Antigen-stimulated Cells Research Articles

The significance of chloride in the inhibitory action of disodium cromoglycate on immunologically-stimulated rat peritoneal mast cells

BackgroundThe microelectrode array (MEA) was used to investigate the pharmacological relevance of chloride (Cl−) ions in antigen-dependent mast cell activation and the inhibitory effect of disodium cromoglycate (DSCG) on mast cell activation. MethodsThe movements of ions across the cellular membrane and the potential relationship between Cl− channels and DSCG during immunological activation were investigated using the MEA. The results were then subsequently compared with the amount of histamine released from anti-IgE activated peritoneal mast cells. ResultsThe inclusion of charybdotoxin (ChTX) in Cl−-free buffer showed that the measured field potentials during antigen-stimulated peritoneal mast cell were a combination of Cl− influx and K+ efflux. The delayed onset time of Cl− influx indicated the presence of a delayed outwardly-rectifying Cl− current in the antigen-stimulated peritoneal mast cells. The use of 5-nitro-2-(3-phenylpropylamino) benzoic acid demonstrated that the activated mast cell membrane potential can be stabilised, thereby reducing the amount of histamine released from the anti-IgE activated mast cells. The correlation between the results of the histamine release assay and the electrophysiological measurements demonstrated the importance of Cl− to anti-IgE dependent mast cell activation. The inhibitory effect of DSCG on anti-IgE activated cells, however, did not correlate with the presumed influx of Cl−. ConclusionsThe MEA data suggest that Cl− influx is crucial to IgE-dependent mast cell degranulation. General significanceWhile the MEA cannot yield information about single channel properties, it is convenient to use and can provide information on the global changes in electrophysiological responses of non-excitable cells.

Epidemic of Hepatitis B with high mortality in India: association of fulminant disease with lack of CCL4 and natural killer T cells

An explosive outbreak of Hepatitis B with high mortality was reported in 2009, in Modasa, Gujarat, India. Mortality was associated with basal core promoter and precore mutant hepatitis B virus (HBV). The current study addresses the role of immunological parameters in the progression to fulminant hepatitis. The study population comprised of 22 acute HBV patients, 13 fulminant HBV liver failure patients and 54 healthy controls. Hepatitis B surface antigen-induced CTL responses by enzyme-linked immunosorbent spot (ELISPOT), cytokine and chemokine quantitation by Bioplex assay, peripheral NK, natural killer T (NKT), CD4 and CD8 T-cell frequencies by flow cytometry were carried out. The median percentage of NK cells in the lymphocytes of the acute and fulminant liver failure patients were significantly lower compared to controls. Acute and fulminant liver failure patients had significantly high and comparable NKT cells compared to controls, respectively. Importantly, NKT cells were significantly lower in fulminant HBV liver failure than acute HBV patients. Circulating peripheral CD4/CD8 T-cell subsets among the patient categories and controls were comparable. In acute HBV patients, a significant increase in IFN-γ release was recorded (ELISPOT) by the unstimulated, antigen-stimulated and mitogen-stimulated cells when compared to controls. Comparisons of cytokines and chemokines among the disease categories revealed significantly lower levels of CCL4 in fulminant liver failure patients. NKT cells and CCL4 might be playing a pivotal role in limiting HBV infection among the patients investigated.

Induction of B Cell Responses upon Experimental Infection of Neonatal Calves with Mycobacterium avium subsp. paratuberculosis

The objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis would invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5(dim) and CD5(bright) markers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25(+) and CD45RO(+) B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. avium subsp. paratuberculosis demonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. avium subsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. avium subsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation.

Insight into the mechanism of STAT5 tyrosine phosphorylation in IgE sensitized, antigen-stimulated mast cells (151.13)

Abstract Previously we established that STAT5 expression is required for IgE-mediated mast cell function. STAT5 tyrosine phosphorylation is swiftly and transiently enhanced by FcϵRI aggregation in an IL-3- and MAPK-independent manner. However, a more detailed understanding of the signaling apparatus responsible in this situation is wanting. Recently, we have observed that STAT5 co-localizes with FcϵRI during mast cell activation and that lipid raft dissipation interrupts STAT5 activation. Downstream of FcϵRI we find that this mode of STAT5 activity is also independent of BTK, PI3K, Akt, Syk, and Lyn, whereas removal of Fyn kinase ablates the effect. Immunoprecipitation demonstrates that STAT5 is associated with Fyn, and this co-IP decreases concomitant with the induction of IgE-mediated STAT5 tyrosine phosphorylation. Examination of other signaling-related factors reveals that SHP-1 phosphatase deficiency enhances STAT5 tyrosine phosphorylation, even prior to stimulation. Loss of GAB2, an adaptor protein that associates with Lyn and SHP-1, also augments STAT5 activation. Targeting of STAT5A or B with knock-outs and specific siRNA pools shows that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether these data implicate Fyn as the major positive mediator of STAT5 after FcϵRI engagement, and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.

2-linoleoylglycerol inhibits allergic response and expression of inflammatory cytokines in mast cells (55.19)

Abstract Mast cell-mediated allergic disorder is associated with many diseases such as allergic asthma, atopic dermatitis and allergic rhinitis. In this study, we investigated the effect of the 2-linoleoylglycerol isolated from Adenophora triphylla on the Immunoglobulin E (IgE)-mediated allergic response and expression of inflammatory cytokines in mast cells. We observed that 2-linoleoylglycerol significantly inhibited β-hexosaminidase release in a dose-dependent manner in antigen-stimulated RBL-2H3 mast cells. 2-linoleoylglycerol also suppressed the expression of TNF-α, IL-4, IL-10 and COX-2 mRNA in mast cells. In addition, we showed that 2-linoleoylglycerol attenuated the activating phosphorylation of AKT, p38MAPK, JNK and ERK1/2. Our findings suggest that 2-linoleoylglycerol inhibits mast cell-derived allergic response and expression of inflammatory cytokines and that this effect is related by inhibition of AKT, p38MAPK, JNK and ERK1/2 phosphorylations.

Inhibitory effect of Pleurotus eryngii extracts on the activities of allergic mediators in antigen-stimulated mast cells

Pleurotus eryngii is an edible mushroom native to Europe, the Middle East, and North Africa, and is also grown in parts of Asia. The present study investigated the anti-allergy potential of P. eryngii extract (PEE) in antigen-stimulated RBL-2H3 mast cells. PEE inhibited allergy markers, including release of hexosaminidase and histamine, in antigen-sensitized RBL-2H3 cells. PEE also suppressed the expression and production of interleukin-4 and reduced antigen-induced NFAT and NF-κB transcriptional activity in antigen-sensitized mast cells. Moreover, PEE decreased the levels of proinflammatory cytokines and COX-2 and iNOS expression in antigen-sensitized mast cells. Finally, PEE suppressed antigen-induced signal protein phosphorylation of Lyn, PLCγ2, PKC, Akt, and MAP kinases. Taken together, these results suggest that P. eryngii extract may provide insight into the prevention and treatment of allergic and inflammatory diseases.

Integrin α 5 interacts with EGFR, is necessary for FcɛRI signaling and is necessary for allergic inflammation in relation with angiogenesis

Recent reports have suggested role for epidermal growth factor receptor (EGFR) in asthma and skin inflammation. Integrin(s) are known to be necessary for the transactivation of EGFR. The roles of EGFR and integrin(s) in allergic inflammation were investigated. Antigen stimulation induced activation of EGFR and interaction between EGFR and integrin α 5 in Rat Basophilic Leukemia (RBL2H3) cells and bone marrow-derived mouse mast cells (BMMCs). Flow cytometry revealed increased phosphorylation of EGFR on cell surfaces. Antigen stimulation induced interaction between EGFR and FcɛRI in both RBL2H3 cells and BMMCs. Blocking of EGFR or integrin α exerted negative effects on rac1 activity and secretion of β-hexosaminidase in both RBL2H3 cells and BMMCs. EGFR and integrin α 5 were found to be necessary for IgE-dependent cutaneous anaphylaxis. FAK (focal adhesion kinase), interacted with EGFR and with FcɛRI upon antigen stimulation, and it was necessary for the increased secretion of β-hexosaminidase in both RBL2H3 cells and BMMCs. EGFR and integrin α 5 were necessary for interactions between activated RBL2H3 cells, BMMCs and rat aortic endothelial cells (RAECs). Conditioned medium of antigen-stimulated RBL2H3 cells promoted RAECs tube formation, rat aortic ring formation and blood vessel formation. Conditioned medium of antigen-stimulated BMMCs also had the same effects on RAECs. This enhanced angiogenic potential of RAECs was dependent on EGFR and integrin α 5. In conclusion, EGFR, via interaction with FcɛRI and integrin α 5, is necessary for allergic inflammation associated with cellular interaction.

Anti-allergic activity of sesquiterpenes from the rhizomes of Cyperus rotundus

From the 70% ethanol extract of the rhizomes of Cyperus rotundus (CRE), several major constituents including the sesquiterpene derivatives (valencene, nootkatone, and caryophyllene α-oxide), monoterpenes (β-pinene, 1,8-cineole, and limonene) and 4-cymene were isolated and examined for their anti-allergic activity in vitro and in vivo. In rat basophilic leukemia (RBL)-1 cells, the sesquiterpenes strongly inhibited 5-lipoxygenase-catalyzed leukotrienes production. In addition, they inhibited β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, with valencene having the highest inhibitory effect. CRE inhibited leukotrienes production and β-hexosaminidase release at 300 μg/mL. It was also found that the most active sesquiterpene (valencene) and CRE inhibited β-hexosaminidase degranulation by inhibiting the initial activation reaction, Lyn phosphorylation, in IgE-stimulated RBL-2H3 cells. Moreover, CRE, valencene and nootkatone significantly inhibited the delayed-type hypersensitivity reaction in mice when administered orally at 50-300 mg/kg. In conclusion, C. rotundus and its constituents, valencene, nootkatone, and caryophyllene α-oxide, exert anti-allergic activity in vitro and in vivo. These sesquiterpenes, but not monoterpenes, certainly contribute to the anti-allergic activity of the rhizomes of C. rotundus.

Coxiella burnetii Antigen-Stimulated Dendritic Cells Mediated Protection against Coxiella burnetii in BALB/c Mice

Coxiella burnetii is the etiological agent of human Q fever. In this study, adaptive transfer of mouse bone marrow-derived dendritic cells (BMDCs) stimulated with C. burnetii antigen, phase I whole-cell antigen (PIAg), lipopolysaccharide (LPS)-removed PIAg (PIIAg), protein antigen Com1, or SecB significantly reduced coxiella burden in recipient mice compared with control mice. Mice that received PIIAg-pulsed BMDCs displayed substantially lower coxiella burden than recipient mice of PIAg-pulsed BMDCs after C burnetii challenge. The protection offered by the antigen-activated BMDCs was correlated with the increased proliferation of helper T (T(H)) T(H)1 CD4(+) cells, preferential development of T(H)17 cells, and impaired expansion of regulatory T lymphocytes. Our results suggest that PIIAg is far superior to PIAg in activating BMDCs to confer protection against C. burnetii in vivo, whereas Com1 and SecB are protective antigens because Com1- or SecB-pulsed BMDCs confer partial protection.

A Novel Imidazo[1,5-b]isoquinolinone Derivative, U63A05, Inhibits Syk Activation in Mast Cells to Suppress IgE-Mediated Anaphylaxis in Mice

Mast cells play a pivotal role in IgE-mediated allergic responses. Development of specific inhibitors against FcεRI-associated proximal signaling molecules in mast cells may represent a promising therapeutic strategy for allergic diseases. We examined whether a novel synthetic compound, 3-butyl-1-chloro-8-(2-methoxycarbonyl)phenyl-5H-imidazo[1,5-b]isoquinolin-10-one (U63A05), could suppress antigen-stimulated degranulation and cytokine secretion in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. U63A05 reversibly and dose-dependently inhibited degranulation of rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow–derived mast cells (BMMCs) stimulated by antigen (IC50 values for RBL-2H3 and BMMCs were 4.1 and 4.8 μM, respectively). The secretion of inflammatory cytokines was also suppressed in antigen-stimulated mast cells. However, degranulation by thapsigargin, a typical calcium inducer, was not inhibited by U63A05. U63A05 exerts its inhibitory effect, to the same extent as in degranulation, on the activating phosphorylation of Syk and downstream signaling molecules, including LAT and SLP-76. Further downstream, the activating phosphorylations of Akt, Erk1/2, p38, and JNK were also inhibited. Finally, antigen-stimulated PCA was dose-dependently suppressed in mice (ED50, 26.3 mg/kg). Taken together, the results suggest that U63A05 suppresses the activation of mast cells and the mast cell–mediated allergic response through the inhibition of Syk activation in mast cells.

Effect of a single and repeated dose of caffeine on antigen-stimulated human natural killer cell CD69 expression after high-intensity intermittent exercise

Several studies investigating the effect of caffeine on immune function following exercise have used one large bolus dose of caffeine. However, this does not model typical caffeine consumption. Therefore, the purpose of this study was to investigate whether small repeated doses of caffeine ingested throughout the day would elicit a similar response as one large bolus dose ingested 1 h prior to exercise on antigen-stimulated NK cell CD69 expression following strenuous intermittent exercise. In a randomized cross-over design, 15 healthy males completed six 15 min blocks of intermittent running consisting of maximal sprinting interspersed with less intense running and walking. Participants had ingested either 0 (PLA), 2 mg kg(-1) body mass (BM) caffeine on three separate occasions during the day (3 × CAF) or one dose of 6 (1 × CAF) mg kg(-1) BM caffeine, 1 h before exercise. At 1-h post-exercise, the number of antigen-stimulated CD3(-)CD56(+) cells expressing CD69 was lower on 1 × CAF compared with PLA [P < 0.05; PLA: 42.0 (34.0) × 10(6) cells L(-1), 1 × CAF: 26.2 (25.0) × 10(6) cells L(-1)], with values on 1 × CAF at this time point remaining close to pre-supplement. 1 × CAF tended to attenuate the exercise-induced increase in geometric mean fluorescence intensity of CD69 expression on antigen-stimulated CD3(-)CD56(+) cells 1-h post-exercise [P = 0.055; PLA: 141 (28)%, 1 × CAF: 119 (20)%]. These findings suggest that although one large bolus dose of caffeine attenuated the exercise-induced increase in antigen-stimulated NK cell CD69 expression 1 h following strenuous intermittent exercise, this attenuation at no point fell below pre-supplement values and caffeine does not appear to depress NK cell CD69 expression.

The Effect of a Phytosphingosine-like Substance Isolated from Asterina pectinifera on Involucrin Expression in Mite Antigen-Stimulated HaCaT Cells

The aim of the present study was to investigate the effect of phytosphingosine (PS) on mite antigen-induced terminal differentiation abnormalities in HaCaT cells. For this purpose, a PS-like substance was isolated from Asterina pectinifera (starfish PS) using high-performance liquid chromatography and was partially characterized through 1H NMR analysis. The level of involucrin expression in HaCaT cell was measured by immunoblotting assay. Our results showed that PS treatments remarkably up-regulated the involucrin expression, which is known as a terminal differentiation marker in the epidermal mite antigen-treated HaCaT cells. This indicates that starfish PS could regulate mite antigen-induced terminal differentiation fluctuation in the epidermis. Taken together, the results suggest that starfish PS might be a useful therapeutic agent for atopic dermatitis.

Effects of propolis from different areas on mast cell degranulation and identification of the effective components in propolis

Propolis is considered to down-regulate type I allergy, but the effective components of propolis remain unknown. In addition, propolis components vary depending on the area from which they are collected due to variations among wild plants in an area. Therefore, we compared the effects of water and ethanol extracts of propolis from Brazil and China on mast cell degranulation and cytokine production, thereby identifying effective components in propolis. The amount of released β-hexosaminidase via high-affinity IgE receptor I (FcεRI) from rat basophilic leukemia (RBL-2H3) cells was used as an index of degranulation. All propolis extracts inhibited degranulation from antigen-stimulated RBL-2H3 cells, but the effective doses differed according to collection areas. The ethanol extract of Chinese propolis, which was the strongest inhibitor of mast cell degranulation, was divided into compounds using normal- and reversed-phase liquid chromatography. The isolated anti-allergic components were identified as chrysin, kaempferol and its derivative, and chrysin was revealed to inhibit IL-4 and MCP-1 production from antigen-stimulated RBL-2H3 cells. HPLC quantification also revealed the Brazilian propolis extract to contain only small amounts of these flavonoids, which suggested that variation in propolis components could affect anti-allergic properties.

The mTOR Kinase Determines Effector versus Memory CD8+ T Cell Fate by Regulating the Expression of Transcription Factors T-bet and Eomesodermin

The mechanisms underpinning integration of instructions that program naive CD8+ T cells for effector and/or memory differentiation are not well understood. Herein, we demonstrate that interleukin-12 (IL-12) enhanced and sustained antigen and costimulatory molecule (B7.1)-induced mTOR kinase activity in naive CD8+ (OT-I) T cells via phosphoinositide 3-kinase and STAT4 transcription factor pathways. Blocking mTOR activity by rapamycin reversed IL-12-induced effector functions because of loss of persistent expression of the transcription factor T-bet. Rapamycin treatment of IL-12-conditioned OT-I cells promoted persistent Eomesodermin expression and produced memory cell precursors that demonstrated enhanced sustenance and antigen-recall responses upon adoptive transfer. The memory cell precursors showed greater tumor efficacy than IL-12-conditioned effector OT-I cells. These results identify mTOR as the central regulator of transcriptional programs that determine effector and/or memory cell fates in CD8+ T cells. Targeting mTOR activity offers new opportunities to regulate CD8+ T cell-mediated immunity.

SHP-1 exhibits a pro-apoptotic function in antigen-stimulated mast cells: Positive regulation of mitochondrial death pathways and negative regulation of survival signaling pathways

Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to act as a negative signal modulator in mast cells but its roles in cell survival and cell death are poorly understood. We previously reported that SHP-1 also positively regulates mast cell activation signaling by acting as an adaptor protein. In the present study, we examined whether SHP-1 plays a role in antigen (Ag)-induced activation-induced mast cell death. Bone marrow-derived mast cells (BMMCs) from SHP-1-deficient motheaten (me) mice (me-BMMCs) were significantly less susceptible to store-operated Ca2+ channel (SOC) activation, Ag-induced cell death and DNA fragmentation than BMMCs from their wild-type littermates (WT-BMMCs). Subsequent experiments revealed that the differences in these cellular susceptibilities to SOC activation and cell death resulted from the extent of the mitochondrial permeability transition pore (mPTP) opening. Specifically, mPTP opening was sufficiently persistent in WT-BMMCs to evoke mitochondrial integrity disruption, while mPTP opening was too transient to cause the minimal mitochondrial integrity collapse in me-BMMCs. In addition, pro-survival signaling including activation of mitogen-activated protein kinases (MAPKs) such as the extracellular signal-regulated protein kinases, c-Jun NH2 terminal kinases and p38 and the expression of Bcl-xL were significantly prolonged in me-BMMCs compared with WT-BMMCs. Taken together, these data demonstrate that a lack of SHP-1 prevents the mPTP-mediated mitochondrial integrity collapse and augments anti-apoptotic signaling such as MAPKs and Bcl-xL. These findings suggest that SHP-1 positively regulates mitochondrial death pathways and negatively regulates pro-survival signaling pathways.

Discrete generations of intracellular hydrogen peroxide and superoxide in antigen-stimulated mast cells: Reciprocal regulation of store-operated Ca 2+ channel activity

Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (FcɛRI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca 2+ channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca 2+ channel (SOC) activity is regulated by superoxide (O 2 •−) and hydrogen peroxide (H 2O 2) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O 2 •− and H 2O 2, respectively, in antigen-stimulated cells. The H 2O 2 generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca 2+, and the FcɛRI β-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O 2 •− generation was strictly dependent on extracellular Ca 2+, but negatively regulated by the SFK and PI3K activities. Inhibition of O 2 •− generation resulted in increased H 2O 2 generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca 2+ store depletion. On the contrary, inhibition of H 2O 2 generation resulted in increased intracellular O 2 •− generation and augmented SOC activity. The findings suggest that O 2 •− and H 2O 2, which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca 2+ signaling in mast cells.

Src-like adaptor protein (SLAP) is upregulated in antigen-stimulated mast cells and acts as a negative regulator

Our studies in the RBL-2H3 mast cell line suggest that responses to antigen (Ag) are negatively modulated through upregulation of Src-like adaptor protein (SLAP). Ag stimulation of RBL-2H3 cells leads to increased levels of SLAP (but not SLAP2) transcripts and protein over a period of several hours. The effects of pharmacologic inhibitors indicate that the upregulation of SLAP is dependent on multiple signaling pathways. Knockdown of SLAP with anti-SLAP siRNA is associated with enhanced phosphorylation of Syk, the linker for activation of T cells (LAT), phospholipase Cγ, MAP kinases, and various transcription factors. Production of IL-3 and MCP-1, but not degranulation, is also enhanced. The upregulation of SLAP may thus serve to limit the duration of cytokine production in Ag-stimulated cells.

Celastrol binds to ERK and inhibits FcεRI signaling to exert an anti-allergic effect

The role of celastrol, a triterpene extracted from the Chinese “Thunder of God Vine,” in allergic inflammation was investigated. Celastrol decreased the secretion of β-hexosaminidase, decreased the release of histamine, decreased the expression of Th2 cytokines and decreased calcium influx and cell adhesion in antigen-stimulated RBL2H3 cells. Exposure to celastrol decreased the phosphorylation of extracellular regulated kinase (ERK) and the ERK kinase activity was decreased in RBL2H3 cells. A molecular dynamics simulation showed binding of celastrol to a large pocket in ERK2, which serves as the ATP-binding site. Exposure to celastrol inhibited the interaction between immunoglobulin Fc epsilon receptor I (FcεRIγ) and ERK and inhibited interaction between FcεRIγ and protein kinase C δ (PKCδ). Antigen stimulation induced an interaction between Rac1 and ERK as well as an interaction between Rac1 and PKCδ. Inhibition of ERK decreased Rac1 activity and inhibition of Rac1 decreased ERK activity in antigen-stimulated RBL2H3 cells. Celastrol regulated the expression of epithelial–mesenchymal transition (EMT)-related proteins through inhibition of PKCα, PKCδ, and Rac1 in antigen-stimulated RBL2H3 cells. Exposure to celatrol inhibited PKCδ activity in antigen-stimulated RBL2H3 cells. Celastrol exerted a negative effect on FcεRIβ signaling by inhibiting the interaction between heat shock protein 90 (hsp90) and proteins, such as, FcεRIβ, Akt and PKCα. Celastrol exerted a negative effect on in vivo atopic dermatitis induced by 2, 4-dinitrofluorobenzene (DNFB), which requires ERK. Celastrol also showed an inhibitory effect on skin inflammation induced by phorbol myristate acetate (PMA) in Balb/c mice. In summary, celastrol binds to ERK and inhibits FcεRI signaling to exert an anti-inflammatory effect.

Neonatal Tolerization with Bacterial Antigens Stimulates an Expansion of CD11b+ Leukocytes with Inhibitory Activity in IL-10 Gene-Deficient Mice (39.38)

Abstract Tolerance to antigens of the endogenous flora can be induced by intra-peritoneal injection of 100 microliter of a sterile lysate solution (2mg/ml protein) prepared from fecal material of conventionally-raised mice with enteric micro flora into neonates. In IL-10 gene-deficient mice this treatment results in delayed onset of intestinal inflammation and reduced levels of pro-inflammatory cytokine release with a concomitant increase in CD11b+ leukocytes in spleen. To investigate whether these CD11b+ leukocytes play an active role in the reduction of bacteria-induced immune responses we isolated CD11b+ leukocytes from spleens of 20 week old mice that were tolerized to bacterial antigens as neonates and added these cells in limited dilutions to spleen cell cultures stimulated with bacterial antigens. We found that in bacterial antigen-tolerized, Il-10 gene-deficient mice the amount of CD11b+ leukocytes in spleen doubled compared to the amount found in untreated mice and mice injected with germ-free lysates. Sorted CD11b+ spleen cells from tolerized mice were able to inhibit antigen-stimulated spleen cell proliferation and release of pro-inflammatory cytokines in a concentration-dependent manner suggesting that CD11b+ cells play a regulatory role in the response to bacterial antigens in this IBD model. This work is supported by the Crohn's and Colitis Foundation of Canada.

T helper cytokine patterns: defined subsets, random expression, and external modulation

Although T cell effector subsets, defined by cytokine patterns, have been recognized for more than 20 years, the functional cytokine expression patterns in vivo are still in considerable doubt, particularly for human T cells. At least three new subsets have been recently identified, but the committed cytokine pattern of a T cell (e.g., Th1 cells produce IL-2, interferon-gamma, and lymphotoxin) may differ from the expression pattern of one cell on one occasion, which may be a subset of its full potential. Recent advances in flow cytometry allowed detailed cytokine patterns of antigen-stimulated cells to be identified directly ex vivo. These patterns are clearly more diverse than the major subsets identified as committed phenotypes. Additional contributions to diversity may include new committed subsets, random expression of only part of the committed pattern, and modification of the expression patterns by cytokines and other mediators.