Isolated Rat Peritoneal Mast Cells Research Articles

Light‐driven Activation of NADP(H) Oxidase 5

Ultraviolet band A (UVA) light irradiation has been shown to induce cytosolic calcium oscillations in the isolated rat peritoneal mast cells via activation of NADPH oxidase (NOX). It has been clarified that UVA-triggered calcium oscillations in mast cells (isolated rat peritoneal mast cells, rat mast cell line RBL-2H3, mouse bone marrow-derived mast cells) was due to NOX2 activation. To elucidate whether UVA light irradiation would also activate NOX5, a monomeric isoform of the NOX family of oxidases not requiring transmembrane p22 or cytosolic subunits for activity and not endogenously expressed in rodents, NOX5 was expressed ectopically in CHO-K1 cells, and the effect of UVA irradiation or photodynamic action on cytosolic calcium concentration was monitored with Fura-2 fluorescent calcium imaging. It was found that NOX5 expressed ectopically in CHO-K1 cells was activated by UVA (LED 380 nm) light irradiation to induce persistent cytosolic calcium oscillations. Blue LED (450 nm) light irradiation of CHO-K1 cells expressing protein photosensitizer miniSOG-NOX5 fusion protein triggered also persistent cytosolic calcium oscillations which were completely blocked by NOX inhibitor diphenyleneiodonium (DPI), and inhibited by singlet oxygen quencher Trolox C. Sulphonated aluminium phthalocyanine (SALPC) photodynamically activated the heterologously expressed NOX5 in CHO-K1 cells to trigger similarly cytosolic calcium oscillations, which were completely inhibited by DPI. These data suggest that both UVA light irradiation and 1O2 generation with photodynamic action could activate NOX5, to ultimately trigger calcium oscillations. It is therefore possible now to realize light-driven NOX5 activation for cell-type or subcellular organelle specific generation of superoxide anion.

IL-37 is protective in allergic contact dermatitis through mast cell inhibition

Allergic contact dermatitis (ACD), characterized predominantly by erythema, vesiculation, and pruritus, is a T cell-mediated skin inflammatory condition. Among immune cells involved in ACD, mast cells (MCs) play an essential role in its pathogenesis. As an inhibitor of proinflammatory IL-1 family members, interleukin 37 (IL-37) has been shown to ameliorate inflammatory responses in various allergic diseases. In this study, we assessed the immunomodulatory effect of IL-37 on allergic inflammation using a 2,4-dinitrofluorobenzene (DNFB)-induced ACD rat model and isolated rat peritoneal mast cells (RPMCs). Systematic application of IL-37 significantly relieved ear swelling, reduced inflammatory cell infiltration, decreased inflammatory cytokine production (TNF-α, IL-1β, IFN-γ, and IL-13), inhibited MC recruitment, lowered IgE levels, and reduced IL-33 production in the local ear tissues with DNFB challenge. Additionally, RPMCs isolated from ACD rats with IL-37 intervention showed downregulation of IL-6, TNF-α, IL-13, and MCP-1 production following IL-33 stimulation, and reduction of β-hexosaminidase and histamine release under DNP-IgE/HSA treatment. Moreover, IL-37 treatment also significantly restrained NF-κB activation and P38 phosphorylation in ACD RPMCs. SIS3, a specific Smad3 inhibitor, abolished the suppressive effects of IL-37 on MC-mediated allergic inflammation, suggesting the participation of Smad3 in the anti-ACD effect of IL-37. These findings indicated that IL-37 protects against IL-33-regulated MC inflammatory responses via inhibition of NF-κB and P38 MAPK activation accompanying the regulation of Smad3 in rats with ACD.

Ameliorative effects of type-A procyanidins polyphenols from cinnamon bark in compound 48/80-induced mast cell degranulation.

Allergic diseases are a significant health concern in developing countries. Type-A procyanidin polyphenols from cinnamon (Cinnamomum zeylanicum Blume) bark (TAPP-CZ) possesses antiasthmatic and antiallergic potential. The present study was aimed at the possible anti-allergic mechanism of TAPP-CZ against the compound 48/80 (C48/80)–induced mast cell degranulation in isolated rat peritoneal mast cells (RPMCs). TAPP-CZ (1, 3, 10, and 30 µg/ml) was incubated for 3 hours with isolated, purified RPMCs. The C48/80 (1 µg/ml) was used to induce mast cell degranulation. The mast cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay whereas histamine, β-hexosaminidase (β-HEX), and interleukin-4 (IL-4) levels were determined in RPMCs. TAPP-CZ (3, 10, and 30 µg/ml) showed significant and dose-dependent decrease in a number of degranulated cells and levels of markers (histamine, β-HEX, and IL-4) as compared with C48/80 control. In conclusion, TAPP-CZ stabilizes mast cell and cause inhibition of the allergic markers such as histamine, IL-4, and β-HEX in IgE-mediated manner. The present study supports mast cell stabilization as a possible mechanism of action of TAPP-CZ against immune respiratory disorders such as asthma and allergic rhinitis.

2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 μmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

Vardenafil ameliorates immunologic- and non-immunologic-induced allergic reactions

Cyclic nucleotides, such as cAMP and cGMP, play a protective role in the modulation of the activity of some inflammatory cells in allergic disorders. Their intracellular concentrations are tightly regulated by the phosphodiesterases (PDEs). The protective efficacy of the selective PDE5 inhibitor vardenafil against mast-cell-mediated allergic reactions in murine models has been investigated. Compound 48/80 was used as a direct mast cell degranulator to induce anaphylaxis. Vardenafil (administered orally at 5, 10, 20, 40, and 80 mg/kg body mass) significantly (P < 0.05, n = 12) increased protection against compound-48/80-induced anaphylaxis in mice to 33.33%, 66.67%, 66.67%, 83.33%, and 66.67% respectively compared with the control (vehicle). In passive cutaneous anaphylaxis (PCA) in rats, vardenafil (10 mg/kg body mass) significantly (P < 0.05, n = 6) decreased Evans' blue dye extravasation (4.6-fold). Pre-incubation of isolated rat peritoneal mast cells (RPMCs) with vardenafil (10 and 100 μmol/L) significantly (P < 0.05, n = 6) reduced compound-48/80-induced histamine release by 2.8- and 3-fold, respectively. Moreover, histamine release by immunogenic stimulation of sensitized RPMCs by egg albumin significantly declined following pre-incubation with vardenafil (10 and 100 μmol/L) by 1.94- and 1.99-fold, respectively. In conclusion, inhibition of PDE5 by vardenafil ameliorated immunologic and non-immunologic mast-cell-mediated allergic reactions and reduced histamine release, providing evidence for the potential anti-allergic properties of vardenafil.

Chronic Pain Treatment: The Influence of Tricyclic Antidepressants on Serotonin Release and Uptake in Mast Cells

The involvement of serotonin (5-HT) in chronic pain mechanisms is established. 5-HT inhibits central painful stimuli, but recent data suggests that 5-HT could also enhance pain stimulus from the periphery, where mast cells play an important role. We aimed in our study to clarify the influence of selected tricyclic antidepressants (TCAs) on mast cell function: secretion, uptake, and reuptake of 5-HT, that could interfere with 5-HT levels and in this way contribute to the generation of pain. As an experimental model, we used isolated rat peritoneal mast cells and incubated them with selected TCAs (clomipramine, amitriptyline, doxepin, and imipramine) under different experimental conditions. 5-HT release, uptake, and reuptake were determined spectrofluorometrically. We showed that TCAs were able to inhibit 5-HT secretion from mast cells, as well as uptake of exogenous 5-HT and reuptake of secreted 5-HT back into mast cells. The effects of TCAs were concentration dependent; higher concentrations of TCAs inhibited the secretion of 5-HT induced by compound 48/80, whereas lower concentrations of TCAs inhibited 5-HT uptake. The most effective TCA was halogenated clomipramine. As TCAs are well introduced in chronic pain treatment, the insight into mechanisms of action is important for an understanding of their effect in various pain conditions.

Polydatin attenuated food allergyviastore-operated calcium channels in mast cell

To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and anti-allergic activity. After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for 24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleukin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca(2+) mobilization was investigated by probing intracellular Ca(2+) with fluo-4 fluorescent dye, with the signal recorded and analyzed. We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mL vs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca(2+) influx through store-operated calcium channels (SOCs) (2.35 ± 0.39 vs OVA 3.51 ± 0.38, P < 0.01). Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca(2+) mobilization, mainly through inhibiting Ca(2+) entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.

Caffeic acid phenethyl ester attenuates IgE-induced immediate allergic reaction

Caffeic acid phenethyl ester (CAPE) is the active component of honey bee propolis extracts. The results of the current study demonstrate that CAPE attenuated immunoglobulin (Ig)E-mediated allergic response in mast cells. Oral administration of CAPE inhibited IgE-mediated passive cutaneous anaphylaxis. CAPE effectively reduced both histamine and serotonin (5-HT)-induced vascular permeability in rats. CAPE also reduced histamine and leukotrienes (LTs) release from isolated rat peritoneal mast cells. Moreover, CAPE suppressed contraction induced by histamine (3 × 10(-8)-3 × 10(-5) M), 5-HT (3 × 10(-9)-10(-6) M) and adenosine (3 × 10(-8)-10(-5) M) in guinea pig tracheal zigzag. These findings provide evidence that CAPE may serve as an effective therapeutic agent for allergic diseases.

Mast cell activation by fibrinogen-related homologous c-terminal peptides (haptides) modulates systemic blood pressure

Haptides are a family of short peptides homologous to C-termini sequences of fibrinogen chains β and γ (haptides Cβ and preCγ, respectively) which were previously shown to penetrate and bind cells. This work investigates the systemic effect of the haptides with possible clinical implications. Intra-arterial monitoring in rats recorded the haptides' effects on systemic blood pressure. In parallel, their effect was also tested in vitro on isolated rat peritoneal mast cells and on human mast cells. Intra-arterial monitoring in rats showed that intravenous administration of low haptides concentrations (35-560 μg/kg rat) caused a shocklike behavior with transient decrease in the systolic and diastolic blood pressure by up to 55% (P< .05) in a dose-dependent manner and a minor increase in their heart rate. Randomly scrambled sequences of the haptides had no such effect, suggesting a specific interaction with receptors. Intravenous administration of blockers to histamine receptors H1 and H2 before haptides administration attenuated this effect. Furthermore, in vitro incubation of human LAD2 mast cell line or isolated rat peritoneal mast cells with the haptides caused degranulation of the mast cells. We found that the haptides Cβ and preCγ activated mast cells causing histamine release, resulting in a steep decrease in blood pressure, comparable to anaphylactic shock. In treating vascular occlusive diseases, massive fibrinolysis is induced, and haptide-containing sequences are released. We suggest that treatment with histamine receptor blockers or with mast cell stabilizing agents in such pathological conditions may overcome this effect.

All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.

Contribution of mast cells and snake venom metalloproteinases to the hyperalgesia induced by Bothrops jararaca venom in rats

Bothrops jararaca venom (Bjv) is known to induce local inflammation and severe pain. Since, mast cells are able to secrete mediators involved in algesic processes, in this study we examined the putative role of these cells in the hyperalgesia triggered by Bjv in the rat paw. We noted that treatment with mast cell stabilizer sodium cromoglicate as well as with histamine and 5-hydroxytriptamine receptor antagonists meclizine and methysergide, respectively, inhibited the Bjv-induced hyperalgesia. In addition, we showed that stimulation of isolated rat peritoneal mast cells with Bjv in vitro resulted in the release of stored and neo-generated inflammatory mediators such as histamine and leukotriene C 4, respectively. Bjv-induced histamine secretion was clearly sensitive to treatment with sodium cromoglicate and sodium nedocromil. We further observed that metalloproteinase inhibitors 1,10-phenantroline and DM43 inhibited mast cell degranulation in vitro, under conditions where inhibitors of phospholipase A 2 as well as of serine- and cysteine-proteinases were inactive. Altogether, our findings indicate that mast cells seem to contribute to the hyperalgesia caused by Bjv in the rat paw, and also provide evidence that this response might be dependent on the ability of the Bjv to activate directly mast cells.

Role of Substance P on Histamine H3 Antagonist-Induced Scratching Behavior in Mice

The purpose of the present study was to investigate the involvement of chemical mediators, other than histamine, in the scratching behavior induced by H(3) antagonists. Scratching behavior was induced by the histamine H(3) antagonists iodophenpropit and clobenpropit (10 nmol/site) when they were injected intradermally into the rostral part of the back of mast-cell-deficient (WBB6F1 W/W(v)) and wild-type (WBB6F1 +/+) mice. Subsequently, the effect of spantide, a tachykinin NK(1) antagonist, was measured for 60 min. The effects of the H(3) antagonists on in vitro histamine release from rat peritoneal mast cells were also investigated. When spantide was injected intradermally at a dose of 0.5 nmol/site, it significantly inhibited the response. Furthermore, iodophenpropit and clobenpropit (10(-6)-10(-8) M) did not induce histamine release in isolated rat peritoneal mast cells. Our results indicate that substance P is involved in the skin responses elicited by the histamine H(3) antagonists. Moreover, the fact that these histamine H(3) antagonists did not induce significant increases in the histamine release from rat peritoneal mast cells suggests that the histamine H(3) receptor may not be present in the peripheral cells considered in this study.

Oedematogenic activity induced by Kunitz-type inhibitors from Dimorphandra mollis seeds

Proteinase inhibitors from plants represent a form of storage protein or may be involved in plant defense mechanisms against pests and diseases. In this study, we have investigated the oedematogenic activity of DMTI (20 kDa) and DMTI-II (23 kDa), two serine proteinases inhibitors isolated from Dimorphandra mollis (Leguminosae-Mimosoideae) seeds, belonging to the Kunitz family. Paw oedema was induced in male Wistar rats, and measured before and selected times after injection of the proteinase inhibitors. Injection of DMTI-II (3–100 μg/paw) induced a dose-dependent rat paw oedema of rapid onset and short duration, whereas DMTI (3–100 μg/paw) caused a discrete response. The histamine/5-HT receptor antagonist cyproheptadine (2 mg/kg) markedly reduced the DMTI-II-induced oedema. The bradykinin B 2 receptor antagonist JE 049 (0.6 mg/kg), the tachykinin NK 1 receptor antagonist SR140333 (100 μg/kg) or the NK 2 receptor antagonist SR48968 (1 mg/kg) all significantly reduced the DMTI-II-induced oedema. Depletion of sensory neuropeptides by capsaicin also resulted in a significant reduction of oedema formation. In rat isolated peritoneal mast cells, DMTI-II failed to directly release histamine. In conclusion, the proteinase inhibitor DMTI-II induces rat paw oedema by triggering the formation of different inflammatory mediators and pathways, where mast cells and sensory fibers seem to play a pivotal role.

Signaling pathway in nerve growth factor induced histamine release from rat mast cells

We investigated a signal transduction pathway involved in NGF induced histamine secretion from mast cells. We compared this mechanism with the exocytosis induced by basic secretagogue compound 48/80. Isolated rat peritoneal mast cells were obtained from male Wistar rats. Histamine release was assayed spectrofluorometrically. We found that tyrosine kinase inhibitor genistein, phospholipase C (PLC) inhibitor U-73122, phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, but not MAP kinase inhibitors, SB203580 and PD98059, reduce histamine secretion in NGF provoked mast cell degranulation. In compound 48/80 mediated degranulation, we confirmed only the involvement of tyrosine kinase and PLC, but not PI-3 kinase, PKC and MAP kinases. Our results indicate that release of histamine from mast cells after stimulation with NGF is regulated by tyrosine kinase, PLC, PI-3 kinase and PKC, but not by MAP kinases. This biochemical pathway differs from that provoked by compound 48/80.

Substance P induces TNF-α and IL-6 production through NFκB in peritoneal mast cells

The neuropeptide Substance P (SP) is an important mediator of neuroimmunomodulatory activity. The aim of this study is to elucidate the mechanism used by SP to promote increased production of pro-inflammatory cytokines in fresh isolated rat peritoneal mast cells (rPMC). We have demonstrated that SP induces production of interleukin-6 (IL-6) in rPMC through the PI-3K, p42/44 and p38 MAP kinase pathways. SP-stimulated rPMC also exhibited an enhanced nuclear translocation of the nuclear factor κ B (NFκB). The tumour necrosis factor-α (TNF-α) and IL-6 production was completely inhibited by using ( E)-4-hydroxynonenal (HNE) as an inhibitor of IκB-α and -β phosphorylation. Further, TNF-α and IL-6 expression was significantly inhibited by the oligonucleotides (ODNs) containing the NFκB element (NFκB decoy ODNs) but not by the scrambled control ODNs. These findings indicate that the NFκB pathway is involved in the transcriptional regulation of the TNF-α and IL-6 overexpression in primary SP-stimulated mast cells.

Evidence against the functional involvement of outwardly rectifying Cl − channels in agonist-induced mast cell exocytosis

In isolated rat peritoneal mast cells, an outwardly rectifying Cl − channel has been described. Influx of Cl − through this Cl − channel (I Cl −(OR) ) causes hyperpolarization, which facilitates Ca 2+ currents through store-operated Ca 2+ channels. The exocytotic effect of nerve growth factor (NGF) in the presence of lyso-phosphatidylserine strictly depends on the presence of extracellular [Ca 2+] o. The aim of the present study was to assess the importance of I Cl −(OR) for exocytosis induced by NGF/lyso-phosphatidylserine. Therefore, we investigated the effects on NGF/lyso-phosphatidylserine-induced exocytosis of [ 3H]5-hydroxytryptamine ([ 3H]5-HT) in rat peritoneal mast cells: (a) of two inhibitors of I Cl −(OR) (4,4′-diisothiocyanatostilbene2,2′-disulfonic acid [DIDS] and diethylstilbestrol), and (b) of replacement of extracellular Cl − by methylsulfate. Additionally, whole-cell patch-clamp experiments (nystatin-perforated patch) were performed. Diethylstibestrol and DIDS, in concentrations sufficient to abolish the I Cl −(OR) (10 μM) and the replacement of (Cl −) o by methylsulfate, were ineffective in impairing the NGF/lyso-phosphatidylserine-induced [ 3H]5-HT-release. These findings argue against a role of outwardly rectifying Cl − channels in exocytosis induced by NGF/lyso-phosphatidylserine in rat peritoneal mast cells.

Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.

Influences of everninomicin, vancomycin and teicoplanin on chemical mediator release from rat peritoneal mast cells.

We examined influences of certain antibiotics on the release of chemical mediators from isolated rat peritoneal mast cells in vitro. Isolated peritoneal mast cells were obtained from male Wistar strain rats. Everninomicin (0.06-1.2 mg/ml), vancomycin (0.05-1.0 mg/ml), teicoplanin (0.07-1.4 mg/ml) and concanavalin A (0.01 mg/ml) were used. Isolated mast cells were incubated in the presence of various concentrations of the test compound at 37 degrees C for 10 min. Histamine contents of the supernatant and cell pellet were measured by an automated fluorometric method. Prostaglandin D2 (PGD2) contents of the supernatant were determined by enzyme immunoassay. Everninomicin (0.06-1.2 mg/ml) had no influence on histamine and PGD2 release from mast cells. On the other hand, vancomycin significantly released both histamine (0.5 and 1.0 mg/ml) and PGD2 (1.0 mg/ml) from mast cells, but vancomycin did not affect concanavalin A-induced histamine and PGD2 release. Teicoplanin (more than 0.07 mg/ml) significantly stimulated histamine and PGD2 release from mast cells and it also significantly potentiated concanavalin A-induced histamine and PGD2 release. These results suggest that everninomicin causes no chemical mediator release from mast cells, different from vancomycin and teicoplanin.

Borrelia burgdorferi spirochetes induce mast cell activation and cytokine release.

The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-alpha release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-alpha-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells.

Intestinal Mucosa-Associated Bacteria Modulate Rat Mast Cell Reactivity

It is well known that in some conditions bacteria of physiological flora of gastrointestinal tract may become pathogenic. Each complaint which causes injury of gastrointestinal wall integrity permits bacteria to penetrate the tissues and affect the tissue cells. Since mast cells represent one of very important and numerous cellular elements of the gastrointestinal tract walls, bacteria can exert the effects on them. Therefore, the aim of our study was to examine the influence of four strains of intestinal mucosa-associated bacteria--Bacteroides thetaiotaomicron, Bacteroides fragilis, Bifidobacterium adolescentis and Escherichia coli on the mast cell reactivity. Our experiments were performed in vitro on isolated rat peritoneal mast cells and the reactivity of these cells was estimated on the basis of histamine release. We used the suspensions of whole bacteria, killed by heating at 65 degrees C. We have noticed that the magnitude of bacteria-induced histamine release from mast cells was very low (up to 6.0%) when compared with histamine release induced with Con A, compound 48/80 and TNF-alpha. However, all studied bacteria changed the reactivity of mast cells in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. After 40 min preincubation with B. thetaioataomicron, B. fragilis, B. adolescentis or E. coli ConA-induced histamine release was diminished up to 25%, 71%, 58% and and 68% of maximal histamine release, respectively. Preincubation of rat mast cells with B. thetaioataomicron, B. fragilis, B. adolescentis or E. coli also changed their reactivity in anaphylactoid reaction with compound 48/80 (histamine release was diminished up to 70%, 63%, 63% and 60%, respectively).