Corticotropin-releasing hormone-receptor 2 is required for acute stress-induced bladder vascular permeability and release of vascular endothelial growth factor.

OBJECTIVE: To investigate the corticotropin-releasing hormone (CRH) receptor (CRH-R) requirement for the effect of acute stress on bladder vascular permeability and release of vascular endothelial growth factor (VEGF), as increasing evidence indicates that acute stress worsens certain inflammatory disorders, including interstitial cystitis/painful bladder syndrome (IC/PBlS), which is characterized by pain, variable bladder inflammation, increased expression of bladder vascular endothelial growth factor (VEGF), and many detrusor mast cells. MATERIALS AND METHODS: Bladders of normal C57BL/6, and C57BL/6- derived CRH-R1, CRH-R2 or double CRH-R1 + 2 knockout (-/-) female mice (10-12 weeks old) were catheterized under anaesthesia. After emptying the urine, normal saline was instilled with or without intravesical CRH-R antagonists in C57BL/6 mice before they were stressed by placing them in a restrainer for 30 min. Evans blue was injected in the tail vein before stress for the permeability experiments. The bladders from C57BL/6 or CRH-R -/- mice were then removed, minced into 1 mm(2) pieces and cultured overnight. Culture media were collected 24 h later for VEGF assay. C57BL/6 bladder was processed for CRH-R immunohistochemistry. RESULTS: Acute stress increased bladder vascular permeability in control C57BL/6 and CRH-R1 -/- mice, but not CRH-R2 -/- or CRH-R1+2 -/- mice. The CRH-R2 antagonist Astressin 2B, but not the CRH-R1 antagonist Antalarmin, inhibited stress-induced VEGF release from C57BL/6 mouse bladder explants. Stress could not induce a VEGF increase from bladder explants of CRH-R2 -/- or CRH-R1+2 -/- mice, but did so in CRH-R1 -/- mice. Bladder CRH-R2 immunoreactivity was detected in C57BL/6 bladders. CONCLUSIONS: Acute stress induces bladder vascular permeability and VEGF release that is dependent on CRH-R2. These findings suggest that CRH and VEGF might participate in the pathogenesis of IC/PBlS and provide for new therapeutic targets.

Mitochondrial uncoupling protein 2 inhibits mast cell activation and reduces histamine content.

Mast cells are immune effector cells that are involved in allergies and inflammation through the release of mediators such as histamine, PGs, and cytokines. Uncoupling protein 2 (UCP2) is a mitochondrial protein that inhibits insulin secretion from beta cells, possibly through down-regulation of reactive oxygen species production. We hypothesized that UCP2 could also regulate mast cell activation. In this study, we show that mouse bone marrow mast cells (BMMCs) and human leukemic LAD2 mast cells express UCP2. BMMCs from Ucp2(-/-) mice exhibited greater histamine release, whereas overexpression of UCP2 in LAD2 cells reduced histamine release after both allergic and nonallergic triggers. Ucp2(-/-) BMMCs also had elevated histamine content and histidine decarboxylase expression. Histamine content was reduced by overexpression of UCP2 or treatment with the mitochondrial-targeted superoxide dismutase-mimetic (TBAP) tetrakis(4-benzoic acid) porphyrin manganese(III). Furthermore, Ucp2(-/-) BMMCs also had greater production of both IL-6 and PGD(2) as well as ERK phosphorylation, which is known to regulate PG synthesis. Intradermal administration of substance P, an activator of skin mast cells, and challenge with DNP-human serum albumin after passive sensitization induced significantly greater vascular permeability in the skin of Ucp2(-/-) mice in vivo. Our results suggest that UCP2 can regulate mast cell activation.

Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor.

Mast cells are critical for allergic reactions, but also for innate or acquired immunity and inflammatory conditions that worsen by stress. Corticotropin-releasing hormone (CRH), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. We investigated the expression of CRH receptors and the effects of CRH in the human leukemic mast cell (HMC-1) line and human umbilical cord blood-derived mast cells. We detected mRNA for CRH-R1alpha, 1beta, 1c, 1e, 1f isoforms, as well as CRH-R1 protein in both cell types. CRH-R2alpha (but not R2beta or R2gamma) mRNA and protein were present only in human cord blood-derived mast cells. CRH increased cAMP and induced secretion of vascular endothelial growth factor (VEGF) without tryptase, histamine, IL-6, IL-8, or TNF-alpha release. The effects were blocked by the CRH-R1 antagonist antalarmin, but not the CRH-R2 antagonist astressin 2B. CRH-stimulated VEGF production was mediated through activation of adenylate cyclase and increased cAMP, as evidenced by the fact that the effect of CRH was mimicked by the direct adenylate cyclase activator forskolin and the cell-permeable cAMP analog 8-bromo-cAMP, whereas it was abolished by the adenylate cyclase inhibitor SQ22536. This is the first evidence that mast cells express functional CRH receptors and that CRH can induce VEGF secretion selectively. CRH-induced mast cell-derived VEGF could, therefore, be involved in chronic inflammatory conditions associated with increased VEGF, such as arthritis or psoriasis, both of which worsen by stress.

Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells.

Mast cells participate in allergies, and also in immunity and inflammation by secreting proinflammatory cytokines. Flavonoids are naturally occurring polyphenolic plant compounds, one group of which -- the flavonols, inhibits histamine and some cytokine release from rodent basophils and mast cells. However, the effect of flavonols on proinflammatory mediator release and their possible mechanism of action in human mast cells is not well defined. Human umbilical cord blood-derived cultured mast cells (hCBMCs) grown in the presence of stem cell factor (SCF) and interleukin (IL)-6 were preincubated for 15 min with the flavonols quercetin, kaempferol, myricetin and morin (0.01, 0.1, 1, 10 or 100 microM), followed by activation with anti-IgE. Secretion was quantitated for IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), histamine and tryptase levels. Release of IL-6, IL-8 and TNF-alpha was inhibited by 82-93% at 100 microM quercetin and kaempferol, and 31-70% by myricetin and morin. Tryptase release was inhibited by 79-96% at 100 microM quercetin, kampferol and myricetin, but only 39% by morin; histamine release was inhibited 52-77% by the first three flavonols, but only 28% by morin. These flavonols suppressed intracellular calcium ion elevations in a dose-response manner, with morin being the weakest; they also inhibited phosphorylation of the calcium-insensitive protein kinase C theta (PKC theta). Flavonol inhibition of IgE-mediated proinflammatory mediator release from hCBMCs may be due to inhibition of intracellular calcium influx and PKC theta signaling. Flavonols may therefore be suitable for the treatment of allergic and inflammatory diseases.

Mast cell tryptase: a new biomarker in patients with stable coronary artery disease.

Mast cells may participate actively in the inflammatory process of atherosclerotic plaques by releasing proteolytic enzymes and various other pro-inflammatory substances. We hypothesized that increased levels of mast cell tryptase, could be an important biomarker in patients with stable coronary artery disease (CAD). We measured tryptase in 102 patients without acute coronary syndromes undergoing cardiac catheterization. Patients with significant CAD [> or =50% stenosis in > or =1 artery (n=66)] had significantly higher serum tryptase than patients with normal angiography (n=13) or non-significant CAD [<50% stenosis (n=23)]. The median, 25th and 75th percentiles for tryptase in these two groups were 8.38 (6.4 and 10.7)mug/L versus 6.78 (5.61 and 9.72) microg/L, p=0.014. Patients in the highest quartile of tryptase levels had a 4.3-fold risk for CAD [Odds ratio (OR): 4.3; 95% confidence interval (CI): 1.08-17.19; p=0.04]. In a multivariate regression analysis, tryptase remained an independent predictor for CAD along with age (OR: 1.178; 95% CI: 1.021-1.359, p=0.025). High circulating tryptase levels may be a result of chronic low-grade inflammatory activity present in atherosclerotic plaques. Tryptase measurements may emerge as a novel way of identifying asymptomatic patients with CAD, and represent a new biomarker of therapeutic efficacy in patients with CAD.

Interleukin-17 and the interleukin-17 family member network.

Interleukin-17 (now known as IL-17A), is a homodimer of two 155 amino acid chains secreted by CD4+ activated memory T cells (CD45+ RO+) and is available as a glycosylated 20- to 30-kDa homodimeric peptide. Human IL-17 shows amino acid sequence identity of 62.5 and 58% to the mouse and rat sequences, respectively. IL-17 can regulate the function of a variety of cell types, plays an important role in the maturation of hematopoietic progenitor cells, and induces production of proinflammatory mediators. Here, for the first time, we summarize the biological effects of IL-17 and its family members as important players of T cell-mediated immune responses and underline the important implications of this cytokine in inflammation and degenerative diseases.

Azelastine inhibits secretion of IL-6, TNF-alpha and IL-8 as well as NF-kappaB activation and intracellular calcium ion levels in normal human mast cells.

BACKGROUND: Mast cells are involved in allergic inflammation by secreting histamine, proteases and several cytokines, including interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and IL-8. Certain histamine-1 receptor antagonists, such as azelastine present in the ophthalmic solution Optivar, have been reported to inhibit histamine and tryptase secretion, but its effect on inflammatory cytokine release from normal human umbilical cord blood-derived cultured mast cells (hCBMC) are not well known. METHODS: We investigated the effect of azelastine on the secretion of IL-6, TNF-alpha and IL-8 from hCBMC, as well as its possible mechanism of action. hCBMC sensitized with IgE were pretreated for 5 min with azelastine at 0.01, 0.1, 1, 3, 6, 12, 24, or 60 microM of Optivar, before stimulation with anti-IgE for 6 h. Optivar vehicle without azelastine was used as control. Cytokines were measured by ELISA, intracellular calcium levels by calcium indicators confocal, and nuclear factor-kappaB (NF-kappaB) by electromobility shift assay. RESULTS: Stimulation with anti-IgE led to substantial secretion of IL-6, TNF-alpha and IL-8. Preincubation for 5 min resulted in almost maximal inhibition with 6 microM azelastine for TNF-alpha (80%), with 24 microM for IL-6 (83%) and 60 microM for IL-8 (99%); the vehicle solution at the same concentrations as Optivar had no effect. Stimulation with anti-IgE increased intracellular Ca2+ level and induced NF-kappaB expression in hCBMC, which was inhibited by 24 microM azelastine. CONCLUSION: Azelastine inhibited hCBMC secretion of IL-6, TNF-alpha and IL-8, possibly by inhibiting intracellular Ca2+ ions and NF-kappaB activation. Azelastine may, therefore, be helpful in treating allergic inflammation.

The isoflavone genistein inhibits proliferation and increases histamine content in human leukemic mast cells.

Mast cells are involved in allergic inflammation and some rare disorders such as systemic mastocytosis and mast cell leukemia. Certain naturally occurring flavonoids have been shown to inhibit mast cell activation and promote maturation of secretory granules. Here, we report that the isoflavone genistein inhibited the growth of human leukemic mast cells (HMC-1) by 68.8, 51.6, and 30.2% at 10(-4), 10(-5), and 10(-6) M, respectively, at day 3 (p < 0.001). Genistein at 10(-4) M increased the histamine content per 2 x 10(5) cells at day 3 from 5.9 +/- 1.2 micrograms/mL to 11.1 +/- 1.3 micrograms/mL (n = 6; p < 0.0001). These results indicate that genistein can inhibit proliferation and induce maturation of HMC-1 cells.

IL-1 induces vesicular secretion of IL-6 without degranulation from human mast cells.

Fc epsilon RI cross-linkage in mast cells results in release of granule-associated mediators, such as histamine and proteases, as well as the production of numerous cytokines, including IL-6. Mast cells have been increasingly implicated in inflammatory processes where explosive degranulation is not commonly observed. Here, we show that IL-1 stimulates secretion of IL-6 without release of the granule-associated protease tryptase in normal human umbilical cord blood-derived mast cells (hCBMCs). IL-6 secretion stimulated by IL-1 in hCBMCs is potentiated by priming with IL-4 and reflects the higher levels of IL-6 secreted from human leukemic mast cell line (HMC-1). Stimulating HMC-1 cells by both IL-1 and TNF-alpha results in synergistic secretion of IL-6. IL-6 is de novo synthesized, as its secretion is blocked by inhibitors of transcription or protein synthesis. IL-1 does not increase intracellular calcium ion levels in either hCBMCs or HMC-1 cells, and IL-6 stimulation proceeds in the absence of extracellular calcium ions. Ultrastructural Immunogold localization shows that IL-6 is excluded from the secretory granules and is compartmentalized in 40- to 80-nm vesicular structures. Selective secretion of IL-6 from mast cells appears distinct from degranulation and may contribute to the development of inflammation, where the importance of IL-6 has been recognized.

Flavones inhibit proliferation and increase mediator content in human leukemic mast cells (HMC-1).

OBJECTIVE: Mast cells are involved in allergic and inflammatory reactions. These cells are also increased in the bone marrow, skin, and other organs in systemic mastocytosis. Flavonoids are naturally occurring molecules with antioxidant, cytoprotective, and anti-inflammatory activities. Some flavonoids, like quercetin, inhibit the growth of certain malignant cells in culture. Quercetin also inhibits histamine release and induces accumulation of secretory granules in rat basophilic leukemia cells. METHOD: We investigated the effect of five flavonoids: flavone, kaempferol, morin, myricetin, and quercetin at 1, 10, and 100 microM on proliferation and secretory mediator content (beta-hexosaminidase, histamine, and tryptase) in human leukemic mast cells (HMC-1), the doubling time of which was about 2 d. RESULTS: Flavone and kaempferol at 100 microM inhibited cell proliferation over 80% on either day 3, 4, or 5 of culture. Quercetin showed this level of inhibition only on day 5, myricetin inhibited by 50% at days 3-5, whereas morin's inhibition was < 20%. All flavonoids (except morin) at 100 microm increased histamine and tryptase content, but not beta-hexosaminidase, equally at days 3 and 4 of culture quercetin also increased the development of secretory granules. CONCLUSION: These results indicate that certain flavonoids can inhibit HMC-1 proliferation, induce secretory granule development and the accumulation of mediators.

Interleukin-6 and mast cells.

Interleukin (IL) 6 is a pleiotropic cytokine (26 kDa) that originally was named interferon beta 2 or B cell-stimulating factor or differentiating B cell factor inducing immunoglobulin production. IL-6 is produced in many diseases. After secretion, IL-6 binds to its receptor IL-6R alpha (gp 80), the IL-6R alpha complex then recruits the signal-transducing beta-subunit (gp 130), which is the functional complex for signal transduction. In addition, activation of Th2 cells or mast cells also produce IL-6, which mediates immune responses, inflammation, acute phase responses, hematopoiesis, cancer, inflammatory bowel disease, etc. IL-6 also is a crucial cytokine for mast cell maturation. Human cord blood CD34+ cells differentiate and grow into mast cells in the presence of stem cell factor (SCF) and IL-6, causing increases in cell size, frequency of chymase positive cells, and intracellular histamine levels when compared with cells treated with SCF alone. Activated mast cells increase IL-6 mRNA associated with protein kinase C (PKC) activity. IL-6 also up-regulates histamine production rather than increases its storage and is an important inducing factor for the expression of immunoglobulin E (IgE) Fc epsilon RI.

Serum interleukin-6 reflects disease severity and osteoporosis in mastocytosis patients.

BACKGROUND: Systemic mastocytosis (SM) is a condition typically characterized by an increased number of mast cells in the bone marrow or in skin areas known as urticaria pigmentosa. Patients may present with flushing, itching, gastrointestinal symptoms, arrhythmias, headaches and osteoporosis. Some patients experience systemic symptoms indicative of SM in the absence of a positive bone marrow or skin biopsy, and are known as 'clinical mastocytosis', but are herein referred to as suspected of having systemic mastocytosis. Serum tryptase has been increasingly used as a biochemical marker of mastocytosis, but is not always elevated. OBJECTIVE: To investigate the association of serum levels of two key mast cell mediators, interleukin-6 (IL-6) and tryptase, to each other and with disease severity in patients with mastocytosis. METHODS: Patients responded to an announcement from the Systemic Mastocytosis Society (USA) and submitted frozen serum samples, but the precise diagnosis made by their own health providers was not known until after the assays were completed. There were 9 suspected systemic mastocytosis (SuSM), 3 cutaneous mastocytosis (CM), 11 indolent systemic mastocytosis (ISM), and 3 aggressive systemic mastocytosis (ASM). Five normal volunteers (3 females/2 males) also submitted samples, as did 33 cardiac patients without coronary artery disease. For 2 days prior to and during the collection period, mastocytosis patients were asked to abstain from any over-the-counter or food products containing biogenic amines, as well as drugs prescribed for this condition. Serum levels of IL-6 and tryptase were measured using established assays. RESULTS: Twenty-six patients (14 females/12 males) submitted serum samples. There were 9 cases of SuSM (6 females/3 males) in whom tryptase values were borderline normal; IL-6 values were slightly elevated with one being high. In 3 cases of CM (2 females/1 male), both tryptase and IL-6 were slightly elevated. In patients with ISM (5 females/6 males), only 6/11 had any tryptase elevated significantly as compared to 9/11 with elevated serum IL-6. Three patients with ASM had significant elevations of both IL-6 and tryptase. The most consistent finding was that of IL-6 elevations in 7/7 patients (3 females/4 males) who reported symptoms of osteoporosis and/or bone pain (1 SuSM, 3 ISM, 3 ASM) in the absence of any coexisting condition involving bone pathology. CONCLUSION: Serum IL-6 is elevated in mastocytosis patients and correlates with severity of symptoms and the presence of osteoporosis. High serum IL-6 may not only signify disease progression, but may also participate in the pathophysiology of mastocytosis.

Azelastine is more potent than olopatadine n inhibiting interleukin-6 and tryptase release from human umbilical cord blood-derived cultured mast cells.

BACKGROUND: Mast cells are involved in early- and late-phase reactions by releasing vasoactive molecules, proteases, and cytokines. Certain histamine-1 receptor antagonists and other antiallergic drugs seem to inhibit the release of mediators from rat and human mast cells. OBJECTIVE: Azelastine and olopatadine are antiallergic agents present in the ophthalmic solutions azelastine hydrochloride (Optivar, Asta Medica/Muro Pharmaceuticals, Tewksbury, MA), and olopatadine hydrochloride (Patanol, Alcon Laboratories, Fort Worth, TX), respectively. We investigated the effect of these drugs on interleukin-6 (IL-6), tryptase, and histamine release from cultured human mast cells (CHMCs). METHODS: CHMCs were grown from human umbilical cord blood-derived CD34+ cells in the presence of stem cell factor and IL-6 for 14 to 16 weeks. Sensitized CHMCs were pretreated with various concentrations of azelastine or olopatadine for 5 minutes. CHMCs were then challenged with anti-immunoglobulin E, and the released mediators were quantitated. RESULTS: The greatest inhibition of mediator release was seen with 24 microM azelastine; this level of inhibition was matched with the use of 133 microM olopatadine. At this concentration, these drugs inhibited IL-6 release by 83% and 74%, tryptase release by 55% and 79%, and histamine release by 41% and 45%, respectively. Activated CHMCs were characterized by numerous filopodia that were inhibited by both drugs as shown by electron microscopy. CONCLUSIONS: These results indicate that azelastine and olopatadine can inhibit CHMCs activation and release of IL-6, tryptase, and histamine. On an equimolar basis, azelastine was a more potent inhibitor than olopatadine.