Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.

BACKGROUND: Leukotrienes (LTs) are potent pro-inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down-regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma. METHODS: Bronchoalveolar lavage fluid exosomes were collected from healthy individuals (n = 13) and patients with mild allergic asthma to birch pollen (n = 12) before and after birch allergen provocation. Exosomes were characterized by flow cytometry and Western blot. Their capacity to induce IL-8 and LT production in the human bronchial epithelial cell (BEC) line 16HB14o- was measured by ELISA and reverse-phase HPLC, respectively. RESULTS: Compared to BALF exosomes from healthy individuals, BALF exosomes from asthmatics displayed higher levels of exosome-associated markers, such as the tetraspanins CD63 and CD81 and the scavenger receptor CD36. No major differences were observed between BALF exosomes from before and after allergen provocation. Furthermore, we show that BALF exosomes contain enzymes for LT biosynthesis. The effect of exosomes to promote LTC(4) and IL-8 release in BEC was significantly increased for exosomes from asthmatics, and the CysLT(1) receptor antagonist Montelukast reduced exosome-induced IL-8 secretion. CONCLUSIONS: Bronchoalveolar lavage fluid exosomes from asthmatic and healthy individuals exhibit distinct phenotypes and functions. BALF exosomes from asthmatics might contribute to subclinical inflammation by increasing cytokine and LTC(4) generation in airway epithelium.

Thromboxane generation by human peripheral blood polymorphonuclear leukocytes.

Human peripheral blood polymorphonuclear leukocytes were stimulated to generate thromboxane B2 in a time- and concentration-dependent fashion upon exposure to serum-treated zymosan particles. Conversion by stimulated PMN of [14C] arachidonic acid to [14C]thromboxane B2 was confirmed by thin-layer radiochromatography, radio-gas chromatography, and mass spectrometry. Generation of thromboxane B2 was independent of platelet contamination and could be inhibited by the cyclooxygenase inhibitor, indomethacin. Cells rendered incapable of ingesting particles by treatment with cytochalasin B generated comparable amounts of thromboxane B2. These results suggest that human peripheral blood polymorphonuclear leukocytes synthesize thromboxanes in response to surface stimulation independently of phagocytosis.

Microsomal prostaglandin E synthase-1 and 5-lipoxygenase: potential drug targets in cancer.

There is strong evidence for a role of prostaglandin (PG)E(2) in cancer cell proliferation and tumour development. In PGE(2) biosynthesis, cyclooxygenases (COX-1/2) convert arachidonic acid to PGH(2), which can be isomerized to PGE(2) by PGE synthases, including microsomal PGE synthase-1 (MPGES-1). Data describing genetic deletions of MPGES-1 are reviewed. The results suggest that MPGES-1 is an alternative therapeutic target for cancer cells and tumours that express this enzyme. Several compounds that target COX-2 or MPGES-1 also inhibit 5-lipoxygenase. This may be advantageous for treatment of some forms of cancer.

Leukotriene A4-hydrolase activity in guinea pig and human liver.

Guinea pig and human liver homogenates transformed leukotriene A4 into leukotriene B4. In both species, the enzymatic activity was recovered in the 105000 X g supernatant, and it was found to be susceptible to heat treatment (56 degrees C, 1 h). Digestion with a proteolytic enzyme also resulted in loss of enzymatic activity. The formation of leukotriene B4 was pH-dependent, with an optimum between pH 7 and pH 8.5. In addition, two other organs from the guinea-pig, lungs and kidneys, contained leukotriene A4-hydrolase activity. The identity of leukotriene B4 was ascertained by high-performance liquid chromatography, ultraviolet spectrometry, gas chromatography-mass spectrometry and bioassay. We have recently demonstrated the presence of leukotriene A4-hydrolase activity in mammalian plasma (Fitzpatrick et al. (1983) Proc. Natl. Acad. Sci. USA 80, 5425-5429). The results of the present study suggest several possible origins of this plasma leukotriene A4 hydrolase.

Hemoprotein catalysis of leukotriene formation.

Incubation of various hemoproteins with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid resulted in formation of epimeric 5(S),12-dihydroxy-6,8,10,14 -eicosatetraenoic acids and epimeric 8,15(S)-dihydroxy-5,9,11,13 -eicosatetraenoic acids, respectively. These dihydroxy acids were earlier recognized as nonenzymatic hydrolysis products of 5(S),6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) and 14,15(S)-oxido-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene A4). These allylic epoxides could be isolated as such from the hemoprotein incubations, and most probably they are intermediates in formation of the dihydroxy acids.

Leukotriene A4: metabolism in different rat tissues.

The transformation of leukotriene A4 into dihydroxyeicosatetraenoic acids and sulfidopeptide leukotrienes was determined in homogenates of rat tissues supplied with glutathione and albumin. The highest production of leukotriene B4 was found in spleen, lung and small intestine, while leukotriene C4 dominated in liver and lung. 5(S),6(R)-Dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5,6-DHETE) was formed in all tissues, most prominently in kidney, heart and brain. We also found another isomer of 5,6-dihydroxyeicosatetraenoic acid produced in the kidney. This compound was derived from 5,6-DHETE by isomerization, probably of the 11-cis double bond to 11-trans, and the process appeared to be catalyzed by a membrane-bound factor.

Enzymatic formation of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid: kinetics of the reaction and stereochemistry of the product.

The enzymatic conversion of leukotriene A4 into 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid, catalyzed by mouse liver cytosolic epoxide hydrolase (EC 3.3.2.3), was recently described (Haeggström, J., Meijer, J. and Rådmark, O. (1986) J. Biol. Chem. 261, 6332-6337). In the present study, we report analytical data confirming the stereochemistry of this novel enzymatic metabolite of leukotriene A4. By steric analysis of the vicinal diol and comparison with synthetic material, the structure was established as (5S,6R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid. Apparent kinetic constants of this reaction were determined and found to be 5 microM and 550 nmol.mg-1.min-1, for Km and Vmax, respectively. Also, a semipurified preparation of human liver cytosolic epoxide hydrolase avidly catalyzed the same hydrolysis of leukotriene A4 (apparent Km was 8 microM). The enzyme was not inactivated by leukotriene A4, as judged by time-course experiments with a second substrate addition.

Serum factors regulate 5-lipoxygenase activity in maturating HL60 cells.

5-Lipoxygenase activity in DMSO-differentiated HL60 cells is regulated by human serum. The serum effect depended on the differentiation state of the cells. For a stimulatory effect to occur, it was required that the cells had been treated with DMSO before addition of serum. After this regimen, the HL60 cells acquired the same high 5-LO activity as found for human neutrophils isolated from peripheral blood (about 9-times higher than for HL60 cells treated only with DMSO). On the other hand, when serum was added together with DMSO and present during the entire differentiation period (seven days), or withdrawn after the first four days, the 5-LO activity did not increase. 5-LO activity of HL60 cells covaried with the expression of the CD14 molecule, a marker for myeloid cell maturation which was recently identified as a receptor for the complex of LPS and LPS-binding protein. These serum effects on 5-LO activity were only observed for intact cells. The prominent increase in 5-LO activity induced by serum was not concomitant with similar changes in the expression of 5-LO or 5-LO-activating protein (FLAP), as judged from analyses of immunoreactive protein and mRNA. Also, the high 5-LO activity induced by serum was rather insensitive to the drug MK886 under our standard assay conditions, which included addition of exogenous arachidonic acid (40 microM). The results indicate that additional cellular components of importance for 5-LO activity in HL60 cells become operative after serum treatment, and that mere expression of 5-LO and FLAP is insufficient for high 5-LO activity in intact cells.

Leukotriene A4 hydrolase: analysis of some human tissues by radioimmunoassay.

Leukotriene A4 hydrolase was quantitated by radioimmunoassay, in extracts from eight human tissues. The enzyme was detectable in all tissues, with the highest level (2.6 mg per g soluble protein) in leukocytes, followed by lung and liver. The polyclonal antiserum did not cross-react with cytosolic epoxide hydrolase purified from mouse or human liver. When incubated with leukotriene A4, formation of leukotriene B4 was evident in all tissues. Furthermore, enzymatic formation of (5S,6R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid from leukotriene A4, was found in extracts from liver, kidney and intestines.

Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities.

Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line.

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.

Albumin stabilizes 14,15-leukotriene A4.

14,15-Leukotriene A4 is a pivotal biosynthetic intermediate in 15-lipoxygenase initiated leukotriene biosynthesis. This compound hydrolyzes instantaneously in phosphate buffer at pH 7.4. However, addition of human or bovine albumin to otherwise identical buffer solutions increases its stability. Intact 14,15-leukotriene A4 then decomposes by first-order kinetics with rate constants inversely proportional to the albumin concentration. Stabilization of 14,15-leukotriene A4 under certain conditions may influence its proportionate transformation by enzymatic vs non-enzymatic processes.

Effects of 1-chloro-2,4,6-trinitrobenzene on 5-lipoxygenase activity and cellular leukotriene synthesis.

5-Lipoxygenase (EC 1.13.11.34) is the key enzyme in the regulation of leukotriene synthesis. Here, the effects of various substituted nitrobenzene compounds on 5-lipoxygenase activity and the formation of leukotriene B4 (LTB4) were studied in polymorphonuclear leukocytes (PMNL), B lymphocytes, and human whole blood. 1-Chloro-2,4,6-trinitrobenzene (TNCB) was found to inhibit calcium ionophore A23187-induced leukotriene synthesis in PMNL in a biphasic manner. Thus, 1.0 microM TNCB caused 50% inhibition of LTB4 formation, but only 16% inhibition was found at 10 times higher concentration. In contrast, this higher concentration of TNCB activated the synthesis of LTB4 when PMNL were stimulated with arachidonic acid alone, demonstrating that TNCB can exert both stimulatory and inhibitory effects on leukotriene synthesis depending on the experimental conditions. The inhibitory effect of 1.0 microM TNCB on ionophore A23187-induced leukotriene synthesis could be circumvented by addition of exogenous arachidonic acid. At high concentrations of TNCB (25-100 microM), the drug blocked ionophore A23187-induced leukotriene synthesis. TNCB also inhibited LTB4 formation in B lymphocytes, as well as in human whole blood. The activity of recombinant 5-lipoxygenase was inhibited by TNCB, and reduced glutathione or beta-mercaptoethanol counteracted this inhibition. This suggests that TNCB might inhibit 5-lipoxygenase by alkylating thiol groups. TNCB possessed a high specificity for 5-lipoxygenase with only modest inhibitory effects on 12-lipoxygenase (EC 1.13.11.31), 15-lipoxygenase (EC 1.13.11.12), and phospholipase A2 (EC 3.1.1.4) activities. Taken together, these results show that TNCB can both specifically inhibit and stimulate leukotriene formation and might be useful in further studies on the regulation of 5-lipoxygenase.

Identification and subcellular localization of leukotriene A4-hydrolase activity in human epidermis.

The purpose of this study was to determine whether normal human epidermis could produce leukotriene B4 (LTB4) from leukotriene A4 (LTA4) ex vivo, and to localize this LTA4-hydrolase activity. Epidermis obtained by suction blister technique incubated with human polymorphonuclear cells, resulted in a 54% increase in LTB4 formation when compared to polymorphonuclear cells incubated alone. Furthermore, human epidermis transformed exogenous LTA4 into LTB4, and this reaction obeyed Michaelis-Menten kinetics with an apparent Km of 6 microM. Subcellular fractionation of homogenized epidermis localized the LTA4-hydrolase activity mainly in the 105,000 x g supernatant fraction (cytoplasmic fraction). This activity was inhibited by two inhibitors of LTA4-hydrolase (bestatin and captopril). Western blot analysis of the 105,000 x g fraction of homogenized epidermis and cultured keratinocytes supported the presence of a LTA4-hydrolase. Thus, normal human epidermis possesses LTA4-hydrolase activity which can transform exogenous LTA4 and polymorphonuclear cell-derived LTA4 into LTB4. The identification of LTA4-hydrolase in the cytoplasmic fraction of human epidermis indicates that epidermal cells may play a more active role in the enzymatic process leading to formation of the proinflammatory compound LTB4 than previously expected.

Formation and effects of leukotriene B4 in human lymphocytes.

Incubation of human tonsillar B lymphocytes or peripheral blood T lymphocytes with leukotriene (LT) A4 led to the formation of LTB4. Stimulation of these cells with ionophore A23187 did not lead to the synthesis of detectable amounts of leukotrienes. Formation of LTB4 was observed in several monoclonal B- and T-cell lines after incubation with LTA4, but not after stimulation with ionophore A23187. The Burkitt lymphoma cell line Raji was found to possess higher LTA4-hydrolase activity than normal lymphocytes. The expression of the LTA4-hydrolase gene but not the 5-lipoxygenase gene was demonstrated on the transcriptional level in Northern blots and on the translational level by Western blots. Stimulation of human monocytes with ionophore A23187 resulted in the release of LTA4. Coincubations of transformed lymphocytes and monocytes stimulated with ionophore A23187 produced increased amounts of LTB4 as compared with monocytes alone. LTB4 influence on lymphocyte activation was studied and CD23 expression was used as a marker. The expression of this antigen was enhanced on resting B lymphocytes in synergy with B-cell growth-promoting factors. LTB4 also augmented DNA synthesis, cell replication and IgG secretion. These results indicate that extracellular LTA4, released from activated monocytes, is converted by lymphocytes into LTB4 which might cause activation and differentiation of B lymphocytes.

EGFR signaling upregulates expression of microsomal prostaglandin E synthase-1 in cancer cells leading to enhanced tumorigenicity.

In this report we describe the contribution of prostaglandin E(2) (PGE(2)) derived from the inducible microsomal PGE-synthase type-1 (mPGES-1) to the epidermal growth factor receptor (EGFR) oncogenic drive in tumor epithelial cells and in tumor-bearing mice. EGFR stimulation upregulated expression of mPGES-1 in HT-29, A431 and A549 cancer cells. Egr-1, a transcription factor induced by EGF, mediated this response. The Egr-1 rise provoked the overexpression of mPGES-1 messenger and protein, and enhanced PGE(2) formation. These changes were suppressed either by silencing Egr-1, or by upstream blockade of EGFR or ERK1/2 signals. Further, in a clonogenic assay on tumor cells, EGF induced a florid tumorigenic phenotype, which regressed when mPGES-1 was silenced or knocked down. EGF-induced mPGES-1 overexpression in epithelial cell reduced E-cadherin expression, whereas enhancing that of vimentin, suggesting an incipient mesenchymal phenotype. Additionally, inhibiting the EGFR in mice bearing the A431 tumor, the mPGES-1 expression and the tumor growth, exhibited a parallel decline. In conclusion, these findings provide novel evidence that a tight cooperation between the EGF/EGFR and mPGES-1 leads to a significant tumorigenic gain in epithelial cells, and provide clues for controlling the vicious association.