2,4,6-Trihydroxy-alpha-p-methoxyphenylacetophenone (Compound D-58) is a potent inhibitor of allergic reactions.

The authors investigated the effects of 2,4,6-trihydroxy-alpha-p-methoxyphenylacetophenone (compound D-58), a potent inhibitor of protein tyrosine kinases SYK and Bruton's tyrosine kinase (BTK), on IgE receptor/FcepsilonRI-triggered mast cell-mediated acute allergic responses in vitro and in vivo. Compound D-58 abrogated IgE receptor/FcepsilonRI-mediated SYK and BTK activation as well as calcium mobilization in mast cells. Mast-cell degranulation and leukotriene (LT) C(4) release was inhibited by compound D-58 in a concentration-dependent fashion. Notably, compound D-58 prevented the mast cell mediator-induced vascular hyperpermeability in an in vivo murine model of passive cutaneous anaphylaxis as measured by the prevention of extravasation of systemically administered Evans blue dye. The results uniquely indicate that compound D-58 has potent antiallergic properties. Therefore, further development of compound D-58 may provide the basis for new and effective treatment programs for severe allergic disorders.

Leflunomide metabolite analogue alpha-cyano-beta-hydroxy-beta-methyl-N-[3-(trifluoromethyl)phenyl]propenamide inhibits IgE/FcepsilonRI receptor-mediated mast cell leukotriene release and allergic asthma in mice.

Mast cell-derived leukotrienes (LTs) play a critical role in the pathophysiology of allergy and asthma. We synthesized 13 analogues of leflunomide (LFM) and examined their in vitro effects on IgE/FcepsilonRI receptor-mediated mast cell LT release. We observed that the novel LFM analogue, alpha-cyano-beta-hydroxy-beta-methyl-N-[3-(trifluoromethyl) phenyl]propenamide (LFM-A8), is a more potent inhibitor than LFM of IgE/FcepsilonRI receptor-mediated LTC4 release from RBL-2H3 rat mast cells. Notably, LFM-A8 showed promising biologic activity in a mouse model of allergic asthma. Treatment of ovalbumin (OVA)-sensitized mice with LFM-A8 prevented the development of airway hyperresponsiveness to methacholine in a dose-dependent fashion. Furthermore, LFM-A8 inhibited the eosinophil recruitment to the airway lumen after the OVA challenge in a dose-dependent fashion. Therefore further development of compound LFM-A8 may provide the basis for new and effective treatment programs for severe allergic disorders, including allergic asthma.

Role of Janus kinase 3 in mast cell-mediated innate immunity against gram-negative bacteria.

Mast cells play a pivotal role in innate host immune response to gram-negative bacteria. We report that Janus kinase 3 plays a role in mast cell-mediated bacterial clearance and neutrophil recruitment by regulating the release of tumor necrosis factor from mast cells. The role of JAK3 in mast cell-facilitated neutrophil recruitment and bacterial clearance was investigated by comparing the neutrophil influxes and bacterial clearance in mast cell-deficient W/W(v) mice reconstituted with JAK3(+/+) or JAK(-/-) mast cells. The neutrophil influx, bacterial clearance, and survival outcome in W/W(v) mice reconstituted with JAK3(+/+) mast cells was better than in W/W(v) mice reconstituted with JAK3(-/-) mast cells. These findings provide evidence that JAK3 is a key regulator of mast cell-mediated innate immunity against gram-negative bacteria.

Mast cell modulation of immune responses to bacteria.

Mast cells are key elements of the immune system. These cells release a wide variety of pro-inflammatory mediators which are responsible for the pathophysiology of many allergic diseases. Recent studies, however, have shown that mast cells have the capacity to modulate the host's innate immune response to gram negative bacteria by their ability to phagocytose bacteria, process and present bacterial antigens to T cells and recruit phagocytic help through the release of physiological amounts of pro-inflammatory mediators. Here, current knowledge of mast cell responses to gram negative bacteria and molecular mechanisms associated with mast cell bacteria interaction is reviewed.

Treatment of allergic asthma by targeting janus kinase 3-dependent leukotriene synthesis in mast cells with 4-(3', 5'-dibromo-4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P97).

4-(3',5'-Dibromo-4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P97) is a rationally designed potent inhibitor of Janus kinase (JAK)-3. Treatment of mast cells with WHI-P97 inhibited the translocation of 5-lipoxygenase (5-LO) from the nucleoplasm to the nuclear membrane and consequently 5-LO-dependent leukotriene (LT) synthesis after IgE receptor/FcepsilonRI crosslinking by >90% at low micromolar concentrations. WHI-P97 did not directly inhibit the enzymatic activity of 5-LO, but prevented its translocation to the nuclear membrane without affecting the requisite calcium signal. WHI-P97 was very well tolerated in mice, with no signs of toxicity at dose levels ranging from 5 microg/kg to 50 mg/kg, and LD(10) was not reached at a 50 mg/kg dose level when administered as a single i. p. or i.v. bolus dose. Therapeutic WHI-P97 concentrations, which inhibit mast cell leukotriene synthesis in vitro, could easily be achieved in vivo after the i.v. or i.p. administration of a single nontoxic 40 mg/kg bolus dose of WHI-P97. Notably, WHI-P97 showed promising biological activity in a mouse model of allergic asthma at nontoxic dose levels. Treatment of ovalbumin-sensitized mice with WHI-P97 prevented the development of airway hyper-responsiveness to methacholine in a dose-dependent fashion. Furthermore, WHI-P97 inhibited the eosinophil recruitment to the airway lumen after the ovalbumin challenge in a dose-dependent fashion. Further development of WHI-P97 may therefore provide the basis for new and effective treatment as well as prevention programs for allergic asthma in clinical settings.

Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis.

Stimulated mast cells release a variety of chemotactic factors such as tumor necrosis factor alpha (TNF-alpha) and leukotriene B4. Recent studies have shown that mast cell-derived TNF-alpha plays a critical role in host defense against Gram negative bacterial infections by the recruitment of neutrophils to the sites of infection. In the present study, we sought to investigate if mast cells release leukotriene (LT) B4 in response to bacteria and, if so, to establish its in vivo relevance. We show that mast cells release significant amounts of LTB4 and LTC4 in response to exposure to FimH-expressing type 1 fimbriated Escherichia coli in vitro. To test the functional significance of mast cell-derived LTs during an E. coli infection in vivo, we examined the effect of a LT-synthesis inhibitor, A-63162, on bacterial clearance and neutrophil influx in an infectious peritonitis model in mast cell-deficient mice (WBB6F1-W/WV) and their normal congenic control (WBB6F1-+/+) mice. Our results show that a treatment with A-63162 reduced neutrophil influx and bacterial clearance in the peritoneal cavities of mast cell-sufficient but not -deficient mice. Thus, mast cell-derived LTs contribute to host defense by mediating early neutrophil influx and bacterial clearance at sites of infection.

Histamine as an autocrine regulator of leukemic cell proliferation.

We examined leukemic lymphocyte precursors from ALL patients as well as immortalized ALL cell lines for cytoplasmic histamine expression. The histamine levels ranged from 10.8 pg/10(6) cells to 82.2 pg/10(6) cells in ALL cell lines (N=4) and from 12.5 pg/10(6) cells to 1235.4 pg/10(6) cells for primary leukemic cells from ALL patients (N=13). The presence of histamine in the cytoplasm of these ALL cells was also confirmed by immunostaining using a polyclonal rabbit anti-histamine antibody. Notably, the histamine receptor blocker diphenhydramine inhibited the clonogenic growth of ALL cells by >90% prompting the hypothesis that histamine may be an autocrine regulator of ALL cell proliferation. Our study suggests that histamine receptor blockers may therefore be useful for the treatment of therapy-refractory ALL.

Anti-inflammatory activity of 2,4, 6-trihydroxy-alpha-p-methoxyphenyl-acetophenone (compound D-58).

BACKGROUND: Active oxygen radicals as well as a variety of cytosolic protein tyrosine kinases play a role in the regulation of prostaglandin E(2) (PGE(2)), a key inflammatory mediator, released by skin cells in response to irradiation with ultraviolet B light (UVB). Identification of chemical compounds that can interrupt such events may provide a basis for the development of potent anti-inflammatory agents. OBJECTIVE: To investigate the effect of a novel genistein analog, 2,4, 6-trihydroxy-alpha-p-methoxyphenylacetophenone, with antioxidant property (compound D-58), on UVB-induced inflammatory responses. METHODS: Epidermal cell cultures were irradiated with UVB both in the presence and absence of compound D-58 and the PGE(2) released in the medium was determined by ELISA. For in vivo studies, skin inflammation was induced in mice either by carrageenan challenge of the air pouch or by an acute exposure of skin to UVB radiation. The resulting inflammatory mediator release, skin edema and the histological changes of the skin were determined both in the presence and absence of compound D-58. RESULTS: Compound D-58 treatment effectively inhibited the development of edema and histological changes in the skin of UVB-irradiated mice as well as the release of PGE(2) in vitro as well as in vivo. CONCLUSION: Compound D-58 (2,4,6-trihydroxy-alpha-p-methoxyphenylacetophenone) has potent anti-inflammatory properties.

Coordinate expression of secretory phospholipase A(2) and cyclooxygenase-2 in activated human keratinocytes.

PGE(2) levels are altered in human epidermis after in vivo wounding; however, mechanisms modulating PGE(2) production in activated keratinocytes are unclear. In previous studies, we showed that PGE(2) is a growth-promoting autacoid in human primary keratinocyte cultures, and its production is modulated by plating density, suggesting that regulated PGE(2) synthesis is an important component of wound healing. Here, we examine the role of phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) enzymes in modulation of PGE(2) production. We report that the increased PGE(2) production that occurs in keratinocytes grown in nonconfluent conditions is also observed after in vitro wounding, indicating that similar mechanisms are involved. This increase was associated with coordinate upregulation of both COX-2 and secretory PLA(2) (sPLA(2)) proteins. Increased sPLA(2) activity was also observed. By RT-PCR, we identified the presence of type IIA and type V sPLA(2), along with the M-type sPLA(2) receptor. Thus the coordinate expression of sPLA(2) and COX-2 may be responsible for the increased prostaglandin synthesis in activated keratinocytes during wound repair.

Targeting Janus kinase 3 in mast cells prevents immediate hypersensitivity reactions and anaphylaxis.

Janus kinase 3 (JAK3), a member of the Janus family protein-tyrosine kinases, is expressed in mast cells, and its enzymatic activity is enhanced by IgE receptor/FcepsilonRI cross-linking. Selective inhibition of JAK3 in mast cells with 4-(4'-hydroxylphenyl)-amino-6, 7-dimethoxyquinazoline) (WHI-P131) blocked the phospholipase C activation, calcium mobilization, and activation of microtubule-associated protein kinase after lgE receptor/FcepsilonRI cross-linking. Treatment of IgE-sensitized rodent as well as human mast cells with WHI-P131 effectively inhibited the activation-associated morphological changes, degranulation, and proinflammatory mediator release after specific antigen challenge without affecting the functional integrity of the distal secretory machinery. In vivo administration of the JAK3 inhibitor WHI-P131 prevented mast cell degranulation and development of cutaneous as well as systemic fatal anaphylaxis in mice at nontoxic dose levels. Thus, JAK3 plays a pivotal role in IgE receptor/FcepsilonRI-mediated mast cell responses, and targeting JAK3 with a specific inhibitor, such as WHI-P131, may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.

The mast cell tumor necrosis factor alpha response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48.

Mast cells are well known for their harmful role in IgE-mediated hypersensitivity reactions, but their physiological role remains a mystery. Several recent studies have reported that mast cells play a critical role in innate immunity in mice by releasing tumor necrosis factor alpha (TNF-alpha) to recruit neutrophils to sites of enterobacterial infection. In some cases, the mast cell TNF-alpha response was triggered when these cells directly bound FimH on the surface of Escherichia coli. We have identified CD48, a glycosylphosphatidylinositol-anchored molecule, to be the complementary FimH-binding moiety in rodent mast cell membrane fractions. We showed that (i) pretreatment of mast cell membranes with antibodies to CD48 or phospholipase C inhibited binding of FimH+ E. coli, (ii) FimH+ E. coli but not a FimH- derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) binding of FimH+ bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected with CD48 cDNA, and (iv) antibodies to CD48 specifically blocked the mast cell TNF-alpha response to FimH+ E. coli. Thus, CD48 is a functionally relevant microbial receptor on mast cells that plays a role in triggering inflammation.

Quantitative high-performance liquid chromatographic method for pharmacokinetic studies of the potent mast cell inhibitor 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P131).

The novel quinazoline derivative 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P131) has recently been identified as a potent mast cell inhibitor capable of preventing IgE/antigen induced cutaneous as well as systemic fatal anaphylaxis in mice. Here we describe a sensitive high-performance liquid chromatography (HPLC)-based quantitative detection method for measurement of WHI-P131 levels in plasma as well as in target mast cells. The average extraction recovery for WHI-P131 was 88.4% for plasma and 75.7% for RBL-2H3 mast cell lysates. Good linearity (r>0.999) was observed throughout the concentration range of 0.1-20 microM in plasma and 0.01-5 nmol in 5 x 10(6) cells (0.5-238 microM per cell) for WHI-P131. Intra- and inter-assay variabilities were <7% and the lowest detection limit of WHI-P131 was 0.05 microM in plasma and 0.005 nmol in 5 million cells, respectively, at a signal-to-noise ratio of approximately 2. The practical utility of this new HPLC method was confirmed in a pilot pharmacokinetic study in BALB/c mice as well as in a cellular drug uptake and disposition study in RBL-2H3 mast cells. After intraperitoneal administration of a non-toxic 40 mg/kg bolus dose of WHI-P131, the estimated maximum plasma concentration was 92.7 microM, which is approximately 1-log higher than the effective in vitro mast cell inhibitory concentrations of WHI-P131. The drug absorption was rapid with an absorption half-life of only 2.9 min and the estimated time to reach the maximum plasma concentration was 8.3 min. WHI-P131 was cleared with an apparent systemic clearance rate of 2586 ml/h/kg and an elimination half-life of 1.8 h. An intracellular exposure level (AUC) of 55 microM x h was obtained after in vitro treatment of RBL-2H3 mast cells with WHI-P131 at a 33.6 microM final concentration in culture medium. The availability of the described quantitative HPLC detection method for WHI-P131 provides the basis for further development of WHI-P131 as an anti-allergic drug through detailed pharmacodynamic studies in preclinical animal models.

Genetic and biochemical evidence for a critical role of Janus kinase (JAK)-3 in mast cell-mediated type I hypersensitivity reactions.

We investigated the role of JAK3 in IgE receptor/FcepsilonRI-mediated mast cell responses. IgE/antigen induced degranulation and mediator release were substantially reduced with Jak3-/- mast cells from JAK3-null mice that were generated by targeted disruption of Jak3 gene in embryonic stem cells. Further, treatment of mast cells with 3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154), a potent inhibitor of JAK3, inhibited degranulation and proinflammatory mediator release after IgE receptor/ FcepsilonRI crosslinking. Thus, JAK3 plays a pivotal role in IgE receptor/ FcepsilonRI-mediated mast cell responses and targeting JAK3 may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.

Pharmacokinetics and biologic activity of the novel mast cell inhibitor, 4-(3-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline in mice.

PURPOSE: The purpose of the present study was to examine the pharmacodynamic and pharmacokinetic features of the novel mast cell inhibitor 4-(3'-Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P180) in mice. METHODS: A high performance liquid chromatography (HPLC)-based quantitative detection method was used to measure plasma WHI-P180 levels in mice. The plasma concentration-time data was fit to a single compartment pharmacokinetic model by using the WinNonlin program to calculate the pharmacokinetic parameters. A cutaneous anaphylaxis model was used to examine the pharmacodynamic effects of WHI-P180 on anaphylaxis-associated vascular hyperpermeability. RESULTS: The elimination half-life of WHI-P180 in CD-1 mice (BALB/ c mice) following i.v., i.p., or p.o. administration was less than 10 min. Systemic clearance of WHI-P180 was 6742 mL/h/kg in CD-I mice and 8188 mL/h/kg in BALB/c mice. Notably, WHI-P180, when administered in two consecutive nontoxic i.p. bolus doses of 25 mg/kg, inhibited IgE/antigen-induced vascular hyperpermeability in a well-characterized murine model of passive cutaneous anaphylaxis. CONCLUSIONS: WHI-P180 is an active inhibitor of IgE-mediated mast cell responses in vitro and in vivo. Further preclinical characterization of WHI-P180 may improve the efficacy of WHI-P180 in vivo and provide the basis for design of effective treatment and prevention programs for mast cell mediated allergic reactions.

Clinical implications of mast cell-bacteria interaction.

Mast cells are traditionally known for mediating allergic reactions. In addition, these cells have been implicated in the pathogenesis of a variety of clinical conditions such as atopic and contact dermatitis, bullous pemphigoid, fibrotic lung disease, neurofibromatosis, psoriasis, scleroderma, rheumatoid arthritis, interstitial cystitis, ulcerative colitis, and Crohn's disease, but their role in host defense was an enigma until recently. Owing to the strategic location of mast cells at the host environment interface, their role in bacterial infections has been studied by a number of investigators. Latest reports show that mast cells have an ability to modulate the host's innate immune response to infectious agents. This review discusses the clinical implications of mast cell-bacteria interactions.

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic.

Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection. Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host's immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization. This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies.