcutaneous IT on numbers of nasal mucosal basophils compared with the effects of IT on neutrophils, eosinophils and mast cells. METHOD: During a randomized, placebo-controlled trial of grass pollen IT in 44 adults with severe summer hay fever, nasal biopsies were taken at baseline, out of the pollen season, and at the peak of the pollen season following 2 years treatment. Biopsies were processed for immunohistochemistry for basophils (2D7+), mast cells (AA1+), eosinophils (MBP+) and neutrophils (neutrophil elastase+). RESULTS: In placebo-treated (PL) patients there were significant seasonal increases in basophils (P < 0.01), mast cells (P < 0.05) and eosinophils (P = 0.002) in the nasal submucosa. In IT-treated patients significant increases in 2D7+ cells (P < 0.01) and eosinophils (P = 0.01) but not AA1+ cells (P = 0.9) were observed. These differences were significant between groups for eosinophils (P < 0.05). In the epithelium there were seasonal increases in AA1+ cells and eosinophils in both groups (PL: P < 0.01, IT: P < 0.05 for both). The between-group difference was significant for eosinophils (P = 0.05). Basophils were observed in the epithelium of six out of 17 in the placebo group and one out of 20 in the IT group (P = 0.03). Neutrophil numbers remained constant in both epithelium and submucosa. CONCLUSION: Successful grass pollen immunotherapy was associated with inhibition of seasonal increases in basophils and eosinophils, but not mast cells or neutrophils within the nasal epithelium. Immunotherapy may act, at least in part, by reducing seasonal recruitment of basophils and eosinophils into the epithelium.     

Effect of topical corticosteroids on seasonal increases in epithelial eosinophils and mast cells in allergic rhinitis: a comparison of nasal brush and biopsy methods

BACKGROUND: Nasal brushing and nasal biopsy are well-tolerated sampling techniques. Seasonal grass pollen-induced rhinitis is characterized by epithelial mast cell infiltration and seasonal increases in both epithelial and sub-mucosal eosinophils. OBJECTIVE: To compare the ability of the nasal brush and nasal biopsy techniques to detect natural seasonal increases in eosinophils and mast cells, and to assess the influence of topical corticosteroid. METHODS: Nasal brush samples and nasal biopsies were collected from 46 grass pollen-sensitive seasonal rhinitis patients before the grass pollen season and at the peak of the pollen season following 6 weeks' treatment with either fluticasone propionate aqueous nasal spray (200 microg, twice daily) or placebo nasal spray. RESULTS: Placebo patients showed seasonal increases in epithelial eosinophils both with nasal brushing (P < 0.0001) and biopsy (P < 0.001). Epithelial mast cell numbers also increased during the pollen season as detectable by brushing (P < 0.0001) and biopsy (P < 0.03). Changes in cell numbers measured by nasal brushing correlated with those observed with nasal biopsy, both for eosinophils and mast cells (P < 0.05). Sub-mucosal eosinophils but not mast cells also increased during the pollen season (P < 0.002). Nasal brushing and biopsy revealed that fluticasone treatment inhibited seasonal increases in epithelial eosinophils (P < 0.00001) and epithelial infiltration by mast cells (nasal brushing P < 0.00001 and nasal biopsy P < 0.01). Fluticasone also inhibited seasonal increases in sub-mucosal eosinophils (P < 0.001) and significantly reduced nasal symptoms (P < 0.001). CONCLUSION: Nasal brushing harvests sufficient inflammatory cells from the surface of the nasal mucosa to be used in lieu of nasal biopsies in observation of the effect of drugs on the nasal epithelium.     

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4

BACKGROUND: Human bronchial epithelial cells synthesize cyclooxygenase and 15-lipoxygenase products, but the 5-lipoxygenase (5-LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro-inflammatory mediators is thought to be restricted to leucocytes. OBJECTIVE: We hypothesized that human bronchial epithelial cells (HBECs) express a complete and active 5-LO pathway for the synthesis of LTB4 and LTC4, either constitutively or after stimulation. METHODS: Flow cytometry, RT-PCR, Western blotting, enzyme immunoassays and reverse-phase high-performance liquid chromatography were used to investigate constitutive and stimulated expression of 5-LO pathway enzymes and the synthesis of LTs B4 and C4 in primary HBECs and in the 16-HBE 14o- cell line. RESULTS: Constitutive mRNA and protein expression for 5-LO, 5-LO-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase were demonstrated in primary HBECs and in the 16-HBE 14o- cell line. In 16-HBE 14o- cells, treatment with calcium ionophore A23187, bradykinin or LPS up-regulated the expression of these enzymes. The up-regulation of 5-LO was blocked by the anti-inflammatory glucocorticoid dexamethasone. Human bronchial epithelial cells were shown to generate bioactive LTs, with primary HBECs generating 11-fold more LTC4 and five-fold more LTB4 than 16-HBE 14o- cells. LT production was enhanced by ionophore treatment and blocked by the FLAP inhibitor MK-886. CONCLUSIONS: Expression of an active and inducible 5-LO pathway in HBEC suggests that damaged or inflamed bronchial epithelium may synthesize LTs that contribute directly to bronchoconstriction and leucocytosis in airway inflammation.     

The effect of natural pollen exposure on eosinophil apoptosis and its relationship to bronchial hyperresponsiveness in patients with seasonal allergic rhinitis.

BACKGROUND: Limited data suggest that there is increased eosinophilic inflammation in the airways of patients with seasonal allergic rhinitis (SAR) during pollen season even if they do not have asthma. OBJECTIVE: To investigate the effect of natural pollen exposure on inflammatory cells and apoptosis of eosinophils and its association with bronchial hyperresponsiveness (BHR) during and out of pollen season in SAR patients sensitized to only grass pollens. METHODS: Forty SAR patients and 10 patients with nonallergic rhinitis (NAR) from Ankara, Turkey, were recruited to participate in the study. Two induced sputum samples were taken from SAR patients during pollen season (May-June) and out of pollen season (November-January), but only 1 induced sputum sample was taken from NAR patients. Slides of induced sputum were evaluated by 2 cytologists with the use of light microscopy after cytocentrifuged and dyed with May-Grünwald-Giemsa stain. Induced sputum samples were sufficient for differential cell counts in 14 SAR and 7 NAR patients. RESULTS: Eosinophil counts in SAR patients were statistically higher in pollen season (19.4% +/- 16.2%) compared with out of season (4.6% +/- 6.9%, P = .003) and with NAR patients (4.7% +/- 9.5%, P = .01). The apoptotic eosinophil counts in SAR patients were statistically higher out of pollen season (3.0% +/- 4.5%) than in pollen season (0.38% +/- 0.80%, P = .02) and higher than those of NAR patients (0.14% +/- 0.26%, P = .005). The apoptotic ratio was statistically higher after pollen season compared with pollen season (0.720% +/- 0.394% vs 0.044% +/- 0.116%, P = .002). Blood eosinophil counts of SAR patients were increased during the pollen season (364 +/- 187/mm3) compared with out of season (278 +/- 219/mm3, P = .04) and with NAR patients (85 +/- 54/mm3, P = .001). The number of SAR patients who had BHR during the pollen season (7/14) was higher than the number who had BHR out of season (2/14, chi2 = 4.2, P = .04). CONCLUSION: Our data indicate that changes in eosinophil counts and eosinophil apoptosis may be related to the changes of natural pollen exposure and seasonal changes of BHR in SAR patients.     

Insulin-containing lipogenic stimuli suppress mast cell degranulation potential and up-regulate lipid body biogenesis and eicosanoid secretion in a PPARγ-independent manner

Lipid bodies are most studied in adipocytes, where the lipogenic action of insulin initiates their formation. Here, we test the hypothesis that insulin may regulate lipid body content in mast cells and hence, modify their proinflammatory potential. Our data show that insulin causes lipid body accumulation in RBL2H3 and BMMCs. Lipid body accumulation in mast cells is associated with enhanced levels of leukotriene-synthesizing enzymes (LTC4S and 5-LO). Increased basal and antigen-stimulated release of LTC4 is observed in insulin-treated mast cells. Concomitantly, the insulin-containing lipogenic stimulus induces a phenotypic change in mast cells, where this enhancement in leukotriene levels is accompanied by a marked down-regulation in secretory granule content and release in response to stimulus. Mast cells exposed to insulin exhibit altered scatter and fluorescence properties, accumulating in a SSC(lo)FSC(hi) population that exhibits decreased BS staining and degranulation responses and is enriched in NR-positive lipid bodies and eicosanoid synthesis enzymes. Lipid body accumulation in mast cells is mechanistically distinct from the process in adipocytes; for example, it is independent of PPARγ up-regulation and does not involve significant accumulation of conjugated glycerides. Thus, chronic exposure to metabolic stimuli, such as insulin, may be a determinant of the proinflammatory potential of the mast cell.     

IL-9 and c-Kit+ mast cells in allergic rhinitis during seasonal allergen exposure: effect of immunotherapy

Background IL-9 is an important stimulus for tissue infiltration by mast cells, a feature requiring concomitant activation of c-Kit. Objectives We assessed IL-9 expression and c-Kit + mast cells in the nasal mucosa of patients with allergic rhinitis during seasonal pollen exposure and observed the effects of allergen immunotherapy. Methods We studied 44 patients with seasonal rhinitis and asthma before and 2 years after a double-blind trial of grass pollen immunotherapy. Nasal mucosal IL-9 + cells and c-Kit + mast cells were assessed by means of immunochemistry. Cell types expressing IL-9 protein were determined by means of dual immunofluorescence. IL-9 mRNA-positive cells were assessed by means of in situ hybridization, and their phenotype was determined by using sequential immunohistochemistry and in situ hybridization. Results Nasal mucosal c-Kit + mast cells were increased during the pollen season ( P = .0001). IL-9 mRNA-positive cells also tended to increase ( P = .1) and correlated with nasal EG2 + eosinophils ( r = 0.47, P = .05) and IL-5 mRNA-positive cells ( r = 0.54, P = .02). The cell sources of IL-9 included T cells, eosinophils, neutrophils, and mast cells. When compared with placebo, successful pollen immunotherapy markedly inhibited seasonal increases in nasal mucosal c-Kit + mast cells ( P = .001) and the seasonal expression of IL-9 mRNA-positive cells ( P = .06). Immunotherapy also inhibited IL-9 protein expression from nonendothelial cell sources ( P = .0007). Conclusion IL-9 is upregulated in the nasal mucosa during the pollen season and correlates with tissue infiltration by eosinophils. Successful pollen immunotherapy is associated with inhibition of seasonal increases in both nasal c-Kit + mast cells and eosinophils. This effect might be explained, at least in part, by the reduced local expression of IL-9.     

A follow-up study of immunotherapy-treated birch-allergic patients: effect on the expression of chemokines in the nasal mucosa

Background Specific immunotherapy (SIT) is the only treatment producing lasting clinical improvement in patients with allergy. We investigated the long‐term effect of SIT treatment on the expression of chemokines: eotaxin, RANTES (regulated upon activation, normal T cell expressed and secreted) and thymus and activation‐regulated chemokine (TARC), and their receptors CCR3 and CCR4 in biopsies of nasal mucosa from birch‐allergic individuals. Methods Sixteen patients who completed a 3‐year treatment programme 3–5 years ago, and 12 untreated, matched controls were included in the study. Patients recorded symptoms and use of rescue medication before and during the pollen season. Nasal mucosa samples obtained before and during the season were stained for eosinophil and mast cell markers and for eotaxin, RANTES, TARC, CCR3 and CCR4. Results During the pollen season, rhinoconjunctivitis symptoms increased in both SIT and control groups (P=0.001 and 0.002, respectively). However, SIT patients had 37% fewer symptoms than controls. Medication use increased in both groups (P=0.002) during the season but the SIT group used 28% less than the controls (P=0.02). The number of eosinophils in the nasal mucosa increased in the control group (P=0.01) and the difference between the groups was significant during the season (P=0.01). No seasonal increase in the numbers of mast cells was seen, but during the pollen season, more (P=0.02) AA⁺ cells were found in the controls than in the SIT group. The number of eotaxin⁺ and RANTES⁺ cells increased in the control group (P=0.01 and 0.03, respectively) and the difference between groups during the season was significant (P=0.01 and 0.01, respectively). The TARC⁺ cell numbers were lower in the SIT group during the season (P=0.003). The CCR3⁺ cells increased only in the control group during the pollen season and remained unchanged in SIT patients, while CCR4⁺ cell numbers increased in both the control (P=0.03) and SIT (P=0.02) groups. Conclusion This study confirmed that decreased numbers of eosinophils in the nasal mucosa is a long‐lasting effect of birch SIT. SIT also prevented seasonal rises in the number of cells expressing the chemokines eotaxin and RANTES.     

Grass pollen immunotherapy: symptomatic improvement correlates with reductions in eosinophils and IL-5 mRNA expression in the nasal mucosa during the pollen season

BACKGROUND: Tissue eosinophilia and infiltration by T(H)2-type T cells are characteristic features of allergic rhinitis both after allergen challenge and during natural allergen exposure. Specific immunotherapy inhibits allergen-induced nasal eosinophilia. OBJECTIVES: We sought to assess, in the context of a randomized trial, the relationships between symptomatic improvement after immunotherapy and eosinophil numbers and IL-5 expression in the nasal mucosa during the pollen season. METHODS: Nasal biopsy specimens were taken from 37 adults with severe summer hay fever at baseline (out of season) and at peak season after 2 years of treatment with a depot grass pollen extract or placebo. Biopsy specimens were processed for immunohistochemistry by using mAbs against eosinophils (EG2), T cells (CD3), and IL-2 receptor-positive cells (CD25), as well as for in situ hybridization by using a sulfur 35-labeled antisense riboprobe directed against IL-5. RESULTS: Immunotherapy significantly reduced symptoms (49%, P =.01) and medication requirements (80%, P =.007) compared with placebo. There was a 400% increase (P =.004) in eosinophils during the pollen season in placebo-treated patients, which was inhibited in the immunotherapy group (20% increase, P =.04 between groups). Seasonal increases were also observed for CD25(+) cells (P =.002), CD3(+) cells (P =.02), and IL-5 mRNA-expressing cells (P =.03) in the placebo group but not in the immunotherapy group. A significant correlation was observed between eosinophils and IL-5 expression (r = 0.5, P <.05). Both eosinophils (r = 0.6, P <.02) and IL-5 (r = 0.6, P <.02) correlated with symptoms after immunotherapy. CONCLUSION: Improvement in symptoms after grass pollen immunotherapy may result, at least in part, from inhibition of IL-5-dependent tissue eosinophilia during the pollen season.     

Upregulation of nasal mucosal eotaxin in patients with allergic rhinitis during grass pollen season: effect of a local glucocorticoid

BACKGROUND: Allergic rhinitis is a common disease characterized by infiltration of eosinophils into the nasal mucosa during the periods of symptoms. Among chemokines, which attract cells to the site of inflammation, eotaxin is relatively specific for eosinophils. OBJECTIVE: We examined the influence of grass pollen season on nasal eotaxin expression in patients with seasonal allergic rhinitis, as well as the effect of a nasal glucocorticoid on this eotaxin expression. METHODS: Nineteen patients with allergic rhinitis received treatment with either nasal beclomethasone (400 microgram/day) or placebo over a grass pollen season. In these patients, nasal biopsies were taken prior to and during the peak of the pollen season and stained immunohistochemically for eotaxin and EG2 + eosinophils. Five healthy subjects served as controls and gave nasal biopsies once prior to the pollen season. RESULTS: Prior to pollen season, there was no significant difference in nasal eotaxin expression between patients with allergic rhinitis and healthy subjects. Grass pollen season induced significant increase in eotaxin expression in placebo-treated (P = 0.04; n = 9) but not in beclomethasone-treated rhinitis patients (P = 0.8; n = 10). During peak grass pollen season, the eotaxin expression in placebo-treated patients was significantly higher compared with healthy subjects outside season (P = 0.03). There was no significant correlation between the expression of eotaxin and the number of EG2 + eosinophils in nasal mucosa. The serum levels of eotaxin in rhinitis patients remained stable over the pollen season. CONCLUSION: Expression of eotaxin in nasal mucosa of grass-pollen allergic rhinitis patients is upregulated during pollen season and treatment with a nasal glucocorticoid protects against this upregulation.     

(9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido [1,2-a] pyrimidin-4-one), AS-35, inhibits leukotriene synthesis

AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had only weak inhibitory action on the release of beta-HEX. The results indicate that anti-allergic action of AS-35 is predominantly attributable to its inhibition of LT synthesis by suppressing three consecutive enzymes for LTC(4) synthesis.     

Fluticasone propionate aqueous nasal spray reduces inflammatory cells in unchallenged allergic nasal mucosa: effects of single allergen challenge

BACKGROUND: Topical corticosteroid therapy reduces symptoms and nasal mucosal inflammatory cells in patients with allergic rhinitis. Usually patients are advised to start their medication (1 week) before the beginning of the pollen season. The effect of pretreatment with a topical corticosteroid on unchallenged nasal mucosa is not well documented. OBJECTIVES: The purpose of this study was to investigate, in a double-blind, placebo-controlled study, the effect of 6 weeks' pretreatment with 200 microg twice daily fluticasone propionate on nasal symptoms and inflammatory cell numbers after nasal allergen provocation in patients with seasonal allergic rhinitis. METHODS: Nineteen patients with grass pollen-induced allergic rhinitis were treated for a 6-week period out of the grass pollen season. After completing the treatment period, patients were challenged with grass pollen. Nasal mucosal biopsy specimens were taken 5 times in every patient. In nasal mucosa changes in numbers of T cells, B cells, mast cells, eosinophils, macrophages, and Langerhans' cells were investigated. RESULTS: After 4 weeks of treatment but before allergen provocation, significantly fewer epithelial Langerhans' cells, macrophages, mast cells, T cells, and eosinophils were found in the fluticasone propionate group compared with those found in the placebo group. In the lamina propria significantly fewer Langerhans' cells and eosinophils were found in the fluticasone propionate group. Cell influx in nasal mucosa after allergen provocation was significantly inhibited in the fluticasone propionate group compared with that in the placebo group for epithelial Langerhans' cells, mast cells, macrophages, and T cells and for lamina propria eosinophils, mast cells, Langerhans' cells, macrophages, and T cells. CONCLUSIONS: Fluticasone propionate is effective in reducing early- and late-phase nasal symptoms. Topical corticosteroid treatment reduces inflammatory cells in unchallenged nasal mucosa.     

Gene expression of 5-lipoxygenase and LTA4 hydrolase in renal tissue of nephrotic syndrome patients.

Leukotrienes (LT) of the 5-lipoxygenase pathway constitute a class of potent biological lipid mediators of inflammation implicated in the pathogenesis of different models of experimental glomerulonephritis. The key enzyme, 5-lipoxygenase (5-LO), catalyses oxygenation of arachidonic acid to generate the primary leukotriene LTA4. This LT, in turn, serves as a substrate for either LTA4 hydrolase, to form the potent chemoattractant LTB4, or LTC4 synthase, to produce the powerful vasoconstrictor LTC4. To investigate the potential role of LT in the pathogenesis of human glomerulonephritis with nephrotic syndrome, we examined the gene expression of 5-LO and LTA4 hydrolase in renal tissue of 21 adult patients with nephrotic syndrome and 11 controls. The patients consisted of 11 cases of membranous nephropathy (MN), seven focal and segmental glomerulosclerosis (FSGS), two non-IgA mesangial glomerulonephritis and one minimal change disease. Total RNA purified from renal tissue was reverse transcribed into cDNA and amplified with specific primers in a polymerase chain reaction (RT-PCR). Eight patients' renal tissue, four MN and four FSGS, co-expressed 5-LO and LTA4 hydrolase. In situ hybridization analysis revealed 5-LO expression and distribution limited to the interstitial cells surrounding the peritubular capillaries. Comparative clinical and immunohistological data showed that these eight patients had impaired renal function and interstitial changes that significantly correlated with 5-LO expression. These findings suggest that leukotrienes may play an important role in the pathogenesis of MN and FSGS. These results are also relevant to elucidating the pathophysiologic mechanisms which underlie progression to renal failure in these diseases.     

A single nasal allergen challenge increases induced sputum inflammatory markers in non-asthmatic subjects with seasonal allergic rhinitis: correlation with plasma interleukin-5

BACKGROUND: Seasonal allergic rhinitis (SAR) is a risk factor for asthma in affected individuals. Nasal allergic inflammation enhances bone-marrow eosinophil production, mainly via IL-5, and rhinitis patients have increased airway inflammation during the pollen season. OBJECTIVE: To assess the impact of nasal allergy on sputum inflammatory markers. METHODS: In an open-labelled, randomized, placebo-controlled cross-over study with 16 non-asthmatic SAR patients (median age 25 years, 56% males), the effect of a single nasal allergen challenge performed out of season on induced sputum inflammatory parameters was evaluated. SAR patients were identified by history, skin-prick test and specific IgE. All patients had normal lung function/bronchial hyper-responsiveness out of season and a negative asthma/wheezing history. Sputum cells and supernatant levels of ECP, sICAM, IL-5 and IL-10, and plasma levels of IL-5 and ECP, were measured before and 24 h after nasal allergen challenge. After a washout period of at least 4 weeks, the procedure was repeated with placebo challenge (diluent). RESULTS: Nasal allergen challenge led to an increase in sputum ECP (pre = 60 +/- 12, post = 212 +/- 63 micro g/L, P = 0.02 vs. placebo), and sICAM (4.8 +/- 2.7 to 6.5 +/- 2.9 ng/mL, P = 0.02 vs. placebo), whereas IL-10 decreased after provocation (44 +/- 11 to 29 +/- 6 pg/mL, P = 0.06 vs. placebo). Sputum IL-5 was undetectable in all patients. The absolute number of blood and sputum eosinophils did not change significantly after allergen or placebo challenge (P > 0.07, both comparisons). Plasma levels of IL-5 increased after allergen challenge (8.7 +/- 2.9 to 14.5 +/- 3.9 pg/mL, P = 0.001), and the increase in plasma IL-5 was positively correlated with the rise in sputum ECP in a subgroup of 'responders' (n = 12, r = 0.71, P = 0.01). CONCLUSIONS: A single nasal allergen challenge in SAR patients increased markers of allergic inflammation in the lower respiratory tract, possibly via pronounced activation of inflammatory cells through circulating immediate-type reaction cytokines like IL-5. These findings may provide additional explanatory data for the high susceptibility of SAR patients to incident asthma.     

The eicosanoid cascade: possible role in gliomas and meningiomas

Eicosanoids constitute a large family of biologically active lipid mediators that are produced by two enzyme classes, cyclooxygenases (COX-1 and COX-2) and lipoxygenases (5-LO, 12-LO, and 15-LO). Increasing evidence suggests that in addition to a variety of epithelial malignancies, the two most common types of human brain tumour, gliomas and meningiomas, aberrantly overexpress eicosanoid producing enzymes and release a spectrum of eicosanoids that may promote tumorigenesis and the development of peritumorous brain oedema. Glioma and meningioma cells are killed in vitro and in animal models when exposed to COX-2 and 5-LO inhibitors, and their effectiveness is under investigation in clinical trials for treatment of patients with malignant brain tumours. However, despite research into the role of the eicosanoid cascade in the tumorigenesis of human brain tumours, many important questions remain unanswered. Current and newer agents that specifically target key players of the eicosanoid cascade could change the approach to treating brain tumours, because their benefits may lie in their synergism with conventional cytotoxic treatments and/or with other novel agents targeted against other procarcinogenic pathways.     

The effect of omalizumab on nasal allergic inflammation

BACKGROUND: In sensitized patients, coupling between IgE and FcepsilonRI receptors on mast cells leads to release of proinflammatory mediators and a subsequent influx of inflammatory cells to the affected organ. Omalizumab (Xolair; formerly rhuMAb-E25) binds to circulating IgE, thus preventing induction of the allergic process. OBJECTIVE: We investigated the effect of treatment with omalizumab on seasonal allergic rhinitis and related changes in inflammatory cell numbers in nasal biopsy specimens. METHODS: Patients were randomized to treatment with omalizumab or placebo before the pollen season; the treatment was started and continued during season. Symptoms and use of medication were recorded, and blood samples and nasal biopsy specimens were obtained before and during season. Immunocytochemistry was performed on biopsy sections through use of the following antibodies: anti-CD4, CD8 (T lymphocytes), EG2, and anti-eosinophil peroxidase (eosinophils), anti-tryptase (mast cells), human neutrophil lipocalin (neutrophils), and antibodies against IgE and FcepsilonRI. RESULTS: During the season, blood eosinophils increased in placebo-treated patients but not in omalizumab-treated patients (P =.01); the difference between the treatment groups was significant (P =.04). Free IgE in serum decreased significantly (P =.0002) in omalizumab-treated patients but not in placebo-treated patients; the difference between the groups was significant (P =.0001). In nasal biopsy specimens, the number of eosinophil peroxidase-positive staining cells increased in the placebo-treated patients (P =.003) but not in the actively treated patients during the season; the difference between the groups was significant (P =.0001). The number of IgE(+) staining cells decreased significantly in the omalizumab group during the season in comparison with the placebo group (P =.04). CONCLUSION: The clinical benefit of treatment with omalizumab is associated with an anti-inflammatory effect on cellular markers in blood and nasal tissue.     

Bronchial mast cells are the dominating LTC4S-expressing cells in aspirin-tolerant asthma

The increased bronchial production of leukotriene C4 (LTC4) in asthma is assumed to derive from infiltrating eosinophils expressing LTC4-synthase (LTC4S). Multicolor immunohistofluorescence examination of bronchial cryosections from 30 treated, untreated, or bronchial antigen-provoked aspirin-tolerant individuals with asthma and nine control subjects revealed that the dominating LTC4S-expressing cells were mast cells (> 80%), and not eosinophils. Whereas 95% of the mast cells expressed high levels of LTC4S, only 8-27% of the eosinophils expressed low levels. Image analysis revealed a significantly higher LTC4S expression levels in mast cells than in eosinophils. The bronchial mRNA levels for LTC4S did not correlate with the densities of LTC4S-positive eosinophils or mast cells. Treated individuals with asthma with more than 12% reversibility had significantly higher density of LTC4S-positive mast cells than those with less reversibility, and it correlated significantly with reduction in lung function (FEV1-predicted), both before and after salbutamol inhalation. Thus, mucosal mast cells, and not eosinophils, were the dominating LTC4S-containing cells in both untreated and treated aspirin-tolerant asthma. The density of LTC4S-positive mast cells correlated, moreover, with both the reduction in lung function and the degree of reversibility in treated asthma.