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PubMed2000

Shift from Th2 to Th1 response in immunotherapy with venoms.

Cytokines released from T lymphocytes regulate antibody production by B lymphocytes and releasability of mast cells and basophils. Immune tolerance in specific allergen (Ag) immunotherapy (SIT) might be a consequence of decreased Th2 or increased Th1 response of Ag specific T lymphocytes. We compared function of T lymphocytes in 11 patients with Hymenoptera venom allergy and in 7 healthy volunteers. We followed function of T lymphocytes in 6 patients during SIT. We measured released IL-2, IFN-gamma, IL-4 and IL-5 from Ag and mitogens (either with phorbol-myristate-acetate (PMA) and ionomycin or anti-CD3 antibodies) stimulated peripheral blood mononuclear cells (PBMC). After stimulation with Ag PBMC of patients released 86 +/- 133 pg/ml IFN-gamma and 19.7 +/- 20.1 pg/ml IL-4 (Th2 response), PBMCs of healthy controls released 184 +/- 116 pg/ml IFN-gamma and 4.8 +/- 8 pg/ml IL-4 (Th1 response). Spontaneous release of IFN-gamma in cultures of PBMC of patients increased after rush SIT (before: 25 +/- 31 pg/ml, after 241 +/- 281 pg/ml, p<0.05). After 6 months of SIT response of PBMCs of patients became similar to response of PBMCs of healthy controls. Time pattern of cytokines secreted from PBMCs after stimulation with PMA and ionomycin was different than after stimulation through T-cell receptor/CD3 complex.

Bee Venoms↗
PubMed2001

Mechanisms of CD23 hyperexpression on B cells from patients with rheumatoid arthritis.

OBJECTIVE: To analyze the mechanisms involved in the characteristic hyperexpression of CD23 on peripheral blood B cells from patients with rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients with active disease and activated during 18 h with an anti-CD3 monoclonal antibody in the presence or absence of blocking antibodies to CD154 or CD40. PBMC were further purified by rosetting and CD23 expression was assessed on B cells by flow cytometry after double staining (CD19/CD23). Lymphocytes were also isolated from synovial fluid (SF). CD154 expression was analyzed on PB or SF CD4+ T cells after double staining (CD4/CD154) by flow cytometry at basal conditions and after different stimuli [anti-CD3 or phorbol myristic acetate (PMA) plus ionomycin]. Co-culture experiments between SF and PB cells were performed to analyze the involvement of the CD40-CD154 interaction on CD23 expression. CD154 and CD23 expression was also analyzed on synovial membrane by immunohistochemical techniques. RESULTS: A high proportion of activated CD23 B cells was detected in patients with RA. Blocking experiments with both anti-CD40 and anti-CD154 Mab showed a significant reduction in the proportion of PB B cells expressing CD23. Following activation with anti-CD3 Mab or PMA plus ionomycin, CD154 expression was mainly induced on PB CD4+ T cells. In co-culture experiments, SF T cells were more efficient than PB T cells in inducing CD40 dependent CD23 expression on PB B cells. In addition, CD4+ T cells from synovial membrane clearly expressed CD154. CONCLUSION: Our results establish a link between CD154-CD40 pathway and CD23 expression on PB B cells from patients with RA. T cells from the synovial microenvironment were active participants in this CD23 expression, presumably in the context of cell recirculation.

Antibodies, Monoclonal↗
PubMed1999

[Ciliary neurotrophic factor prevents NO mediated cytotoxicity in cultured hippocampal neurons].

The effects of ciliary neurotrophic factor on nitric oxide (NO)-induced cytotoxicity were examined in primary cultures of rat hippocampal neurons. The neurotoxicity was induced by NO donors, such as sodium nitroprusside (SNP), s-nitroso-N-acetylpenicillamine (SNAP), NOS substrate, L-arginine (L-Arg) and ionomycin (a calcium ionophore). Preincubation of the cultures with ciliary neurotrophic factor (CNTF) for 24 h promoted neuronal survival and decreased the LDH efflux and the level of NO induced by ionomycin. The protective effects of CNTF against NO cytotoxicity were concentration-dependent. The above findings suggest that CNTF promotes the survival of neurons in the hippocampus by preventing NO mediated neurotoxicity.

Animals↗
PubMed2001

Effects of propofol on mitochondrial function and intracellular calcium shift in bovine aortic endothelial model.

BACKGROUND: Hypotension was commonly encountered in clinical practice during induction of anesthesia with propofol. The purpose of this study is to investigate the effect of propofol on mitochondrial membrane potential and morphology so as to infer its relation with intracellular calcium mobilization in bovine aortic endothelium. METHODS: In this study, we used the cultured bovine aortic endothelial cells (Gm 7372a) to elucidate the impact of propofol upon the membrane potential and morphology of mitochondria in correlation with its effect on intracellular calcium shift. The intracellular calcium mobilization within the cells preincubated with or without propofol was evaluated using a fluorescent spectrophotometer (confocal microscope) after being treated with Fluo-3. The mobilization of intracellular calcium was demonstrated by the appearance of "hot spots" released from intracellular stores after the addition of an ionophore, ionomycin, to the incubation system. The membrane potential of mitochondria was measured by DiOC6 and the morphology of the mitochondria was evaluated by the treatment of TM Ros and compared with that by the treatment of the uncoupler, FCCP, as control. RESULTS: The release of calcium "hot spots" from the intracellular stores (e.g. mitochondria) after the addition of ionomycin was visualized to decrease dramatically within the endothelial cells after preincubation with propofol. The membrane potential of mitochondria was significantly inhibited by pretreatment of propofol at 0.01 mM, 37 degrees C for 30 min. Morphologically, the integrity of mitochondria was distorted and fragmented in the presence of propofol as compared with that of control. CONCLUSIONS: Our data showed that propofol in clinical concentration, 0.01 mM, could inhibit intracellular calcium shift from the intracellular stores and decrease the membrane potential and distort the morphology of mitochondria in bovine aortic endothelial cells. These inhibitions of the function and disfiguration of the morphology of mitochondria signify that the clinical hypotension induced by propofol might be of a potential mechanism.

Anesthetics, Intravenous↗
PubMed1992

Luminal Ca2+ increases the sensitivity of Ca2+ stores to inositol 1,4,5-trisphosphate.

Ca2+ within intracellular stores has been proposed to act with cytosolic inositol 1,4,5-trisphosphate (InsP3) to cause opening of the integral Ca2+ channel of the InsP3 receptor, leading to mobilization of intracellular Ca2+ stores [FEBS Lett. 263:5-9 (1990)]. We have tested that suggestion in saponin-permeabilized rat hepatocytes by manipulating the Ca2+ content of the stores and then determining their sensitivity to InsP3, while keeping the cytosolic Ca2+ concentration constant. Stores depleted of Ca2+ by incubation with ionomycin were significantly less sensitive to InsP3, an effect thought likely to result from the decrease in luminal free Ca2+ concentration rather than from direct effects of ionomycin on InsP3 binding or Ca2+ permeability. The luminal free Ca2+ concentration of stores loaded in the presence of pyrophosphate appeared to be substantially reduced, and again there was a significant inverse correlation between the estimated free Ca2+ concentration of the stores and their sensitivity to InsP3. By following the kinetics of 45Ca2+ uptake into empty stores in the presence of inositol trisphosphorothioate, a stable InsP3 analogue, we demonstrated that stores respond to inositol trisphosphorothioate only after their luminal free Ca2+ concentration exceeds a critical level. We conclude that InsP3 and luminal Ca2+ together regulate Ca2+ mobilization from intracellular stores, and we discuss some of the implications of this interaction for the complex Ca2+ signals evoked by extracellular stimuli.

Animals↗
PubMed1992

Signal transduction in glycosaminoglycan (GAG) synthesis by cultured chondrocytes and its inhibition by inflammatory cell-derived hydrogen peroxide.

Signal transduction in glycosaminoglycan (GAG) synthesis by chondrocytes has been studied. The activity of various subspecies of protein kinase C (PKC) in chondrocytes derived from rodent costal cartilage and bovine articular cartilage has been determined and the role of PKC in GAG synthesis as well as the possible interactions of PKC with calcium- or cyclic AMP (cAMP)-dependent systems in the synthesis of GAG. To investigate GAG synthesis in inflammatory conditions, the effects of hydrogen peroxide on PKC activity of the chondrocytes and PKC-mediated GAG synthesis have been studied. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, increased GAG synthesis in a dose-dependent fashion. This suggests that PKC up-regulates the synthesis of GAG in cultured chondrocytes. This increase was not significantly affected by simultaneous addition of the calcium ionophore, ionomycin, or dibutyryl cAMP (db-cAMP), a cAMP analogue. Ionomycin and db-cAMP, when used alone, did not significantly alter GAG synthesis by chondrocytes. Thus there appears to be no interaction between PKC and calcium- or cAMP-mediated systems in GAG synthesis. The increase in GAG synthesis induced by TPA was significantly (P less than 0.01) reduced by simultaneous addition of hydrogen peroxide (10(-6) M), without affecting cell viability. The activity of PKC in chondrocytes pretreated with 10(-6) M hydrogen peroxide was also significantly inhibited. Thus hydrogen peroxide which is generated by inflammatory cells may be important in suppression of GAG synthesis in inflammatory conditions.

Animals↗
PubMed1992

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.

Animals↗
PubMed1992

Inhibition of interleukin-5 gene expression by dexamethasone.

The effect of glucocorticoids on interleukin-5 (IL-5) gene expression was assessed in human peripheral blood mononuclear cells. IL-5 expression was stimulated by phytohaemagglutinin (PHA), IL-2, phorbol myristate acetate (PMA) or Ionomycin. A semi-quantitative assay for IL-5 gene expression was developed, based on RNA extraction and the polymerase chain reaction. IL-5 expression in response to PHA was profoundly inhibited by 10(-6) M dexamethasone, and significant inhibition was detected at doses of dexamethasone as low as 10(-9) M. When dexamethasone was added to the cells at the same time as PHA, the inhibitory effect could be detected as early as 3 hr. Dexamethasone at 10(-6) M also profoundly inhibited the IL-5 response to PMA and to IL-2, but the IL-5 response to Ionomycin was not significantly affected. These results suggest that dexamethasone may be capable of interfering with a pathway involving protein kinase C. There is increasing evidence that IL-5 may play a pathogenic role in asthma and other manifestations of acute hypersensitivity. The present findings indicate that inhibition of IL-5 expression may be one of the mechanisms whereby glucocorticoids exert their beneficial effects in diseases such as asthma.

Base Sequence↗
PubMed2002

[Sheep transgenic embryos produced by intracytoplasmic sperm injection].

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.

Animals↗
PubMed1992

[Studies of pepsinogen release in guinea pig gastric chief cells based on changes of cytosolic free Ca2+ concentration].

In order to evaluate the role of secretagogue-induced changes of cytosolic free calcium concentration ([Ca2+]i) in pepsinogen release, the effects of cholecystokinin-octapeptide (CCK-8) on pepsinogen release and on [Ca2+]i in gastric chief cells isolated from guinea pig stomach were investigated. [Ca2+]i was measured with fura-2, Ca(2+)-sensitive fluorescent probe, using dual-wavelength excitation fluorospectrophotometer. In the presence of extracellular Ca2+, CCK-8 induced a rapid and transient increase in [Ca2+]i followed by a sustained plateau of [Ca2+]i increase which remained 20-50 nM above basal level until antagonist was added. Both in the absence of external Ca2+ and in the presence of inorganic Ca2+ channel blocker Ni2+(2 nM), plateau phase but not initial transient peak of [Ca2+]i was diminished. On the other hand, nicardipine, an organic Ca2+ channel blocker, did not affect the [Ca2+]i transient induced by CCK-8. Time course of CCK-8-stimulated pepsinogen release was biphasic; the initial phase of pepsinogen release was unaffected by Ca2+ removal from the medium but was inhibited by Ca2+ chelation with an intracellular Ca2+ chelator BAPTA/AM, while the late phase of pepsinogen release was dependent on extracellular Ca2+. Artificially introduced Ca2+ into the chief cells with ionomycin elicited the increase in [Ca2+]i and pepsinogen release. However, ionomycin-induced release of pepsinogen was transient and several times smaller than that induced by CCK-8. Ni(2+)-sensitive Ca2+ influx across the plasma membrane was also necessary for reloading of agonist-sensitive Ca2+ pool. These results suggest that the initial, transient increase in [Ca2+]i from the intracellular Ca2+ store mediates the initial pepsinogen release, while the sustained increase in [Ca2+]i which is dependent on Ca2+ influx is responsible for the late phase of pepsinogen release. Factors other than the changes in [Ca2+]i may also be required in producing optimal pepsinogen release.

Animals↗
PubMed2005

Role of Ca2+ in the effects of shear stress and TNF-alpha on caveolin-1 expression.

The aim of this work was to study the influences of Ca2+ (medium free calcium, with BAPTA, with 100 mM Ca2+, 100 mM Ca2++10 microM ionomycin) on the expression of caveolin-1 (structural protein of caveolae) of endothelial cells (ECs) submitted to mechanical forces (shear stress) or biochemical stimulation (TNF-alpha). We found that shear stress enhanced the caveolin-1 expression. Simultaneously, the caveolin-1 expression is dependant on [Ca2+]i: [Ca2+]free medium+100 microM BAPTA<[Ca2+]free medium<[Ca2+]100 mM<[Ca2+]100 mM+10 microM ionomycin. In contrast, TNF-alpha induced a decrease of caveolin-1 in the cells, whatever the [Ca2+]i. These results suggest that there could be a synergistic effect between shear stress and Ca2+ on caveolin-1 expression but an inflammatory stimulation (TNF-alpha) induces a down regulation of caveolin-1 expression.

Calcium↗
PubMed2005

Inhibitory effects of berberine on the activation and cell cycle progression of human peripheral lymphocytes.

The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin (PHA) alone or phorbol dibutyrate (PDB) plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry. The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide (PI) and 7-aminoactinomycin D (7-AAD), respectively. The results showed that 100 micromol/L and 50 micromol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 micromol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased. Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 micromol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes, suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.

Adolescent↗
PubMed1991

Disassociation of the macrophage-maturational effects of vitamin D from respiratory burst priming.

During the process of enhancing monocytic differentiation of the human leukemia line HL-60, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) also "primes" the cell for respiratory burst by increasing the uptake of Ca2+ across the plasma membrane (Hruska, K.A., Bar-Shavit, Z., Malone, J.D., and Teitelbaum, S.L. (1988) J. Biol. Chem. 263, 16039-16044). The present study asked if the maturational effect of vitamin D is dependent upon this "priming" phenomenon. To this end, we exposed HL-60 to either 1,25(OH)2D3 or its synthetic analogue (1 alpha, 3 beta, 5Z, 7E)-9-10-Secocholesta-5,7,10(19)-triene-1, 3, 25-triol (22-oxa). We found that 22-oxa induced HL-60 maturation as effectively as does the natural steroid. As expected, 48 h of 1,25(OH)2D3 exposure more than doubles (p less than 0.005) HL-60 basal cytosolic Ca2+ and increases inositol triphosphate-sensitive Ca2+ stores approximately 4-fold (p less than 0.01). 22-oxa in contrast alters neither Ca(2+)- nor inositol triphosphate-mobilizable deposits. Moreover, 1,25(OH)2D3 treatment prompts a transient Ca2+ "spike" in response to formyl-methionyl-leucyl-phenylalanine (fMLP) and a marked increase in superoxide (O-2) generation when exposed to the chemotactic peptide (p less than 0.01) or phorbol ester (p less than 0.02). Treatment with 22-oxa does not enable HL-60 to respond to fMLP with a Ca2+ spike or prime the cell for respiratory burst unless it is co-incubated with the Ca2+ ionophore, ionomycin. Similarly, phorbol ester impacts more profoundly on O-2 generation by 1,25(OH)2D3 than 22-oxa preincubated cells (p less than 0.02), unless the latter is added with ionomycin. Our findings indicate that the maturational effects of vitamin D sterols are independent of their capacity to prime cells for respiratory burst and that the Ca2+ ionophoretic effects of 1,25(OH)2D3 play a major role in such priming.

Antineoplastic Agents↗
PubMed1991

Subpopulations of fetal thymocytes defined by expression of T cell receptor/CD3 and IL-2 receptor. CD3 and IL-2 receptor alpha-chain are expressed on reciprocal cell populations.

The expression of the TCR/CD3 complex and the IL-2R alpha chain (p55) on fetal thymocytes has been analyzed by flow cytometry (FCM). Two-parameter immunofluorescence identified three subpopulations which were respectively IL-2R alpha-/CD3+, IL-2R alpha+/CD3-, or IL-2R alpha-/CD3-; no detectable population of IL-2R alpha+/CD3+ cells was found in unstimulated fetal thymocytes. Fractionation by "panning" and by sterile flow cytometric separation was used to characterize the functional responsiveness of these three subpopulations to a variety of stimuli. All three populations proliferated in response to PMA + ionomycin + rIL-2. In contrast, stimulation with anti-CD3 + IL-2 induced proliferation in IL-2R alpha-/CD3+ and IL-2R alpha-/CD3- but not in IL-2R alpha+/CD3- thymocytes. IL-2R alpha- cells, including sorted IL-2R alpha-/CD3- thymocytes, underwent a phenotypic change in response to in vitro stimulation with anti-CD3 + IL-2, resulting in the appearance of an IL-2R alpha+/CD3+ population that was not detected in freshly isolated thymocytes. The ability of fractionated fetal thymocytes to produce lymphokine in response to PMA + ionomycin was also evaluated. Only the IL-2R alpha-/CD3- fraction generated detectable IL-2. These findings demonstrate for the first time that CD3 and IL-2R alpha are expressed in a mutually exclusive fashion in fetal thymocytes and define three subpopulations of thymocytes that differ significantly in their proliferative and differentiative responses to TCR-mediated, IL-2R-mediated, and pharmacologic stimulation.

Animals↗
PubMed1991

Calcium-dependent eicosanoid metabolism by concanavalin A-stimulated human monocytes in vitro. Synergism with phorbol ester indicates separate regulation of leukotriene B4 synthesis and release.

Human monocytes obtained by counter-current centrifugal elutriation released arachidonic acid when challenged in vitro with Con A, as well as with other soluble (PMA or ionomycin) or particulate stimuli (serum-treated zymosan). Cyclo-oxygenase metabolites were the principal eicosanoids detected in the supernatants of Con A-stimulated, [3H]arachidonate-labeled monocytes, 5-Lipoxygenase (5-LO) products, such as leukotriene B4 (LTB4), were conspicuously absent. Release of arachidonate and its metabolites in response to Con A was dependent on the presence of extracellular Ca2+, but not Mg2+. In contrast to serum-treated zymosan challenge, which resulted in increased inositol trisphosphate and LTB4 release, Con A-induced inositol phospholipid hydrolysis in monocytes was limited to phosphatidylinositol or phosphatidylinositol monophosphate. Despite an inability to augment LTB4 release, Con A or PMA induced a loss of 5-lipoxygenase from a cytosolic compartment that was similar to that achieved with a calcium ionophore (ionomycin), a potent stimulus for LTB4 generation. When cell-associated LTB4 was evaluated, evidence for increased LTB4 production was obtained in response to either stimulus (PMA greater than Con A). In combination, however, PMA and Con A treatment resulted in monocyte LTB4 release comparable with that observed with the calcium ionophore or STZ. LTB4 release in response to all stimuli tested was inhibited by MK-886, a drug that binds to 5-lipoxygenase-activating protein. These results indicate the following: 1) Phospholipase A2 activation and attendant arachidonic acid release induced by agents that increase intracellular Ca2+ and/or generate diacylglycerol results in increased synthesis and release of PG and increased synthesis of leukotrienes, but not necessarily leukotriene release. 2) 5-LO translocation, which may occur independently of increased intracellular Ca2+, may be necessary for LTB4 generation but is insufficient for its release. 3) 5-Lipoxygenase-activating protein activity is necessary for 5-LO activation and LTB4 release in response to all stimuli investigated here. 4) Phorbol ester, an activator of protein kinase C, may synergize with agents such as Con A (which by themselves induce a minimal intracellular Ca2+ rise), so as to result in the release of LTB4. Thus, Con A may represent a class of surface receptor-aggregating agents that initiates inflammatory changes or immunomodulation associated with liberation of PG and might predispose to release of other inflammatory mediators, such as leukotrienes, in the presence of additional signals including protein kinase activation.

Arachidonate 5-Lipoxygenase↗
PubMed1991

Expression of the Blast-1 activation/adhesion molecule and its identification as CD48.

We have analyzed the induction and expression of Blast-1 at the mRNA and protein levels and demonstrated its identity with CD48. Blast-1/CD48 is expressed on a wider range of cell types, notably T cells and monocytes, than previously thought, but appears to be restricted to lymphoid and myeloid cells. Resting B and T cells express Blast-1/CD48 molecules at the cell surface; however, they lack the epitope recognized by the 17D6 mAb. Resting B cells express no detectable Blast-1/CD48 mRNA. Induction by EBV infection or stimulation with PMA, IL-4, or PHA results in increased levels of Blast-1/CD48 protein (both 6.28 and 17D6 epitopes) at the cell surface. Detailed analysis of EBV-induced expression revealed that it is due to increased steady-state levels of Blast-1/CD48 mRNA induced by transforming but not nontransforming strains of the virus. Induction by IL-1 beta, ionomycin, or suboptimal levels of PMA plus ionomycin results in increased expression of the 17D6 epitope only. In transfected Cos-7 cells Blast-1/CD48 at the cell surface expresses only the 6.28 epitope, whereas cytoplasmic molecules express both 17D6 and 6.28 epitopes. We suggest that these results are most consistent with the idea that Blast-1/CD48 molecules are complexed at the surface of resting cells and Cos-7 cells, resulting in masking of the 17D6 epitope. Activation causes dissociation of the complex, revealing the 17D6 epitope. The existence of 17D6+6.28- Blast-1/CD48 molecules was demonstrated by immunoprecipitation analysis, which also revealed that, unlike the rest of the molecules, this subset was resistant to digestion with glyosylphosphatidylinositol-specific phospholipase C.

Antibodies, Monoclonal↗
PubMed1991

Differential regulation of the tumor suppressor molecules, retinoblastoma susceptibility gene product (Rb) and p53, during cell cycle progression of normal human T cells.

We have activated resting human T lymphocytes to study the roles of the putative cell cycle control gene products, retinoblastoma susceptibility gene product (Rb) and p53, in regulating cell proliferation. After stimulation with phorbol, 12,13, dibutyrate and the calcium ionophore, ionomycin, which triggers a rapid entry of cells into G1 phase, we demonstrated Rb phosphorylation 24 h later, well before the onset of DNA synthesis. This finding, in contrast to reports using proliferating cell lines, implies that Rb phosphorylation is not a proximal event regulating the G1 to S transition. The production of p53 became detectable 3 to 6 h after addition of phorbol, 12,13,-dibutyrate and ionomycin, and peaked at 30 to 42 h. To further delineate the relationship of the synthesis and metabolism of the proteins to cell cycle progression, we used three agents to arrest progression of activated T cells at various points in the cell cycle. Aphidicolin arrested the cells at the G1/S boundary, whereas deferoxamine, an iron chelator, arrested the cells at an earlier stage of the cell cycle. Cyclosporin A blocked T cell activation at the earliest point in the cell cycle. In the presence of aphidicolin, Rb phosphorylation and p53 production proceeded normally whereas cyclosporin A inhibited both events. Although deferoxamine completely prevented Rb phosphorylation, p53 production was unaffected, suggesting a differential regulation of the two molecules. Our results place Rb phosphorylation and p53 production in the hierarchy of genetic events that are thought to regulate T lymphocyte progression through the cell cycle.

Aphidicolin↗
PubMed1991

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease.

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.

Antigens, CD↗