Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.     

Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation

Fresh arterial tissue generates an unstable substance (prostaglandin X) which relaxes vascular smooth muscle and potently inhibits platelet aggregation. The release of prostaglandin (PG) X can be stimulated by incubation with arachidonic acid or prostaglandin endoperoxides PGG₂ or PGH₂. The basal release of PGX or the release stimulated with arachidonic acid can be inhibited by previous treatment with indomethacin or by washing the tissue with a solution containing indomethacin. The formation of PGX from prostaglandin endoperoxides PGG₂ or PGH₂ is not inhibited by indomethacin. 15-hydro-peroxy arachidonic acid (15-HPAA) inhibits the basal release of PGX as well as the release stimulated by arachidonic acid or prostaglandin endoperoxides (PGG₂ or PGH₂). Fresh arterial tissue obtained from control or indomethacin treated rabbits, when incubated with platelet rich plasma (PRP) generates PGX. This generation is inhibited by treating the tissue with 15-HPAA. A biochemical interaction between platelets and vessel wall is postulated by which platelets feed the vessel wall with prostaglandin endoperoxides which are utilized to form PGX. Formation of PGX could be the underlying mechanism which actively prevents, under normal conditions, the accumulation of platelets on the vessel wall.     

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs

Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.     

Macrophage procoagulant activity as a measure of cell-mediated immunity in the mouse

Thioglycollate-induced peritoneal exudate cells (TG-PEC) developed increased procoagulant activity after incubation with lymphokine and lipopolysaccharide (LPS). Dilutions of up to 1/1000 for insoluble Con A and 1/200 for periodate-induced lymphokine supernatants were active in enhancing macrophage procoagulant activity (MPCA), which was detected after a 2-hr incubation period and steadily increased over 20 hr. MPCA could also be induced by antigen; peritoneal cells from sensitized B6AF1 mice with strong footpad reactions to ovalbumin (OVA) responded to as little as 0.1 microgram/ml OVA in the MPCA test in an antigen-specific manner. By contrast, PEC from sensitized CBA/J mice that gave poor in vivo responses to OVA only reacted with high concentrations of the antigen in vitro. Production of the lymphokine responsible for induction of MPCA required an Ly-1+2- T cell, a nylon wool-adherent cell, and an la-17-bearing adherent cell. The MPCA induced by lymphokine or LPS did not appear to be a serine esterase and was not inhibited by phospholipase C. Coagulation of human factor-deficient plasma with activated TG-PEC indicated a requirement for Factor X.     

P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma

Leukotriene E4 (LTE4) that plays a key role in airway inflammation is expressed on platelets and eosinophils. We investigated whether blocking of the P2Y12 receptor can suppress eosinophilic inflammation in a mouse model of asthma because platelets and eosinophils share this receptor to be activated. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. On each challenge day, clopidogrel, a P2Y12 antagonist was administered 30 min. before each challenge. Forty‐eight hours after the last OVA challenge, mice were assessed for airway hyperresponsiveness (AHR), cell composition and cytokine levels, including chemokine ligand 5 (CCL5), in bronchoalveolar lavage (BAL) fluid. EOL cells were treated with LTE4, with or without clopidogrel treatment, and intracellular and extracellular eosinophil cationic protein (ECP) expressions were measured to find the inhibiting function of P2Y12 antagonist on eosinophilic activation. The levels of P2Y12 expression were increased markedly in the lung homogenates of OVA‐sensitized and ‐challenged mice after platelet depletion. Administration of clopidogrel decreased AHR and the number of airway inflammatory cells, including eosinophils, in BAL fluid following OVA challenge. These results were associated with decreased levels of Th2 cytokines and CCL5. Histological examination showed that inflammatory cells as well as mucus‐containing goblet cells were reduced in clopidogrel‐administered mice compared to vehicle‐treated mice. Clopidogrel inhibited extracellular ECP secretion after LTE4 stimulation in EOL‐1 cells. Clopidogrel could prevent development of AHR and airway inflammation in a mouse model of asthma. P2Y12 can be a novel therapeutic target to the suppression of eosinophils in asthma.     

Decreased antigen-induced eicosanoid release in conjugated linoleic acid-fed guinea pigs

This study investigated the capacity of conjugated linoleic acids (CLA) to reduce ex vivo antigen-induced release of eicosanoids in a type I hypersensitivity model. Guinea pigs were fed a diet containing 0.25% safflower oil (control) or 0.25% CLA [43% trans (t)10, cis (c)12; 41% c9, t11/t9, c11 18:2] for 2 wk before and during sensitization to ovalbumin (OVA). Lungs, tracheas, and bladders were incubated in physiological saline solution (PSS) for 1 h (basal mediator release) and challenged with OVA (0.01 g/l PSS) for 1 h (mediator release in response to antigen). Eicosanoids were quantified by HPLC/tandem mass spectrometry or enzyme immunoassay. CLA feeding resulted in no change in basal release but decreased eicosanoid release from sensitized tissues in response to antigen challenge in the following manner: thromboxane B(2), 6-keto-prostaglandin (PG)F(1alpha), PGF(2alpha), PGD(2), PGE(2) by 57-75% in lung, 45-65% in trachea, and 38-60% in bladder; and leukotriene C(4)/D(4)/E(4) by 87, 90, and 50% in lung, trachea, and bladder, respectively. These data indicate that feeding CLA reduces lipid-derived inflammatory mediators produced by this type I hypersensitivity model.     

Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.     

Suppression of Ag-induced release of EP (IL 1) by spleen cells of specifically desensitized donors: evidence for the role of a suppressor cell

Monocytes or macrophages may be induced to produce IL 1 by activators (e.g., lipopolysaccharide endotoxin) that act directly or by antigens/mitogens (e.g., Con A) that stimulate inducer lymphocytes to release a lymphokine that stimulates macrophages. Using guinea pigs (GP) rendered delayed hypersensitive to ovalbumin (OVA), we investigated the role of spleen cells from normal, sensitized, and specifically desensitized GP in suppressing release of IL 1, measured as endogenous pyrogen (EP), from peritoneal exudates of sensitized GP when incubated with OVA in vitro. Co-cultivation of all three sources of spleen cells with GP peritoneal exudate cells and OVA suppressed EP release as measured in the rabbit fever assay, the effect being most marked with cells from desensitized GP, intermediate with cells from sensitized GP, and least with normal cells. This suppressor activity of spleen cells on in vitro EP release was not explained by nonspecific absorption of EP by the added cells and did not affect EP release by a stimulus that activates macrophages directly (heat-killed staphylococci). It required both lymphocytes and macrophages for its effect, but unlike some other suppressor factors, it was not modified by indomethacin, an inhibitor of prostaglandin release. This appears to be the first reported evidence for cell-mediated suppression of lymphokine-mediated release of IL 1, an important modulator of the immune system through its combined role as a lymphocyte-activating factor and an inducer of fever (EP).     

Compliance and stability of the bronchial wall in a model of allergen-induced lung inflammation.

Airway wall remodeling in response to inflammation might alter load on airway smooth muscle and/or change airway wall stability. We therefore determined airway wall compliance and closing pressures in an animal model. Weanling pigs were sensitized to ovalbumin (OVA; ip and sc, n = 6) and were subsequently challenged three times with OVA aerosol. Control pigs received 0.9% NaCl (n = 4) in place of OVA aerosol. Bronchoconstriction in vivo was assessed from lung resistance and dynamic compliance. Semistatic airway compliance was recorded ex vivo in isolated segments of bronchus, after the final OVA aerosol or 0.9% NaCl challenge. Internally or externally applied pressure needed to close bronchial segments was determined in the absence or presence of carbachol (1 microM). Sensitized pig lungs exhibited immediate bronchoconstriction to OVA aerosol and also peribronchial accumulations of monocytes and granulocytes. Compliance was reduced in sensitized bronchi in vitro (P < 0.01), and closing pressures were increased (P < 0.05). In the presence of carbachol, closing pressures of control and sensitized bronchi were not different. We conclude that sensitization and/or inflammation increases airway load and airway stability.     

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and incorporation of [14C]-leucine into proteins were quantified and expressed per microgram DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P less than .05) and for releasing PGFM was increased (P less than .05) on day 20 compared to day 16 of pregnancy. Neither superovulation nor day of pregnancy altered trophoblastic activity for releasing PGF2 alpha. Superovulation increased (P less than .05) endometrial release of PGF2 alpha. Endometrial release of PGF2 alpha was less (P less than .05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 micrograms) was added at the start of incubation, trophoblastic release of PGF2 alpha changed (P less than .05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, trophoblastic release of PGF2 alpha increased linearly (P less than .01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P less than .05) trophoblastic protein synthesis and release of PGF2 alpha. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from superovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Glutamate-stimulated ATP release from spinal cord astrocytes is potentiated by substance P

ATP has recently emerged as a key molecule mediating pathological pain. The aim of this study was to examine whether spinal cord astrocytes could be a source of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from these astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamate-induced inward currents, but did not cause such currents alone. When glutamate was applied alone it acted exclusively through alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors to stimulate Ca(2+) influx-dependent ATP release. However, when substance P was co-applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. Activation of neurokinin receptor subtypes, protein kinase C and phospholipases A(2), C and D were needed for substance P to bring about its effects. These results suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that release substance P and glutamate.     

Substance P-Induced Release of Prostaglandins from Astrocytes: Regional Specialisation and Correlation with Phosphoinositol Metabolism

Addition of substance P (SP) to astrocytes cultured from rat neonatal spinal cord evoked a time- and concentration-dependent increase in the accumulation of phosphoinositol and the release of prostaglandin (PG) D2 and PGE2. Both basal and stimulated releases were reduced to similar levels by indomethacin. In contrast, astrocytes cultured from cerebral cortex and cerebellum showed no SP-stimulated increase in phosphoinositol accumulation or release of PGs. Release of PGD2 and PGE2 was, however, stimulated by the calcium ionophore A23187, and both phosphoinositol accumulation and PG release were stimulated from cortical astrocytes incubated in the presence of serum. The results from this study suggest that SP-stimulated phosphoinositol accumulation and release of PGs from cultured rat neonatal astrocytes are regionally specialised in favour of cells derived from spinal cord.     

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.     

A comparison of reactive oxygen species generation by rat peritoneal macrophages and mast cells using the highly sensitive real-time chemiluminescent probe pholasin: inhibition of antigen-induced mast cell degranulation by macrophage-derived hydrogen peroxide

Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another's function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 microM) or ionomycin (1 microM), but not OVA (1 microg/ml), released high-level ROS, levels of which peaked after 3-7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degranulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell activation and promote resolution of IgE-mediated inflammation.     

In vitro release of PGF(2alpha) from the caprine uterus and the effect of ovarian steroids

The purpose of the study was to determine the in vitro release of prostaglandin F(2)alpha (PGF(2alpha)) from the uterine tissues of ovariectomized goats after steroid treatment. Strips of endometrial tissues (separated by trypsin treatment) and intact uterine tissues (with endometrium and myometrium) were maintained in organ culture and exposed to estradiol-17beta(E) at 50 ng/ml and to progesterone (P) at 250 pg/ml alone or in combination. The endometrial tissues, in general, continued to release PGF(2alpha) without steroids treatment throughout the 96-h incubation period; their output was consistently higher (based on ng/gm of wet tissue) than the corresponding intact uterine tissues. However, exposure to E reversed this trend and produced a three-fold increase from the intact uterine strips at 24 h. A combined E-plus-P treatment blocked the stimulatory response, whereas P treatment alone evoked a delayed stimulatory response at 48 h. Endometrial tissue reaction was similar to that of P treatment, but it exhibited a more moderated response to E, which could not be blocked by a combined E-plus-P treatment. The results suggest that both endometrial and myometrial tissues release PGF(2alpha) and that this release is regulated by ovarian steroid hormones.     

A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model.

Nitric oxide (NO), an important cell signaling molecule, is considered a marker of inflammatory response and is elevated in asthmatics. This study investigated the effects of montelukast (a leukotriene receptor antagonist) on iNOS expression and activity in a Brown Norway (BN) rat allergic inflammation model and in L2 lung epithelial cells. Allergic inflammation was induced by ovalbumin (OVA) injection in BN rats followed by treatment with either montelukast or dexamethasone (DX). Allergen inhalation was performed, and post-allergen Penh was measured 5 min after the challenge. Cysteinyl leukotriene levels were measured in bronchoalveolar lavage (BAL) fluid and lung iNOS expression and activity determined. These parameters were also measured in cytokine stimulated L2 lung epithelial cells. iNOS expression was significantly higher in OVA challenged rats compared to the naive, DX, and montelukast treated groups, as confirmed by immunohistochemistry and Western blot analysis. However, no significant differences in NOS activity were found. Cysteinyl leukotriene measured in BAL was significantly higher in all OVA challenged rats compared to naive controls. Incubation of L2 cells with a mixture of interferon gamma (IFNgamma), lipopolysaccharide (LPS), and tumor necrosis factor (TNFalpha) resulted in high levels of nitrite formation resulting from iNOS induction. Treatment of cytokine stimulated cells with DX or montelukast significantly decreased iNOS expression and activity. No detectable cysteinyl leukotrienes were found in the supernatant fluid of L2 cells. This study confirms the ability of montelukast to modulate iNOS function and raises the possibility that changes in iNOS expression and activity may occur via pathways independent of cysteinyl leukotrienes.     

Oral administration of ovalbumin after sensitization attenuates symptoms in a mouse model of food allergic enteropathy

Oral immunotherapy (OIT) is a promising treatment of food allergy. To administer an appropriate oral dose of an allergenic component as OIT to individuals sensitized with a food allergen may prevent inducing food allergic inflammation in them. So we attempted to establish a mouse model to evaluate efficacy for oral administration of food allergen after sensitization. In BALB/c mice sensitized by injecting ovalbumin (OVA) with alum twice, OVA was administered before inducing inflammation by feeding the mice with egg white (EW) diet. Severe inflammatory responses, such as enteropathy, weight loss, IL-4 production, and increase of IgE antibody levels, were suppressed by administration with 4 mg of OVA 7 times before feeding EW diet. OVA administration alone induced a slight Th2 response, but no symptoms. The current study demonstrated that severe food allergic enteropathy could be prevented by pre-administration with appropriate dose of OVA to sensitized mice.     

'Antiflammins': two nonapeptide fragments of uteroglobin and lipocortin I have no phospholipase A2-inhibitory and anti-inflammatory activity

The 'antiflammin' nonapeptides P1 and P2 [(1988) Nature 335, 726-730] were synthesized and tested for inhibition of phospholipase A2 and release of prostaglandin E2 and leukotriene C4 in stimulated cells in vitro, and in vivo for anti-inflammatory activity in rats with carrageenan-induced paw oedema. Porcine pancreatic phospholipase A2 was not inhibited at concentrations of 0.5-50 microM. Prostaglandin E2 and leukotriene C4 release by mouse macrophages stimulated with zymosan or ATP was not affected up to a concentration of 10 microM, nor was prostaglandin release by interleukin 1 beta-stimulated mesangial cells and angiotensin II-stimulated smooth muscle cells. Both peptides exhibited no anti-inflammatory activity in carrageenan-induced rat paw oedema after topical (250 micrograms/paw) or systemic administration (1 or 4 mg/kg s.c.). These results do not support the claim of potent phospholipase A2-inhibitory and anti-inflammatory activity of the 'antiflammins' P1 and P2.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Enzyme-modified cheese exerts inhibitory effects on allergen permeation in rats suffering from indomethacin-induced intestinal inflammation

We have found that an enzyme-modified cheese (EMC) inhibited the permeation of allergens such as ovalbumin (OVA), using Caco-2 cells as an in vitro intestinal epithelial model. In addition, NPWDQ (Asn-Pro-Trp-Asp-Gln, aa 107-111 of alphas(2)-casein) was isolated from EMC and identified as one of the responsible peptides for this inhibitory activity (Tanabe et al., J. Agric. Food Chem., (2007)). In this study, we aimed to clarify the mechanism by which NPWDQ inhibited allergen permeation in vitro, and also to evaluate the effects of EMC on allergen permeation in vivo. Intestinal permeability for both fluorescein isothiocyanate conjugated dextran and horseradish peroxidase was decreased in Caco-2 cells by the addition of NPWDQ, indicating that NPWDQ might inhibit both paracellular and transcellular transports. Next, intestinal inflammation was induced by subcutaneous injections of indomethacin to rats. When OVA was injected into the jejunal and ileac loops of indomethacin-administered rats with and without NPWDQ, it was found that the addition of NPWDQ effectively diminished OVA permeation from both loops. Although the plasma OVA concentration of indomethacin-administered rats after oral OVA challenge was markedly elevated over that of normal rats, supplemental administration of EMC to the rats effectively suppressed OVA permeation. These results suggest that EMC is useful for the prevention of food allergy by inhibiting allergen permeation probably by enforcing the intestinal barrier.