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F T Liu

Publications and source records attributed to F T Liu.

At least 127 records · Page 7Linked to original sources
PubMed1984

IgE class-restricted tolerance induced by neonatal administration of soluble or cell-bound IgE. Cellular mechanisms.

Certain aspects of the phenomenon of IgE class-restricted tolerance induced in mice by neonatal treatment with monoclonal IgE, either in soluble form or coupled to syngeneic spleen cells, were examined. The present studies document that this tolerance results from exposure to IgE molecules, irrespective of their antigen specificity, and the resulting effects are polyclonal in nature since IgE responses directed against antigenic determinants unrelated to the tolerance-inducing IgE molecules are affected. Moreover, such findings indicate that the molecular subregion(s) responsible for inducing IgE class-restricted tolerance resides in the epsilon heavy chain constant region domain(s) of IgE. When soluble IgE is employed, tolerance induction results from neonatal treatment with doses as low as 2.5 micrograms per injection per mouse; cell-bound IgE is considerably more potent, in terms of total dose required, since tolerance results from treatment with as few as 1 X 10(6) cells per injection (per mouse), equivalent to an absolute quantity of 0.2 ng of IgE per injection. This long-term class-specific tolerance appears to be a unique feature of the IgE antibody system, since treatment of mice with monoclonal antibodies of the IgA, IgG1, or IgG2b isotypes, either in soluble or cell-bound form, does not perturb antibody responses of their corresponding isotypes or in the IgE class. By analyzing the lymphoid cells of IgE-tolerant mice after they reached adulthood, the following observations were made: (a) lymphoid cells from such tolerant mice fail to develop FcR epsilon + cells upon in vitro stimulation with IgE, as is characteristically observed with lymphoid cells from nontolerant mice; and (b) mice rendered tolerant by neonatal treatment with soluble IgE possess IgE class-restricted suppressor T cells, demonstrable in adoptive transfer experiments, whereas no such suppressor cells are evident in mice in which cell-bound IgE was used for neonatal treatment. The latter observations could mean that two different mechanisms underlie the IgE class-restricted tolerance, or both mechanisms operate coordinately to varying degrees depending upon which regimen is used for tolerance induction, as discussed herein.

Animals↗
PubMed1984

Synthesis of surface immunoglobulin E receptor in Xenopus oocytes by translation of mRNA from rat basophilic leukemia cells.

Immunoglobulin E-binding activity was expressed in Xenopus oocytes injected with mRNA from rat basophilic leukemia cells which possess abundant immunoglobulin E (IgE) receptor. Such activity was demonstrated with intact oocytes by their binding of 125I-labeled mouse monoclonal IgE. Binding activity was specific as shown by the total inhibition of 125I-IgE binding by unlabeled IgE but not by unlabeled IgG1. The relevance of the IgE-binding activity to the IgE receptor was also supported by the absence of this activity in oocytes injected with mRNA from cells lacking surface IgE receptors. mRNA coding for the IgE-binding activity was enriched in fractions sedimenting at 13.5 S in sucrose density gradients. From oocytes injected with rat basophilic leukemia mRNA, two major polypeptides were isolated by affinity purification on IgE immunoadsorbent. One (Mr = 31,000) is equivalent in size to the previously identified "receptor-associated protein;" the other (Mr = 40,000) is speculated to be a partially glycosylated or unglycosylated form of the alpha subunit of the IgE receptor. The binding of IgE-coated fluorescent microspheres by oocytes injected with rat basophilic leukemia mRNA demonstrated the surface expression of the IgE-binding proteins.

Animals↗
PubMed1984

Expression of a biologically active fragment of human IgE epsilon chain in Escherichia coli.

cDNA corresponding to human IgE heavy (epsilon) chain mRNA was cloned from human IgE-secreting myeloma U266 cells. Partial nucleotide sequence analysis demonstrated that the cloned cDNA contained the coding region for about two-thirds of the CH2 and all of the CH3 and CH4 domains as well as the 3'-untranslated region. This epsilon cDNA was inserted into expression vector pUC7 and expression of an epsilon-chain fragment in Escherichia coli was demonstrated by protein blot analysis using 125I-labeled goat anti-human IgE as probe. The expression product was purified on a column of goat anti-human IgE-conjugated Sepharose 4B and the polypeptide was found to retain binding activity to human basophils.

Amino Acid Sequence↗
PubMed1984

Mouse monoclonal IgE antibodies specific for ragweed pollen antigens.

In order to obtain monoclonal IgE antibody specific for the major allergens in ragweed pollen extracts, hybridomas were constructed from spleens of mice immunized with ragweed antigen E (AgE). Two hybridomas were selected for thorough study, both secreting antibodies of the IgE class. Large quantities of IgE antibodies were isolated by affinity purification using Sepharose 4B conjugated with ragweed pollen proteins (Fraction A). Both MAbs were found to bind to a high molecular weight and heterogeneous population of proteins, but not to monomeric AgE as demonstrated by protein blot analysis. It is suspected that both MAbs react with low affinity with AgE determinants and binding could be demonstrated only with aggregated forms of AgE. Although the specific antigens with which they react are unknown, these monoclonal IgE antibodies should be useful reagents, complementing the previously obtained monoclonal anti-dinitrophenyl IgE antibody, for studying various aspects of the mouse and human IgE antibody systems.

Animals↗
PubMed1984

Biologically active molecules regulating the IgE antibody system: biochemical and biological comparisons of suppressive factor of allergy (SFA) and enhancing factor of allergy (EFA).

Studies in recent years directed at unraveling the complex regulatory mechanisms controlling IgE antibody production have demonstrated the existence of soluble factors that exert selective regulatory effects on the IgE antibody system. In addition, the demonstration of IgE-specific Fc receptors (FcR epsilon) on B and T lymphocytes, especially after exposure to high concentrations of IgE either in vivo or in vitro, has provided increasingly strong indications of an important role for such cells in the overall control of the IgE system. In our own laboratory, we have been studying soluble regulatory factors known as suppressive factor of allergy (SFA) and enhancing factor of allergy (EFA), which were initially identified by their selective, and opposing, regulatory effects on in vivo IgE responses in inbred mice. More recently, in an in vitro system in which it is possible to induce the de novo expression of FcR epsilon on lymphocytes cultured in the presence of monoclonal IgE, we reported that concomitant exposure of such cultured cells to SFA selectively blocked the induction of FcR epsilon expression. In the present study, we have extended these investigations by making a direct comparison between certain biological properties and biochemical characteristics of SFA and EFA. We found that SFA and EFA can be distinguished biochemically on the basis of size, SFA falling in the range of 30,000 daltons or so, and EFA falling in the range of 15,000 daltons. In examining their biological properties, we unexpectedly found that although SFA-enriched and EFA-enriched fractions exert dramatically distinct biological effects on in vivo IgE antibody synthesis (as implied by their names), the two respective active fractions are totally indistinguishable in their inhibitory effects on IgE-mediated induction of FcR epsilon + lymphocytes in vitro when intact spleen cell populations are exposed to monoclonal IgE. That the active entities in SFA and EFA responsible for inhibition of FcR epsilon induction do not exhibit IgE-binding properties was shown by the fact that adsorption on IgE-coupled affinity columns had no appreciable effect on their inhibitory capacities. This distinguishes these molecules from the IgE-binding factors described by Ishizaka and colleagues. Moreover, as previously documented for SFA, we found that EFA neither possesses immunoglobulinlike determinants nor binds to other immunoglobulin class molecules. Finally, chromatographically fractionated EFA was shown to exert its enhancing activity on in vivo IgE antibody production across strain barriers, thereby ruling out any genetic restriction on its functional capacity. A similar absence of genetic

Animals↗
PubMed1984

An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells.

The quantitative relationships between the secretion of a granule-associated mediator, beta-hexosaminidase, and the oxidative metabolism of arachidonic acid by the 5-lipoxygenase pathway were analyzed for a homogeneous population of T cell-dependent, bone marrow-derived, murine mast cells. The mast cells were either sensitized with a monoclonal IgE and challenged with specific antigen, or to bypass a transmembrane signal, were stimulated with calcium ionophore A23187. The released products of the 5-lipoxygenase pathway were quantitated by integrated ultraviolet absorbance after resolution by reverse phase-high performance liquid chromatography in the case of 5-hydroxy-eicosatetraenoic acid (5-HETE), and by separate radioimmunoassays for leukotriene C4 (LTC4) and leukotriene B4 (LTB4). The activation-release response of the cells was perturbated by the introduction of three pharmacologic agents, each directed to different steps in the 5-lipoxygenase pathway of arachidonic acid metabolism, and the action of each agent was determined for separate cell samples while present and after its removal by washing. 5,6-Dehydroarachidonic acid (5,6-DHA), an irreversible inhibitor of 5-lipoxygenase, prevented formation of 5-HETE from exogenous [14C]arachidonic acid and from membrane-derived arachidonic acid in a dose-related fashion when sensitized mast cells, preincubated with drug, were washed before antigen activation. Release of 5-HETE, LTC4, and LTB4 was inhibited by 5,6-DHA in a corresponding dose-related fashion, with a minimal preincubation period of 1 to 5 min before the cells were washed and subjected to antigen-dependent activation. In contrast, the inhibitory effect of 5,6-DHA on beta-hexosaminidase release was lost after three washes and was not evident after one wash unless the preincubation period was extended to 15 min. The capacity of 5,6-DHA to prevent leukotriene generation without altering beta-hexosaminidase release was also observed with ionophore-activated mast cells. Preincubation of sensitized cells with diethylcarbamazine (DEC), followed by a wash before antigen-dependent activation, produced inhibition of leukotriene generation, no effect on beta-hexosaminidase release, and augmentation of 5-HETE release at the maximum dose studied; thus, DEC interrupts the pathway distal to the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) from arachidonic acid by 5-lipoxygenase. Preincubation of sensitized cells with incremental amounts of the prostacyclin analog U-60,257, followed by washing and antigen challenge, inhibited LTC4 release without altering the release of LTB4 or beta-hexosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)

8,11,14-Eicosatrienoic Acid↗
PubMed1984

Cloned mouse mast cells derived from immunized lymph node cells and from foetal liver cells exhibit characteristics of bone marrow-derived mast cells containing chondroitin sulphate E proteoglycan.

Cloned mouse mast cells which were T cell growth-dependent were derived both from immunized lymph node and from foetal liver, and were found to be morphologically and biochemically similar to mast cells previously differentiated in vitro from mouse bone marrow (BMMC). These two T cell growth-dependent mouse mast cell clones were identical to the BMMC in their preferential synthesis of chondroitin sulphate E proteoglycan rather than heparin proteoglycan. The hydrodynamic size of the cell-associated proteoglycan from each of the three mast cell sources was 150,000-250,000 mol. wt.; and that of the covalently bound glycosaminoglycans was 13,000-25,000 mol. wt. Chondroitinase ABC digestion of the [35S]proteoglycans from both cloned mast cells, as well as the BMMC, yielded only two disaccharides which comigrated on ascending thin layer chromatography with delta Di-4S and delta Di-diSE standards, respectively. Quantification of the radioactivity in the enzyme digests revealed that one-sixth to one-half of the resulting disaccharides were disulphated, similar to that found in BMMC containing chondroitin sulphate E. When sensitized with monoclonal IgE, washed, and subsequently challenged with specific antigen, each of the two cloned mast cells generated more than 100 ng of leukotriene C4 (LTC4)/10(6) cells, but only 3-12 ng leukotriene B4 (LTB4)/10(6) cells, characteristics also observed for the BMMC. Based upon these observations, it is concluded that the cloned mast cells from lymph node and liver and the bone marrow-derived mast cell belong to a distinct subclass of mast cells. These mast cells have been designated E-mast cells (E-MC) in order to distinguish them from heparin-containing mast cells (H-MC).

Animals↗
PubMed1983

IgE-mediated chemotaxis of rat basophilic leukemia cells towards specific antigen.

We evaluated chemotactic properties of four sublines of rat basophilic leukemia cells using blindwell Boyden chamber assays. After sensitization with a mouse monoclonal IgE directed against dinitrophenyl (DNP), cells from sublines 2H3-C and 926a underwent chemotaxis toward DNP-bovine serum albumin (BSA) and sublines RBL-1 and 4A did not. Chemotactic responses required specific IgE and were determined by the IgE antigen specificity used for sensitization. The threshold for chemotaxis was on the order of 10(-10) M DNP-BSA. Release of incorporated [3H]-serotonin did not always parallel chemotactic responses, which suggests that chemotaxis and secretion may be two unlinked processes that occur during basophil activation. Our results predict a possible in vivo mechanism whereby specific chemotactic responses of basophils and other FcR epsilon-bearing cells are mediated via specific IgE bound to membrane FcR epsilon.

Animals↗
PubMed1983

IgE-mediated release of leukotriene C4, chondroitin sulfate E proteoglycan, beta-hexosaminidase, and histamine from cultured bone marrow-derived mouse mast cells.

Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation with the release of histamine, beta-hexosaminidase, chondroitin sulfate E proteoglycan, and leukotriene C4 (LTC4). The chondroitin sulfate E nature of the glycosaminoglycan side chain was established by demonstrating that the chondroitinase ABC disaccharide digestion products were composed of equal quantities of 4-sulfated and 4,6-disulfated N-acetyl-galactosamine. The single immunoreactive sulfidopeptide leukotriene, released and quantitated with a class-specific antibody, was identified as LTC4 by its retention time on reverse-phase high-performance liquid chromatography and by its specific spasmogenic activity on the guinea pig ileum. The release of the preformed mediators, as well as of LTC4, was related in a dose-response fashion to the concentration of monoclonal IgE used during the sensitization step and to the concentration of specific antigen used to initiate the activation-secretion response. The optimal concentrations of IgE for sensitization and of antigen for challenge were the same for the release of preformed mediators and of LTC4. In addition, the time courses of their release were superimposable, with a plateau at 5 min after antigen challenge. The release of three preformed mediators and of LTC4 after fixation of IgE, washing of the sensitized cells, and antigen challenge unequivocally indicates a bone marrow-derived mast cell origin for these products. Linear regression analyses of the net percent release of beta-hexosaminidase to histamine and of 35S-chondroitin sulfate E to beta-hexosaminidase yielded straight lines that intersected at the origin, which indicates that the three preformed mediators are localized in the secretory granules of the bone marrow-derived mast cells. The concomitant generation of 23 ng of LTC4/10(6) sensitized bone marrow-derived mast cells represents the first example of IgE-dependent release of substantial amounts of LTC4, a component of slow reacting substance of anaphylaxis, from a mast cell population of greater than 95% purity. The IgE-dependent generation of LTC4, rather than prostaglandin D2, by the chondroitin sulfate E proteoglycan-containing bone marrow-derived mast cells contrasts with the predominant generation of prostaglandin D2 by heparin proteoglycan-containing mast cells. These differences together support the existence of two phenotypically different mast cell subclasses.

Animals↗
PubMed1983

Monoclonal antibodies against vasoactive intestinal polypeptide: studies of structure and related antigens.

Hybridomas secreting monoclonal anti-vasoactive intestinal polypeptide (VIP) antibodies were constructed from spleen cells sensitized to VIP in vitro. The secreted antibodies were characterized by binding to VIP in indirect radioimmunoassays and enzyme-linked immunosorbent assays. Two monoclonal antibodies, characterized for their binding activities with synthetic fragments of VIP, were found to bind different sites on the VIP molecule. These monoclonal antibodies may recognize tertiary structures of the VIP. A search was conducted for antigens recognized by the monoclonal antibodies in brain: brain proteins separated on polyacrylamide gels were electroblotted onto nitrocellulose filters and were reacted first with the mouse antibody and then with goat anti-mouse immunoglobulin coupled to horseradish peroxidase as a means of detection. The monoclonal antibodies were found to react with a protein of molecular weight 60,000, which was also recognized by polyclonal antibodies, although the latter reacted with a number of additional proteins. The relationship of the protein of molecular weight 60,000 to VIP is discussed.

Adrenal Gland Neoplasms↗
PubMed1983

Synthesis and myocardial kinetics of N-13 and C-11 labeled branched-chain L-amino acids.

Glutamate dehydrogenase (GDH), immobilized on CNBr-activated Sepharose supports, was used with N-13 ammonia to aminate alpha-ketoisocaproic acid (KIC), and alpha-ketoisovaleric acid (KIV) to produce N-13-labeled branched-chain L-amino acids with radiochemical yields ranging from 29% to 35%. From kinetic and practical considerations, pH 7.5-8.0 was established to be optimal for the synthesis of N-13-labeled branched-chain-L-amino acids. Myocardial time-activity curves in dogs at control, during low-flow ischemia, reperfusion, and after transaminase inhibition following intracoronary bolus injection of the N-13-labeled amino acids were biexponential. Higher retention of N-13 activity was observed in ischemic segments both during low-flow ischemia (29.2%) and reperfusion (23.2%) when compared with controls (20.0%), (n = 4). On the other hand, transaminase inhibition decreased residue fractions from 21.0% at control to 13.9% (n = 4). The residual activity with L-[1-11C]leucine allows for the calculation of protein synthesis rates.

Amino Acids, Branched-Chain↗
PubMed1982

Secretion of leukotriene C and other arachidonic acid metabolites by macrophages challenged with immunoglobulin E immune complexes.

Resident mouse peritoneal macrophages release the slow-reacting substance leukotriene C (LTC) on exposure to particulate IgE immune complexes. Because these cells lose their responsiveness to an IgE stimulus after 4 h in culture, maximum release of 20:4 metabolites is observed before this time. However, a similar diminution in 20:4 metabolism was not observed with a zymosan stimulus. Freshly explanted cells are deficient in intracellular glutathione (GSH) (12.4 +/- 0.4 pmol/micrograms cell protein), but GSH increases to a steady state value of 30-35 pmol/micrograms of cell protein between 3 and 9 h of culture. Because GSH is required for the synthesis of LTC and prostaglandin (PG)E2, cultures challenged immediately after explanation have a diminished capacity to synthesize these 20:4 metabolites and release prostacyclin as the major product. By 4-5 h in culture, macrophages form significant amounts of LTC and PGE2. Under optimum conditions of maximum responsiveness to an IgE stimulus and GSH content (after 4 h of culture), macrophages challenged with latex beads coated with IgE immune complexes synthesize 1.0 +/- 0.3 pmol of LTC/microgram cell protein (60 +/- 18 pmol/10(6) cells) in addition to prostacyclin (8.2 +/- 0.8 pmol/micrograms cell protein) and PGE2 (4.7 +/- 1.5 pmol/micrograms cell protein). These amounts are quantitatively similar to the arachidonic acid metabolites produced by macrophages challenged with IgG immune complex-coated latex beads or zymosan. These data demonstrate that macrophages produce large quantities of LTC and other 20:4 metabolites in response to particle-bound IgE and antigen, provided that the appropriate in vitro conditions are met. The macrophage might, therefore, be a major source of slow-reacting substance and other 20:4 metabolites generated during IgE-mediated reactions in vivo.

Animals↗
PubMed1982

IgE immune complexes stimulate arachidonic acid release by mouse peritoneal macrophages.

Resident mouse peritoneal macrophages were labeled with [3H]arachidonic acid and challenged with Sephadex beads coated with immune complexes of IgE and antigen. Arachidonic acid release by the cells was assessed by the quantity or radiolabel recovered from the culture medium. Freshly isolated macrophages responded to IgE immune complexes with a release of [3H]arachidonic acid that was linear for 1-2 hr. The magnitude of the response was dependent on both the number of immune complex-coated beads and on the degree of opsonization of the beads. Under conditions of maximal stimulation, macrophages challenged with IgE immune complex-coated Sephadex released 23 +/- 4.5% of their incorporated radiolabel. This is compared to values of 34.2 +/- 0.5% and 38.1 +/- 3.3% for cultures that received IgG immune complex-coated Sephadex or zymosan, respectively. Macrophages did not release arachidonic acid upon exposure to soluble IgE and antigen given sequentially or simultaneously, and soluble IgE did not inhibit the cells' response to IgE immune complexes. Incubation of macrophages for longer than 3 hr prior to challenge resulted in a selective loss in the cells' ability to respond to IgE immune complexes. After 16 hr of culture, macrophages released only 3.9 +/- 0.3% of their incorporated 3H on exposure to IgE immune complexes; however radiolabel release in response to zymosan (42.0 +/- 0.8%) was identical to that of freshly isolated cells. These data indicate that macrophages are capable of releasing arachidonic acid in response to preformed particulate immune complexes of IgE and antigen. Because Sephadex beads are too large to be interiorized by the cells, this response results from the interaction of the immune complexes with the macrophage plasma membrane.

Animals↗
PubMed1982

Cloning and nucleotide sequence of mouse immunoglobulin epsilon chain cDNA.

cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.

Amino Acid Sequence↗
PubMed1981

A clonal analysis of the IgE response and its implications with regard to isotope commitment.

In a clonal analysis of the IgE response, it was found that a small proportion of primary or nonimmune B cells in spleen and mesenteric lymph nodes can be stimulated by antigen to produce IgE-secreting clones. In addition, there appears to be no substantial difference in the frequency of such cells between the classical low and high IgE responder strains. An analysis of immune, or memory, B cells revealed substantial increases in the frequency of B cells secreting IgE as compared with primary B cells, although the actual proportion of B cells secreting IgE remained relatively low. When the IgE-secreting clones derived from either primary or secondary B cells were reanalyzed for the presence of other isotypes, it was found that all clones secreting IgE were secreting at least one other isotype, with the majority secreting two or three other isotypes in addition to IgE. This demonstrates that there is no distinct subpopulation of B cells committed to IgE expression per se.

Animals↗
PubMed1981

A sensitive enzyme-linked immunosorbent assay for the quantitation of antigen-specific murine immunoglobulin E.

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of 2,4-dinitrophenyl (DNP)-specific murine immunoglobulin (Ig) E is described. The assay uses beta-galactosidase, which is conjugated to goat anti-rabbit gamma-globulin (GARG) via a method using a mild heterobifunctional cross-linking reagent, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). The assay has a sensitivity for detection of about 200 pg/ml of anti-DNP IgE. Analyses of murine serum samples using this ELISA correlate well with those obtained using the passive cutaneous anaphylaxis (PCA) reaction in rats. With the use of automated 96-well reader, data acquisition is rapid and, therefore, this ELISA is ideal for analyses of large numbers of samples. The assay can be easily modified for the measurement of other Ig classes and of IgE of other antigen specificities.

Animals↗
PubMed1981

Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles.

Thirteen peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the hepatitis B surface antigen were synthesized chemically. The free or carrier-linked synthetic peptides were injected into rabbits, and 7 of the 13 elicited an antipeptide response. Antisera against four of the six soluble peptides longer than 10 amino acids were reactive with native antigen and specifically precipitated the 23,000- and 28,000-dalton forms from Dane particles. As the hepatitis molecule had not been chosen for study because of any structural feature suggesting unique opportunities for success, these results suggest that the strategy is general and should work for any protein as long as enough domains are studied. Peptides such as these could prove to be ideal vaccines.

Amino Acid Sequence↗